CN115819484A

CN115819484A - Double-stranded oligonucleotide, composition containing double-stranded oligonucleotide, conjugate, preparation method and application

Info

Publication number: CN115819484A
Application number: CN202210873191.7A
Authority: CN
Inventors: 梁子才; 杨志伟; 刘国成; 李海涛; 王晨钰
Original assignee: Beijing Ruibo Kaifa Pharmaceutical Technology Co ltd; Suzhou Ruibo Biotechnology Co ltd
Current assignee: Beijing Ruibo Kaifa Pharmaceutical Technology Co ltd; Suzhou Ruibo Biotechnology Co ltd
Priority date: 2021-07-23
Filing date: 2022-07-22
Publication date: 2023-03-21

Abstract

A double-stranded oligonucleotide comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, each nucleotide in the double-stranded oligonucleotide being independently a modified or unmodified nucleotide, a portion of the sense strand and the antisense strand being reverse-complementary to form a double-stranded region, wherein the 5 'terminal nucleotide of the antisense strand is a nucleotide having a ribose 5' modification, the modified nucleotide having a structure according to formula (101). The double-stranded oligonucleotides, pharmaceutical compositions and/or oligonucleotide conjugates of the present disclosure have better activity and have lower toxicity.

Description

Double-stranded oligonucleotide, composition containing double-stranded oligonucleotide, conjugate, preparation method and application

Technical Field

The present disclosure relates to a modified double-stranded oligonucleotide and pharmaceutical compositions and oligonucleotide conjugates containing the same. The disclosure also relates to methods of making and uses of these double-stranded oligonucleotides, pharmaceutical compositions and oligonucleotide conjugates.

Background

Double-stranded oligonucleotides are known as pharmaceutically active ingredients. In recent years, considerable progress has been made in the development of double-stranded oligonucleotide pharmaceuticals.

In the research of double-stranded oligonucleotide drug, the safety of double-stranded oligonucleotide itself is always the focus of research. For example, WO2019/105437A1 in the prior art discloses an siRNA or a pharmaceutical composition or an siRNA conjugate comprising the siRNA, which shows a better target gene expression inhibition activity in a mouse, but in subsequent studies, it is found that the siRNA still shows a certain hepatotoxicity, thereby hindering further application thereof in drug development. Therefore, how to obtain double-stranded oligonucleotides having both good pharmaceutical activity and low toxicity is still a need for further exploration in the art and there is still a related unsolved need in actual development.

Disclosure of Invention

The inventors have surprisingly found that a modified double-stranded oligonucleotide formed by subjecting the first nucleotide at the 5 '-end of the antisense strand of the double-stranded oligonucleotide to a specific ribose 5' chemical modification, as well as pharmaceutical compositions and oligonucleotide conjugates comprising the same, have high stability, good activity in vivo and low toxicity. Thus, the inventors have made the following invention.

In a first aspect, the present disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, each nucleotide in the double-stranded oligonucleotide being independently a modified or unmodified nucleotide, a portion of the sense and antisense strands being reverse-complementary to form a double-stranded region, wherein the 5 '-terminal nucleotide of the antisense strand is a nucleotide having a ribose 5' modification, the modified nucleotide having a structure according to formula (101), the group R being ₀ Constituting the 5' overhang of the antisense strand, R ₀ Has a structure as shown in formula (102):

wherein:

R ₂₀₁ is a hydroxyl or phosphate group:

G ₁ is OH, O ^- Or OJ ₁ Wherein J ₁ Is C1-C6 alkyl, substituted C1-C6 alkyl, C3-C6 cycloalkyl or substituted C3-C6 cycloalkyl;

Bx ₁ is hydrogen, a heterocyclic base or a base substitution group, and the 3' end of the sense strand, if including an overhang, bx ₁ (ii) does not base pair with the overhang, and the base substitution group is phenyl or substituted phenyl; bx ₂ Is a heterocyclic base;

z is a group having one of divalent linking groups represented by the structures represented by the following formulae (Z1) to (Z5), or Z is 1,2-cycloalkylene or heterocyclylene group having 3 to 6 carbon atoms, or substituted 1,2-cycloalkylene or heterocyclylene group having 3 to 6 carbon atoms:

wherein, P ₁ And P ₂ Each independently is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, or substituted C2-C6 alkenyl;

P ₃ is O or S;

X ₁ is H or hydroxy, X ₂ Selected from H, halogen, hydroxy, C1-C6 alkoxy or substituted C1-C6 alkoxy;

T ₂ is a phosphate subunit or a phosphorothioate subunit; y is ₁ 、Y ₂ 、Y ₃ 、Y ₄ 、Y ₅ 、Y ₆ 、Y ₇ And Y ₈ Each independently is H, halogen, hydroxy, methyl, ethyl, n-propyl, or isopropyl;

indicates the site at which the group is covalently attached.

In a second aspect, the present disclosure also provides a pharmaceutical composition comprising the double-stranded oligonucleotide provided by the present disclosure and a pharmaceutically acceptable carrier.

In a third aspect, the present disclosure also provides an oligonucleotide conjugate comprising a double-stranded oligonucleotide provided in the present disclosure and a conjugate group conjugated to the double-stranded oligonucleotide, wherein the conjugate group comprises a linker and a pharmaceutically acceptable targeting group and/or a delivery assisting group, and the double-stranded oligonucleotide, the linker and the targeting group or the delivery assisting group are covalently or non-covalently linked in sequence, each targeting group is selected from a ligand capable of binding to a cell surface receptor, and each delivery assisting group is selected from a group capable of increasing biocompatibility of the oligonucleotide conjugate in a delivery target organ or tissue.

In a fourth aspect, the present disclosure also provides the use of a double-stranded oligonucleotide, pharmaceutical composition and/or oligonucleotide conjugate of the present disclosure in the manufacture of a medicament for the treatment and/or prevention of a disease or condition associated with mRNA levels expressed by a target moiety.

In a fifth aspect, the present disclosure also provides a method of treating and/or preventing a disease or condition associated with mRNA levels of target gene expression, the method comprising administering to a subject in need thereof a double-stranded oligonucleotide, pharmaceutical composition and/or oligonucleotide conjugate of the present disclosure.

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

In a seventh aspect, the present disclosure also provides a kit comprising a double-stranded oligonucleotide, a pharmaceutical composition and/or an oligonucleotide conjugate of the present disclosure.

Is incorporated by reference

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

Advantageous effects

The double-stranded oligonucleotides, pharmaceutical compositions and/or oligonucleotide conjugates of the present disclosure exhibit significantly lower toxicity while maintaining good stability, better target gene expression modulating activity. The concrete description is as follows:

first, the double-stranded oligonucleotides, pharmaceutical compositions, and/or oligonucleotide conjugates of the present disclosure exhibit lower toxic reactions in vivo. For example, when the double-stranded oligonucleotide of the present disclosure is siRNA, the siRNA conjugate provided by the present disclosure is observed for 14 days continuously at a single subcutaneous administration dose of 30mg/kg for each rat, the rat is dissected, and the liver histopathological section is observed under an optical microscope, and it is found that the rat administered with the conjugate of the present disclosure has a clear liver cable structure, a tight arrangement of liver cells, a clear boundary, abundant cytoplasm, uniform staining, a circular nucleus, a normal size, a complete and normal venous endothelium, and no obvious abnormality of tissues, while the rat administered with the reference conjugate has extensive edema and degeneration of liver cells, cell swelling, loose staining of cytoplasm, and a large number of liver cells are also accompanied with steatosis, a variable number of circular vacuoles are visible in the rat, and a plurality of inflammatory focal infiltrations are visible in lobules. Indicating a significant reduction in hepatotoxicity of the siRNA conjugates of the present disclosure compared to the reference siRNA conjugate.

Second, the double-stranded oligonucleotides, pharmaceutical compositions and/or oligonucleotide conjugates of the present disclosure exhibit excellent target gene expression modulating activity in vitro cell experiments. For example, when the double-stranded oligonucleotide of the present disclosure is an siRNA, the siRNA conjugate provided by the present disclosure shows excellent target gene inhibitory activity in vitro cell experiments. In some embodiments, the siRNA conjugates provided by the present disclosure exhibit a target mRNA expression level inhibition of 81.63% -84.04% in HBV transgenic mouse liver primary cells at an siRNA concentration of 10 nM.

In addition, the double-stranded oligonucleotides, pharmaceutical compositions and/or oligonucleotide conjugates of the present disclosure also have greater stability and/or greater activity in vivo. For example, where the double-stranded oligonucleotide of the present disclosure is an siRNA, the siRNA conjugate provided by the present disclosure may have higher target gene inhibitory activity in vivo. For example, the siRNA conjugates provided by the present disclosure exhibit a high HBV mRNA inhibition rate of 60.85% -69.73% in HBV transgenic mice at an administration dose of 0.1 mg/kg. As another example, the siRNA conjugate of the present disclosure showed high inhibition rate of FXI mRNA in mouse liver tissue at day 8, day 15 and day 29 at siRNA administration dose of 3mg/kg, up to 82%, and the FXI mRNA inhibition rate did not show a decreasing trend during the experiment, suggesting that it is possible to stably and efficiently inhibit FXI mRNA over a longer period of time. As another example, the siRNA conjugates of the present disclosure exhibited high inhibition rates of ANGPTL3mRNA in mouse liver tissue at day 8, day 15, and day 29 at a siRNA administration dose of 3mg/kg, up to 92%, and still exhibited at least 63% inhibition rate of ANGPTL3mRNA at day 29.

Therefore, the double-stranded oligonucleotide and oligonucleotide conjugate provided by the disclosure can effectively regulate the expression of a target gene in vivo and in vitro for a long time, and have low toxicity, so that the double-stranded oligonucleotide and oligonucleotide conjugate can effectively treat and/or prevent diseases and/or symptoms related to the mRNA level expressed by the target gene while having significantly higher safety, and have good application prospects.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:

FIG. 1 is a line graph showing the relative expression levels of FXI mRNA at days 8, 15 and 29 after administration of 3mg/kg of different siRNA conjugates in liver tissues of mice compared to a blank control.

Figure 2 is a line graph showing the relative expression levels of ANGPTL3mRNA at day 8, day 15, and day 29 post-administration in liver tissue of mice after administration of 3mg/kg of different siRNA conjugates compared to a blank control.

Detailed Description

The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

In the above and following, unless specific meanings are provided, in connection with the analytical chemistry, synthetic organic chemistry and medicinal chemistry described herein used nomenclature and its procedures and techniques are those well known and commonly used in the art. Standard calculations can be used for chemical synthesis and chemical analysis.

Definition of

In the above and below, the capital letters C, G, U, T, A denote the base composition of nucleotides, unless otherwise specified; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates a phosphorothioate-based linkage between two nucleotides adjacent to the left and right of the letter s; the letter combination VP indicates that the adjacent nucleotide on the right side of the letter combination VP is a vinylphosphate (5' - (E) -vinylphosphonate, E-VP) modified nucleotide.

In the above and the following, the "fluorine-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group of the nucleotide is substituted with fluorine, and the "non-fluorine-modified nucleotide" refers to a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorine group. "nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Such as a heteronucleotide, a bridged nucleotide (BNA for short) or an acyclic nucleotide. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group.

In the present context, the expressions "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to the skilled person, i.e. in a double stranded nucleic acid molecule, the bases of one strand each pair with bases on the other strand in a complementary manner. 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 comprises a purine and a pyrimidine. Two strands are considered to be complementary to each other when adenine on one strand always pairs with thymine (or uracil) on the other strand and guanine always pairs with cytosine, and the sequence of that strand can be deduced from the sequence of its complementary strand. Accordingly, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at the corresponding positions are not paired in a complementary fashion.

In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that no more than 1 base mismatch exists between two nucleotide sequences; "completely reverse complementary" means that there is no base mismatch between two nucleotide sequences.

In the above and the following, particularly in describing the method for preparing the double-stranded oligonucleotide, the pharmaceutical composition or the oligonucleotide conjugate of the present disclosure, unless otherwise specified, the Nucleoside monomer (Nucleoside monomer) means a modified or unmodified Nucleoside phosphoramidite monomer (sometimes referred to as Nucleoside phosphoramidites) used in solid phase synthesis of phosphoramidites, depending on the kind and order of nucleotides in the double-stranded oligonucleotide or oligonucleotide conjugate to be prepared. Solid phase phosphoramidite synthesis is a method used in RNA synthesis well known to those skilled in the art. Nucleoside monomers for use in the present disclosure are all commercially available.

In the above or below, unless otherwise stated, a "substituted" group refers to a group in which a hydrogen atom in the group is replaced with one or more substituents. For example, "substituted C1-C5 hydrocarbyl" refers to a C1-C5 hydrocarbyl group in which one or more hydrogen atoms have been replaced with a substituent. It will be appreciated by those skilled in the art that compounds useful in the application of the present disclosure may contain various substituents, and that the introduction of such substituents may be useful in the present disclosure as long as the introduction does not interfere with the function of the present disclosure and the purpose of the present disclosure can be achieved. In some embodiments, the substituents are selected from the group consisting of: c ₁ -C ₁₀ Alkyl radical, C ₆ -C ₁₀ Aryl radical, C ₅ -C ₁₀ Heteroaryl, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl, -C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkyl, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl, -C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ Haloalkyl, halogen substituents, -OH, -SH, -NH ₂ 、-C ₁ -C ₁₀ alkyl-NH ₂ 、-N(C ₁ -C ₁₀ Alkyl) (C ₁ -C ₁₀ Alkyl), -NH (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) (C ₁ -C ₁₀ Alkylphenyl), -NH (C) ₁ -C ₁₀ Alkylphenyl), cyano, nitro, -CO ₂ H、-C(O)O(C ₁ -C ₁₀ Alkyl), -CON (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkyl), -CONH (C) ₁ -C ₁₀ Alkyl), -CONH ₂ ，-NHC(O)(C ₁ -C ₁₀ Alkyl), -NHC (O) (phenyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ Alkyl, -C (O) C ₁ -C ₁₀ Alkylphenyl, -C (O) C ₁ -C ₁₀ Haloalkyl, -OC (O) C ₁ -C ₁₀ Alkyl, -SO ₂ (C ₁ -C ₁₀ Alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ Haloalkyl), -SO ₂ NH ₂ 、-SO ₂ NH(C ₁ -C ₁₀ Alkyl), -SO ₂ NH (phenyl), -NHSO ₂ (C ₁ -C ₁₀ Alkyl), -NHSO ₂ (phenyl) and-NHSO ₂ (C ₁ -C ₁₀ Haloalkyl). In some embodiments, the substituent is C ₁ -C ₃ Alkyl radical, C ₆ -C ₈ Aryl, -OC ₁ -C ₃ Alkyl, -OC ₁ -C ₃ One of alkyl phenyl, halogen substituent, cyano or nitro. It will be understood by those skilled in the art that for any group containing one or more substituents, these groups are notIt is intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically non-feasible, and/or inherently unstable.

As used herein, "halogen" refers to F, cl, br, or I. As used herein, "alkyl" refers to straight and branched chain saturated hydrocarbon groups having the specified number of carbon atoms, typically 1 to 20 carbon atoms, for example 1 to 10 carbon atoms, such as 1 to 8 or 1 to 3 carbon atoms. For example, C1-C6 alkyl groups contain straight and branched chain alkyl groups of 1 to 6 carbon atoms. When referring to a residue having a particular number of alkyl groups, it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to the same residue as alkyl, but with two points of attachment.

As used herein, "alkenyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double bond obtained by the removal of one hydrogen molecule from the adjacent carbon atom of the parent alkyl group. The group may be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to: a vinyl group; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl; butenyl, e.g., but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl, and the like. In certain embodiments, alkenyl groups have 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Cycloalkenyl refers to a monocyclic hydrocarbon group having a specified number of carbon atoms and at least one carbon-carbon double bond.

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

As used herein, "cycloalkyl" refers to saturated or unsaturated and non-aromatic hydrocarbon ring groups having 3 to 14 carbon atoms, unless specifically stated otherwise. Examples of cycloalkyl groups include, but are not limited to: cyclopropyl, methylcyclopropyl, 2,2-dimethylcyclobutyl, 2-ethylcyclopentyl, or cyclohexyl. The cycloalkyl group may contain a plurality of spiro or fused rings. Cycloalkyl is optionally mono-, di-, tri-, tetra-or penta-substituted at any position as allowed by conventional valency.

As used herein, "alkoxy" refers to an alkyl group of the indicated number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-methylpentyloxy, and the like. Alkoxy groups typically have 1 to 10,1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through an oxygen bridge.

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

"heteroaryl" refers to a group derived from a3 to 18 membered aromatic ring radical containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, heteroaryl may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., it comprises a cyclic delocalized (4 n + 2) pi-electron system according to Huckel theory. Heteroaryl includes fused or bridged ring systems. Alternatively, the heteroatom in the heteroaryl group is an oxidized heteroatom. Alternatively, one or more nitrogen atoms (if present) are quaternized. The heteroaryl group is attached to the rest of the molecule through any ring atom. Examples of heteroaryl groups include, but are not limited to: <xnotran> , , , , 3584 zxft 3584- , , , [ d ] , , [ b ] [ 4284 zxft 4284 ] (benzo [ b ] [1,4]dioxepinyl), [ b ] [ 5325 zxft 5325 ] (benzo [ b ] [1,4]oxazinyl), 5623 zxft 5623- (6262 zxft 6262-benzodioxanyl), , , (benzodioxolyl), (benzodioxinyl), , , , , , [ 3256 zxft 3256-d ] , , [ 3456 zxft 3456 ] [ 3838 zxft 3838-a ] , , (cinnolinyl), [ d ] , 5749 zxft 5749- -5H- [ 6595 zxft 6595 ] [ 6898 zxft 6898-d ] , 3428 zxft 3428- [ h ] (3476 zxft 3476-dihydrobenzo [ h ] quinazolinyl), 3734 zxft 3734- [ h ] (5,6dihydrobenzo[h]cinnolinyl), 3757 zxft 3757- -5H- [ 5852 zxft 5852 ] [ 3575 zxft 3575-c ] , , , , , [ 3625 zxft 3625-c ] , 3826 zxft 3826- [ d ] , 3828 zxft 3828- [ d ] , 3925 zxft 3925- [ d ] , , , (indazolyl), , , </xnotran> Indolinyl, isoindolinyl, isoquinolinyl, indolizinyl, isoxazolyl, 5,8-methanol-5,6,7,8-tetrahydroquinazolinyl (5,8-methano-5,6,7,8-tetrahydroquinazolinyl), naphthyridinyl, 1,6-naphthyridinyl (5749-naphthyridinyl), oxadiazolyl, 2-oxazepinyl (2-oxoazepinyl) oxazolyl, oxacyclopropanyl (oxaraninyl), 5,6,6a,7,8,9,10 a-octahydrobenzo [ H ] quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl (phthalazinyl), pteridinyl (pteridinyl), purinyl, pyrrolyl, pyrazolyl, pyrazolo [ 6595 zft 6595-d ] pyrimidinyl, pyridinyl, pyrido [ 6898 zft 6898-d ] pyrimidinyl, o-substituted pyrido [ pteridinyl ] quinolinyl, pyrido [ 1H ] quinolinyl, and pyrido [ 1H ] quinolinyl pyrido [3,4-d ] pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl (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-cyclohepta [4,5] thieno [2,3-d ] pyrimidinyl, 5,6,7,8-tetrahydropyrido [4,5-c ] pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno [2,3-d ] pyrimidinyl, thieno [ 5678-d ] pyrimidinyl, thieno [ 39-74zxft 7439-c ] pyridinyl (8624 zienyl) and thienyl [ 8624 zienyl/8624 z/thienyl).

"heterocyclyl" refers to a stable 3-to 18-membered non-aromatic ring group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless stated otherwise in the specification, a heterocyclic group is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, and may include fused or bridged ring systems. Alternatively, the heteroatom in the heterocyclic group is an oxidized heteroatom. Alternatively, one or more nitrogen atoms (if present) are quaternized. Heterocyclyl groups are partially or fully saturated. The heterocyclyl group may be attached to the remainder of the molecule through any ring atom. Examples of such heterocyclic groups include, but are not limited to: dioxanyl, thienyl [1,3] dithioyl (thienyl [1,3] dithianyl), decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithioacyl (trithiofuranyl), tetrahydropyranyl, thiomorpholinyl (thiomorpholinyl), 1-oxothiomorpholinyl (1-oxo-thiomorpholinyl), and 1,1-dioxothiomorpholinyl (1,1-dioxothiomorpholinyl).

As used herein, "heterocyclic base" refers to a nucleobase or a modified nucleobase. In some embodiments, the heterocyclic base is a pyrimidine, substituted pyrimidine, purine or 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 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 substituted pyrimidine.

Various protecting groups, such as hydroxyl protecting groups, can be used in the present disclosure. In general, protecting groups render a chemical functional group insensitive to particular reaction conditions, and can be added to and removed from that functional group in a molecule without substantially damaging the remainder of the molecule. Representative hydroxyl protecting Groups are disclosed in Beaucage et al, tetrahedron 1992,48,2223-2311, and Greenand Wuts, protective Groups in Organic Synthesis, chapter 2,2d, john Wiley &sons, new York,1991, each of which is incorporated herein by reference in its 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 hydroxy protecting groups that may be used herein include Dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl), and 9- (p-methoxyphenyl) xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4 '-dimethoxytrityl), and TMTr (4,4', 4 "-trimethoxytrityl).

The term "subject", as used herein, refers to any animal, e.g., a mammal or a marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, rabbits, sheep, rats, and any species of poultry. As used herein, "treatment" refers to a method of obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.

As used herein, "prevention" refers to a method of obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," a double-stranded oligonucleotide, pharmaceutical composition, or oligonucleotide conjugate can be administered to a subject at risk for developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.

Double-stranded oligonucleotides

In a first aspect, the present disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, each nucleotide in the double-stranded oligonucleotide being independently a modified or unmodified nucleotide, a portion of the sense strand and the antisense strand being reverse-complementary to form a double-stranded region, wherein the 5 'terminal nucleotide of the antisense strand is a nucleotide having a ribose 5' modification, the modified nucleotide having a structure as shown in formula (101), the group R being ₀ Constituting the 5' overhang of the antisense strand, R ₀ Has a structure as shown in formula (102):

wherein:

R ₂₀₁ is hydroxyl or phosphate;

G ₁ is OH, O ^- Or OJ ₁ Wherein, J ₁ Is C1-C6 alkyl, substituted C1-C6 alkyl, C3-C6 cycloalkyl or substituted C3-C6 cycloalkyl;

P ₁ and P ₂ Each independently is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, or substituted C2-C6 alkenyl;

P ₃ is O or S;

T ₂ is a phosphate subunit or a phosphorothioate subunit; y is ₁ 、Y ₂ 、Y ₃ 、Y ₄ 、Y ₅ 、Y ₆ 、Y ₇ And Y ₈ Each independently H, halogen, hydroxy, methyl, ethyl, n-propyl or isopropyl;

indicates the site at which the group is covalently attached.

As will be understood by those skilled in the art, bx ₁ Is hydrogen, a heterocyclic base or a base substitution group, and the 3' end of the sense strand, if including an overhang, bx ₁ Not base-pairing with the overhang, the base-substitution group being phenyl or substituted phenyl, B _x2 The object of the present invention can be achieved without changing the properties of the siRNA of the present disclosure in the case of heterocyclic bases. In the context of the present disclosure, heterocyclic base refers to a nucleobase or a modified nucleobase. In some embodiments, the heterocyclic base is a pyrimidine, a substituted pyrimidinePurine or substituted purine. For ease of synthesis, in some embodiments, the heterocyclic base is uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.

In some embodiments, the base substitution group is phenyl or substituted phenyl, meaning that one or more hydrogens on the phenyl group are each independently replaced with F, cl, CH ₃ 、CH ₂ F or CF ₃ Substitution; in some embodiments, the substituted phenyl group means that one or more hydrogens on the phenyl group are each independently substituted with F, cl or CH ₃ The resulting group is substituted.

In some embodiments, Z is a divalent linking group represented by any one of formulas (Z1) to (Z5) that enables linking of two nucleotide molecules in formula (101). In some embodiments, Z has a structure represented by formula (Z1) or (Z2), and P in formulae (Z1) and (Z2) ₁ And P ₂ Each independently is H.

According to the invention, Z can also be 1,2-cycloalkylene or heterocyclylene with 3-6 carbon atoms, or substituted 1,2-cycloalkylene or heterocyclylene with 3-6 carbon atoms, and the structure does not change the properties of the double-stranded oligonucleotide of the disclosure, and the invention purpose of the disclosure can also be achieved. In some embodiments, Z is 1,2-cyclopropylene, in view of ease of synthesis, structure/process cost, and the like.

According to the invention, X ₁ And X ₂ The choice of (a) may affect the structure and properties of the double-stranded oligonucleotide. In some embodiments, X ₁ Independently selected from H or hydroxy, X ₂ Selected from hydroxyl or OCH ₃ . In some embodiments, X ₁ Is H, X ₂ Is OCH ₃ 。

For synthetic convenience, in some embodiments, Y ₁ 、Y ₂ 、Y ₃ 、Y ₄ 、Y ₅ 、Y ₆ 、Y ₇ And Y ₈ Are all H.

In some embodiments, formula (101) is selected from one of the following structures:

wherein, base refers to C, G, U, T or A. The double-stranded oligonucleotide comprising a ribose 5' -modified nucleotide represented by one of formulae (501) to (507) at the 5' -end of the nucleotide sequence II in the antisense strand, so that the overhang at the 5' -end of the antisense strand can have a further good balance of the target gene expression regulatory activity and low toxicity.

As previously mentioned, in the double-stranded oligonucleotides of the present disclosure, each nucleotide is a modified or unmodified nucleotide. In the context of the present disclosure, the term "modified nucleotide" is used to refer to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with other groups, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotide does not result in a significant impairment or loss of the function of the double-stranded oligonucleotide to regulate gene expression. For example, one can select the modified nucleotides disclosed in J.K.Watts, G.F.Delevay, and M.J.Damha, chemical modified siRNA: tools and applications.drug discovery Today,2008, 13 (19-20): 842-55.

In some embodiments, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, wherein the 5 'terminal nucleotide of the nucleotide sequence II is a nucleotide having a ribose 5' modification, the modified nucleotide having a structure represented by formula (101); the nucleotide sequence I consists of 19 nucleotides, the group shown in the formula (101) is counted as 2 nucleotides, the nucleotide sequence II consists of 20 nucleotides, and the group R ₀ Form a reverseAn overhang at the 5' end of the sense strand, and the rest of the nucleotide sequence II and the nucleotide sequence I form a double-stranded region, wherein the nucleotide sequence II is at least partially reverse-complementary to a first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence with the length of 19 nucleotides in mRNA expressed by a target gene; in the direction from the 5 'end to the 3' end, the 7 th to 9 th nucleotides of the nucleotide sequence I are fluoro-modified nucleotides; according to the direction from the 5 'end to the 3' end, the 3 rd, 7 th, 15 th and 17 th nucleotides of the nucleotide sequence II are fluorine modified nucleotides.

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

In some embodiments, nucleotides 2 to 20 of the nucleotide sequence II is substantially reverse complementary, or fully reverse complementary to the first stretch of nucleotide sequence in a5 'end to 3' end direction.

In some embodiments, at least the nucleotides at positions 3-20 of the nucleotide sequence II are fully reverse complementary to the nucleotides at positions 1-18 of the first stretch of nucleotide sequence in the 5 'to 3' direction.

In some embodiments, the 2 nd to 20 th nucleotides of the nucleotide sequence II are substantially reverse complementary, or fully reverse complementary to the nucleotide sequence I in the 5 'end to 3' end direction.

In some embodiments, the 2 nd to 20 th nucleotides of the nucleotide sequence II are fully reverse complementary to the nucleotide sequence I in the 5 'end to 3' end direction, or there is a base mismatch between the 3 rd nucleotide of the nucleotide sequence II and the 2 nd nucleotide of the nucleotide sequence I in the 3 'end to 5' end direction in the 5 'end to 3' end direction.

In some embodiments, the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, each nucleotide of the nucleotide sequence III and the nucleotide sequence IV is independently one of non-fluorinated modified nucleotides, the nucleotide sequence III is 1,2, 3 or 4 nucleotides in length, the nucleotide sequence IV and the nucleotide sequence III are equal in length, and the nucleotide sequence IV and the nucleotide sequence III are substantially reverse complementary or fully reverse complementary, the nucleotide sequence III is linked at the 5 'end of the nucleotide sequence I, the nucleotide sequence IV is linked at the 3' end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or fully reverse complementary to a second nucleotide sequence that is adjacent to the first nucleotide sequence and has the same length as the nucleotide sequence IV in the mRNA expressed by the target gene. Thus, a double-stranded oligonucleotide of the present disclosure may have a double-stranded complementary region of 19-23 nucleotides in length.

In some embodiments, the double-stranded oligonucleotide further comprises a nucleotide sequence V, each nucleotide of said nucleotide sequence V being independently one of the non-fluorinated modified nucleotides, said nucleotide sequence V being 1 to 3 nucleotides in length, attached to the 3 'terminus of the antisense strand, thereby constituting a 3' overhang of the antisense strand. Thus, the ratio of the lengths of the sense and antisense strands of a double-stranded oligonucleotide provided by the present 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.

In some embodiments, the nucleotide sequence V is 2 nucleotides in length and is, in the direction from the 5' end to the 3' end, 2 consecutive thymidylate nucleotides, 2 consecutive uracil ribonucleotides, or is fully reverse complementary to a third nucleotide sequence that is adjacent to the 5' end of the first or second nucleotide sequence and is equal in length to the nucleotide sequence V in an mRNA expressed by the target gene. Thus, in some embodiments, the sense and antisense strands of the double-stranded oligonucleotides of the disclosure each have a length of 19/21 nucleotides or 21/23 nucleotides, at which point the double-stranded oligonucleotides of the disclosure have better target gene expression modulating activity.

In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group.

In some embodiments, each of the non-fluorinated modified nucleotides is a methoxy modified nucleotide, which refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group. In the context of the present disclosure, "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with fluorine, which has a structure represented by the following formula (7). "non-fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group, or a nucleotide analog. In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group.

The nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group is substituted with a non-fluorine group is known to those skilled in the art, and the nucleotide may be one selected from the group consisting of a 2' -alkoxy-modified nucleotide, a 2' -alkyl-modified nucleotide, a 2' -substituted alkyl-modified nucleotide, a 2' -amino-modified nucleotide, a 2' -substituted amino-modified nucleotide, and a 2' -deoxynucleotide.

In some embodiments, the 2 '-alkoxy modified nucleotide is a methoxy modified nucleotide (2' -OMe), as shown in formula (8). In some embodiments, 2 '-amino modified nucleotides (2' -NH) ₂ ) As shown in formula (9). In some embodiments, 2' -deoxynucleosidesAcid (DNA) is represented by formula (10):

a nucleotide analog refers to a group that can replace a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, the nucleotide analog can be a heteronucleotide, a bridged nucleotide (BNA for short), or an acyclic nucleotide.

BNA refers to a constrained or inaccessible nucleotide. BNAs may contain five-, six-, or seven-membered ring bridged structures with "fixed" C3' -endo-sugar pull-down. The bridge is typically incorporated at the 2'-, 4' -position of the ribose to provide a 2',4' -BNA nucleotide. In some embodiments, BNA may be LNA, ENA, cET BNA, etc., where LNA is as shown in equation (12), ENA is as shown in equation (13), and cET BNA is as shown in equation (14):

acyclic nucleotides are a class of nucleotides in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide can be an Unlocked Nucleic Acid (UNA) or a Glycerol Nucleic Acid (GNA), wherein UNA is represented by formula (15) and GNA is represented by formula (16):

in the above formulae (15) and (16), R is selected from H, OH or an alkoxy (O-alkyl) group.

An isonucleotide is a compound formed by changing the position of a base in a nucleotide on a ribose ring. In some embodiments, the isonucleotides can be compounds in which the base moves from the 1' -position to the 2' -position or the 3' -position of the ribose ring, as shown in formula (17) or (18).

In the compounds of the above-mentioned formula (17) -formula (18), base represents a nucleic acid Base, for example A, U, G, C or T; r is selected from H, OH, F or a non-fluorine group as described above.

In some embodiments, the nucleotide analog is selected from one of a heteronucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each of the non-fluorinated modified nucleotides is a methoxy modified nucleotide, which refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group, both supra and infra.

In the above and hereinafter, "fluoro-modified nucleotide", "2 '-fluoro-modified nucleotide", "nucleotide in which 2' -hydroxyl group of ribose group is substituted with fluorine" and "nucleotide having 2 '-fluoro-ribosyl group" have the same meaning, and all refer to a compound having a structure represented by formula (7) in which 2' -hydroxyl group of nucleotide is substituted with fluorine; the terms "methoxy-modified nucleotide", "2 '-methoxy-modified nucleotide", "nucleotide in which 2' -hydroxyl group of ribose group is substituted with methoxy group" and "nucleotide having 2 '-methoxy ribosyl group" are the same, and refer to a compound having a structure represented by formula (8) in which 2' -hydroxyl group of ribose group of nucleotide is substituted with methoxy group.

The double-stranded oligonucleotide with the modification is low in cost, and can ensure that the double-stranded oligonucleotide is not easily cut by ribonuclease in blood, so that the stability of the double-stranded oligonucleotide is improved, and the double-stranded oligonucleotide has stronger resistance to nuclease hydrolysis. Meanwhile, the modified double-stranded oligonucleotide has higher activity of regulating the expression of a target gene.

In some embodiments, at least 1 of the phosphate groups in the phosphate-sugar backbone of at least one of the sense strand and the antisense strand is a phosphate group having a modifying group. In some embodiments, the phosphate group having a modifying group is a phosphorothioate group formed by substituting at least one oxygen atom in a phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, the phosphate group having a modifying group is a phosphorothioate group having a structure as shown in formula (121):

the modification can stabilize the double-stranded structure of the double-stranded oligonucleotide, and maintain high specificity and high affinity of base pairing.

In some embodiments, the phosphate group having a modifying group is present in at least one of the group consisting of:

between the 1 st and 2 nd nucleotides at the 5' terminal end of the sense strand;

between the 2 nd and 3 rd nucleotides at the 5' terminal end of the sense strand;

the 3' terminal end of the sense strand is between the 1 st nucleotide and the 2 nd nucleotide;

between the 2 nd and 3 rd nucleotides at the 3' terminal end of the sense strand;

between the 3 rd and 4 th nucleotides of the 5' terminal end of the antisense strand;

between the 1 st and 2 nd nucleotides at the 3' terminal end of the antisense strand; and

the 3' terminal end of the antisense strand is between the 2 nd and 3 rd nucleotides.

The double-stranded oligonucleotides of the present disclosure can be a variety of double-stranded oligonucleotides that modulate gene expression. In some embodiments, can be a double-stranded oligonucleotide that inhibits or downregulates gene expression, such as siRNA; in some embodiments, it may be a double-stranded oligonucleotide that activates or upregulates gene expression, e.g., saRNA.

The use of the disclosed double-stranded oligonucleotides unexpectedly reduces toxicity while also exhibiting excellent target gene expression modulating activity.

The modified double-stranded oligonucleotides, pharmaceutical compositions and oligonucleotide conjugates provided by the present disclosure can be used to modulate the abnormal expression of various genes, and treat various pathological conditions or diseases caused by the abnormal expression of genes. Alternatively, the double-stranded oligonucleotides, pharmaceutical compositions, and nucleotide conjugates provided by the present disclosure can be used to treat or ameliorate a pathological condition or disease associated with gene expression by modulating the gene expression. These genes may be various endogenous genes in the human body or animal body, or may be genes of pathogens which propagate in the human body or animal body. Double-stranded oligonucleotides having specific nucleotide sequences and the modification schemes can be designed and prepared based on the mRNA expressed from the target gene. In some embodiments, the mRNA expressed by the target gene is selected from one of 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, SOD, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT.

In some embodiments, the double-stranded oligonucleotide is an siRNA and the mRNA expressed by the target gene is selected from the mRNA expressed by the hepatitis b virus gene (HBV). In some embodiments, the double-stranded oligonucleotide is a siRNA and the mRNA expressed by the target gene is selected from the group consisting of plasma coagulation Factor XI (Factor XI, FXI) expressed mRNA. In some embodiments, the double-stranded oligonucleotide is an siRNA and the mRNA expressed by the target gene is selected from the group consisting of an angiopoietin-like protein 3 (ANGPTL 3) expressed mRNA.

In some embodiments, the siRNA is an siRNA having the sequence shown in table 1 below. Wherein, for siRNA1-7, the mRNA expressed by the target gene is selected from mRNA expressed by hepatitis B virus gene (HBV); for siRNAs 8 to 14, the mRNA expressed by the target gene is selected from mRNA expressed by plasma coagulation Factor XI (Factor XI, FXI); for siRNA15-21, the mRNA expressed by the target gene is selected from mRNA expressed by angiopoietin-like protein 3 (ANGPTL 3).

TABLE 1 siRNA sequences

Wherein the capital letters C, G, U, A represent the base composition of nucleotides; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that the two nucleotides to the left and right of the letter are phosphorothioate-based linkages, of siRNAs 1-7VPDThe sequences are respectively the structures shown as formula (501) to formula (507), siRNA8 to 14VPDThe sequences are respectively the structures shown in the formulas (501) to (507), siRNA15 to 21VPDThe structures shown in the formulae (501) to (507) are shown in this order.

Methods of making double-stranded oligonucleotides of the disclosure

The method for preparing the double-stranded oligonucleotide comprises the steps of synthesizing a sense strand and an antisense strand of the double-stranded oligonucleotide according to a double-stranded oligonucleotide sequence expected by a solid phase phosphoramidite method, and annealing the sense strand and the antisense strand to form an oligonucleotide double strand. The difference is that in the case of connecting the last nucleotide at the 5 '-end of the antisense strand, a 5' -ribose-modified phosphoramidite monomer is used as a nucleoside phosphoramidite monomer for the ligation and the sulfurization reaction is carried out. The 5' ribose-modified phosphoramidite monomer is described below. In some embodiments, the method of making further comprises isolating and purifying the double-stranded oligonucleotide.

In some embodiments, when the double-stranded oligonucleotide is an siRNA, the method of making an siRNA of the present disclosure comprises:

(1) Synthesizing a sense strand of the siRNA by a phosphoramidite solid phase synthesis method according to the 3'-5' direction;

(2) Synthesizing an antisense strand of siRNA by a phosphoramidite solid phase synthesis method according to the 3'-5' direction;

(3) The sense and antisense strands of the siRNA are isolated and annealed to obtain the siRNA of the present disclosure.

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

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

The coupling reaction conditions include a temperature of 0-50 ℃, in some embodiments 15-35 ℃, and a molar ratio of nucleic acid sequence attached to the solid support to nucleoside monomer of 1:1-1, in some embodiments 1:5-1; the molar ratio of nucleic acid sequence attached to the solid support and coupling reagent can be 1:1-1, in some embodiments 1.

Capping reaction conditions include a temperature of 0-50 deg.C, in some embodiments 15-35 deg.C, and a reaction time of 5-500 seconds, in some embodiments 10-100 seconds, with the same selection of capping reagents as previously described. The molar ratio of the total amount of capping reagent to nucleic acid sequence attached to the solid support can be 1. In the case where equimolar amounts of acetic anhydride and N-methylimidazole are used as capping reagents, the molar ratio of acetic anhydride, N-methylimidazole and nucleic acid sequence attached to the solid support can be 1.

The oxidation reaction conditions include a temperature of from 0 to 50 deg.C, in some embodiments from 15 to 35 deg.C, a reaction time of from 1 to 100 seconds, in some embodiments from 5 to 50 seconds, and the oxidizing agent, in some embodiments, being iodine (in further embodiments, provided in the form of iodine water). The molar ratio of oxidizing reagent to nucleic acid sequence attached to the solid support in the coupling step can be 1:1-100, in some embodiments 5:1-50. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran, water, pyridine = 3. The sulfurization reaction conditions include a temperature of from 0 to 50 deg.C, in some embodiments from 15 to 35 deg.C, a reaction time of from 50 to 2000 seconds, in some embodiments from 100 to 1000 seconds, and a sulfurizing agent, in some embodiments hydrogenated flavonones. The molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling step may be 10. In some embodiments, the sulfidation reaction is carried out in a mixed solvent of acetonitrile pyridine = 1:3-3:1.

The method of annealing in step (3) is also well known to those skilled in the art. For example, the synthesized sense strand and antisense strand can be simply mixed in equimolar ratio in water for injection and heated to 70-95 ℃ followed by cooling at room temperature to allow formation of a double-stranded structure by hydrogen bonding. This gives the siRNA of the present disclosure.

The purity and molecular weight of the nucleic acid sequence can be readily determined during synthesis to better control the quality of the synthesis, methods of detection being well known to those skilled in the art. For example, nucleic acid purity can be detected by ion exchange chromatography and molecular weight determined by liquid chromatography-mass spectrometry (LC-MS).

After obtaining the sirnas of the present disclosure, in some embodiments, the synthesized sirnas can also be characterized by molecular weight detection, using methods such as liquid chromatography-mass spectrometry, and the like, to determine the sirnas designed for the target, e.g., corresponding to one of the sequences listed in table 1 above.

The 5' -modified phosphoramidite monomer has a structure represented by the following formula (301):

wherein, X ₁ 、X ₂ 、Z、Bx ₁ 、Bx ₂ 、Y ₁ 、Y ₂ 、Y ₃ 、Y ₄ 、Y ₅ 、Y ₆ 、Y ₇ And Y ₈ The definition and selection ranges of (a) are as described above. In some embodiments, bx in the compound of formula (301) ₁ Group and Bx ₂ The groups are respectively Bx in the group shown in the formula (101) ₁ Group and Bx ₂ Reactive functional groups (e.g., amino groups) in the group, if any, are all protected with a protecting group. When Bx1 is a heterocyclic base, commonly used protecting groups for reactive functional groups in nucleoside heterocyclic bases are well known to those skilled in the art and can be used in the double stranded oligonucleotides of the present disclosure. In some embodiments, X in the compound of formula (301) ₁ Group and X ₂ The groups are respectively X in the group shown in the formula (101) ₁ Group and X ₂ The reactive functional groups (e.g., hydroxyl groups) in the group, if any, are all protected with a protecting group. In some embodiments, X in the group of formula (101) ₁ The group is a hydroxyl group, in which case X in the compound represented by the formula (301) ₁ Is a protected hydroxy group, for example a hydroxy group protected with a silane protecting group (e.g., TBDMS or TBDPS). The protective groups can be removed in the subsequent synthesis of the double-stranded oligonucleotide, and active functional groups are released again, so that the prepared double-stranded oligonucleotide has the expression regulation activity of a target sequence.

R _k To provide R ₂₀₁ A group of (1). In some embodiments, R _k Is a group R ₂₀₁ Wherein all the active groups are protected by protecting groups. In some embodiments, R ₂₀₁ Is hydroxy, R _k Is a hydroxyl protecting group. The hydroxyl protecting group may be one or more of Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4 ' -bismethoxytrityl), TMTr (4,4 ',4' -trimethoxybenzyl). In some embodiments, R _k May be DMTr, i.e., 4,4 '-bismethoxytrityl (4,4' -dimethoxytrityl).

Each B ₁ Independently selected from substituted or unsubstituted C1-C5 hydrocarbyl; each B ₂ Independently selected from one of C1-C5 alkyl, cyanoethyl, cyanopropyl and cyanobutyl. In some embodiments, each B is ₁ Are each isopropyl or tert-butyl, each B ₂ Are each 2-cyanoethyl or 3-cyanopropyl.

Compounds of formula (301) are commercially available or one skilled in the art can prepare compounds of formula (301) using any reasonable synthetic route.

For example, the compound represented by formula (301) can be obtained by a method comprising the steps of: contacting a compound represented by formula (302) with a compound represented by formula (303) in an organic solvent under condensation reaction conditions and in the presence of a condensation reaction auxiliary agent, and isolating to obtain a compound represented by formula (301):

wherein R is _k 、X ₁ 、X ₂ 、Z、Bx ₁ 、Bx ₂ 、Y ₁ 、Y ₂ 、Y ₃ 、Y ₄ 、Y ₅ 、Y ₆ 、Y ₇ 、Y ₈ 、B ₁ And B ₂ The respective definitions and alternative ranges are as described above.

B ₃ Is N (B) ₁ ) ₂ Or halogen, in some embodiments, B ₃ Is diisopropylamino (N (iPr) ₂ ) Or chlorine.

The condensation reaction conditions comprise that the reaction temperature is 0-50 ℃ and the reaction time is 0.5-5h. In some embodiments, the reaction temperature is 15-35 ℃ and the reaction time is 1-3h. The reaction pressure may be normal pressure.

The organic solvent is one or more of epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N-dimethylformamide and N, N-diisopropylethylamine. In some embodiments, the epoxy-based solvent is dioxane and/or tetrahydrofuran, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether, and the haloalkane-based solvent is one or more of dichloromethane, chloroform, and 1,2-dichloroethane. In some embodiments, the organic solvent is N, N-diisopropylethylamine. The organic solvent is used in an amount of 1 to 50L/mol, and in some embodiments, 3 to 20L/mol, relative to the compound represented by the formula (302).

The condensation reaction auxiliary may be any substance that can promote the condensation reaction between the compound represented by formula (302) and the compound represented by formula (303) to produce the compound represented by formula (301). In some embodiments, B ₃ Is diisopropylamino, and the condensation reaction auxiliary agent is a mixture of N-methylimidazole and tetrazole. In some embodiments, B ₃ Is chlorine, and the reaction auxiliary agent is N, N-diisopropylethylamine. The molar ratio of the total amount of condensation reaction aid to the compound of formula (302) is 1:1-10, in some embodiments 1:1-5:1.

The molar ratio of the compound of formula (303) to the compound of formula (302) is 1:1-10, in some embodiments 1:1-5:1. The compound represented by formula (303) can be obtained commercially, or can be synthesized by a method known to those skilled in the art. In some embodiments, the compound of formula (303) is a readily commercially available bis (diisopropylamino) (2-cyanoethoxy) phosphine or 3- ((chloro (diisopropylamino) phosphono) oxy) propionitrile.

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

The compounds of formula (302) are commercially available or can be prepared by one skilled in the art by a reasonable synthetic route. In some embodiments, Z in formula (302) is a structure represented by any one of formulae (Z1) to (Z5), and the compound represented by formula (302) can be obtained by the following production method: the process comprises contacting a compound of formula (304) with a compound of formula (305) in an organic solvent under condensation reaction conditions in the presence of a condensation reaction aid and isolating to provide a compound of formula (302):

wherein, X ₁ 、X ₂ 、R _k 、Bx ₁ 、Bx ₂ 、Y ₁ 、Y ₂ 、Y ₃ 、Y ₄ 、Y ₅ 、Y ₆ 、Y ₇ 、Y ₈ And B ₂ The respective definitions and alternative ranges are as described above; z is a structure represented by any one of formulas (Z1) to (Z5), and W is Cl, br or I.

The condensation reaction conditions comprise that the reaction temperature is 20-90 ℃ and the reaction time is 0.5-5h. In some embodiments, the reaction temperature is 40-80 ℃ and the reaction time is 1-8h. The reaction pressure may be normal pressure. In some embodiments, the reaction may be carried out under microwave heating, for example, for 1 to 5 hours at 50 to 70 ℃ in a microwave.

The organic solvent is one or more of epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N-dimethylformamide and N, N-diisopropylethylamine. In some embodiments, the epoxy-based solvent is dioxane and/or tetrahydrofuran, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether, and the haloalkane-based solvent is one or more of dichloromethane, chloroform, and 1,2-dichloroethane. In some embodiments, the organic solvent is tetrahydrofuran. The organic solvent is used in an amount of 1 to 50L/mol, and in some embodiments, 3 to 20L/mol, relative to the compound represented by the formula (304).

The molar ratio of compound of formula (305) to compound of formula (304) is 1:1-10, in some embodiments 1:1-5:1.

The condensation reaction auxiliary agent is a mixture of a ligand, propylene oxide and a palladium-containing compound, or a mixture of 1,1' -bis-diphenylphosphino ferrocene palladium dichloride and propylene oxide. The ligand is selected from 1,1' -bisdiphenylphosphine ferrocene, 4,5-bisdiphenylphosphine-9,9-dimethylxanthene, 1,1' -binaphthol, triphenylphosphine and 2-dicyclohexylphosphine-2 ',4',6' -triisopropylbiphenyl. The palladium compound is selected from one of palladium acetate, palladium chloride, tetratriphenylphosphine palladium, bistriphenylphosphine palladium dichloride and palladium acetylacetonate, and is palladium acetate in some modes. In the condensation reaction auxiliary agent, the molar ratio of the ligand, the propylene oxide and the palladium-containing compound is as follows: (1-10) (40-80) (0.1-10), or 1,1' -bisdiphenylphosphinoferrocell dichloropalladium and propylene oxide in a molar ratio of 1:1-1, in some embodiments 1:1-5:1 in the catalyst. The molar ratio of the total amount of condensation reaction promoter to the compound of formula (304) is 1:1-10, in some embodiments 1:1-5:1. In some embodiments, the condensation reaction aid further comprises a tertiary amine, in some embodiments triethylamine. The molar ratio of triethylamine to the compound of formula (304) is 1:1-10, in some embodiments 1:1-5:1.

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

Formula (304) may be prepared by synthetic methods commonly used in the art or may be obtained commercially. For example, when R in formula (304) _k Is DMTr, Y ₅ 、Y ₆ 、Y ₇ And Y ₈ Are all H, B ₂ Is 2-cyanoethyl, B _x1 Is thymine, X ₁ In the case of 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, commercial Availabel 5' -DMT Phosphoramides as Reagents for the Synthesis of vinyliphonate-Linked Oligopeic Acids, scheme 2.

The compound represented by formula (305) can be obtained commercially, or can be synthesized by a method known to those skilled in the art. In some embodiments, the compound of formula (305) is commercially available

In some embodiments, formula (305) may be obtained by methods of preparation known in the art. For example, when Z in the formula (305) is a linking group represented by (Z1), W is Br, Y ₁ 、Y ₂ 、Y ₃ 、Y ₄ Are all H, B _x2 Is uracil, X ₂ Is OCH ₃ In The case of compounds of formula (305), they can be prepared according to The methods described in Collis, alana e.c. (2008) The synthesis of vinyliphonate-linked rna. Phd synthesis, university of Nottingham, scheme 81, with The only difference that The corresponding 2' -methoxy-modified nucleoside is used instead of a deoxyribonucleoside.

In some embodiments, the nucleoside phosphoramidite compounds having a ribose 5' modification represented by formula (301) have one of the structures represented by the following formulae (3011) to (3016):

pharmaceutical composition

In a second aspect, the present disclosure provides a pharmaceutical composition comprising a double-stranded oligonucleotide provided by the present disclosure, and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier may be a carrier conventionally used in the art of double-stranded oligonucleotide administration, such as, but not limited to, magnetic nanoparticles (e.g., fe-based) ₃ O ₄ Or Fe ₂ O ₃ Nanoparticles of (a), carbon nanotubes (carbon nanotubes), mesoporous silicon (mesopore silicon), calcium phosphate nanoparticles (calcium phosphate nanoparticles), polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer), polylysine (L-lysine), PLL (PLL), chitosan (chitosan), 1,2-dioleoyl-3-trimethylammonium propane (1,2-dioleoyl-3-trimethyommine-propane, DOTAP), poly (D-or L-type lactic acid/glycolic acid copolymer (poly (D-D)&L-lactic/glycolic acid) copolymer, PLGA, poly (2-aminoethylethylene phosphate), PPEEA, and poly (N, N-dimethylaminoethyl methacrylate), PDMAEMA, and derivatives thereof.

In some embodiments, the content of the double-stranded oligonucleotide and the pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly limited, and in some embodiments, the weight ratio of the double-stranded oligonucleotide to the pharmaceutically acceptable carrier may be 1 (1-500), and in some embodiments, the above weight ratio is 1 (1-50).

In some embodiments, the pharmaceutical composition may further comprise other pharmaceutically acceptable excipients, which may be one or more of various formulations or compounds conventionally employed in the art. For example, the pharmaceutically acceptable additional excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

The pH buffer may be a tris hydrochloride buffer at a pH of 7.5 to 8.5 and/or a phosphate buffer at a pH of 5.5 to 8.5, for example a phosphate buffer at a pH of 5.5 to 8.5.

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

The osmotic pressure regulator may be sodium chloride and/or potassium chloride. The osmolality adjusting agent is present in an amount such that the osmolality of the pharmaceutical composition is 200-700 milliosmoles per kilogram (mOsm/kg). The content of the osmolality adjusting agent can be easily determined by the skilled person, depending on the desired osmolality. In some embodiments, the dosage of the preparation prepared from the pharmaceutical composition during the administration process can be adjusted according to different administration modes.

In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection solution; or can be lyophilized powder for injection, and can be mixed with liquid adjuvant to make into liquid preparation. The liquid preparation can be used for subcutaneous, intramuscular or intravenous injection, and can also be used for delivering the pharmaceutical composition in a mode of spraying to the lung, or spraying to other organ tissues (such as liver), or oral pharynx inhalation, nasal administration and the like. In some embodiments, the pharmaceutical composition is for administration by nebulization.

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 liposome formulation comprises an amine-containing transfection compound (which may also be referred to hereinafter as an organic amine), a helper lipid, and/or a pegylated lipid. Wherein the organic amine, helper lipid, and pegylated lipid can be selected from one or more of the amine-containing transfection compounds described in chinese patent application CN103380113A (which is herein incorporated by reference in its entirety), or pharmaceutically acceptable salts or derivatives thereof, helper lipid, and pegylated lipid, respectively.

In some embodiments, the organic amine can be a compound described in chinese patent application CN103380113A as shown in formula (601) or a pharmaceutically acceptable salt thereof:

wherein:

X ₁₀₁ and X ₁₀₂ Each independently is O, S, N-A or C-A, wherein A is hydrogen or C ₁ -C ₂₀ A hydrocarbon chain;

Y ₁₀₁ and Z ₁₀₁ Each independently is C = O, C = S, S = O, CH-OH or SO ₂ ；

R ₁₀₁ 、R ₁₀₂ 、R ₁₀₃ 、R ₁₀₄ 、R ₁₀₅ 、R ₁₀₆ And R ₁₀₇ Each independently is hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or linear aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic group, a substituted or unsubstituted, branched or linear acyl group, a substituted or unsubstituted, branched or linear aryl group, a substituted or unsubstituted, branched or linear heteroaryl group;

x is an integer from 1 to 10;

n is an integer of 1 to 3, m is an integer of 0 to 20, p is 0 or 1; wherein if m = p =0, then R ₁₀₂ Is hydrogen;

and, if at least one of n or m is 2, then R ₁₀₃ And the nitrogen in formula (601) forms a structure as shown in formula (602) or formula (603):

wherein g, e and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each x N represents a nitrogen atom in formula (601).

In some embodiments, R ₁₀₃ Is a polyamine. In other embodiments, R ₁₀₃ Is a ketal. In some embodiments, R in formula (601) ₁₀₁ And R ₁₀₂ Each of which is independently any substituted or unsubstituted, branched or straight chain alkyl or alkenyl group having from 3 to about 20 carbon atoms, such as from 8 to about 18 carbon atoms, and from 0 to 4 double bonds, such as from 0 to 2 double bonds.

In some embodiments, if each of n and m independently has a value of 1 or 3, then R ₁₀₃ May be any one of the following formulae (604) to (613):

wherein, in formula (604) -formula (613), g, e and f are each independently an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, and each indicates R ₁₀₃ A possible point of attachment to the nitrogen atom in formula (601), wherein each H at any position may be replaced to achieve attachment to the nitrogen atom in formula (601).

The compound of formula (601) can be obtained by any reasonable method by one skilled in the art. In some embodiments, the compound of formula (601) can be prepared as described in chinese patent application CN 103380113A.

In some embodiments, the organic amine is an organic amine according to formula (614) and/or an organic amine according to formula (615):

the helper lipid is cholesterol, cholesterol analogue and/or cholesterol derivative;

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

In some embodiments, the molar ratio among the organic amine, the helper lipid, and the pegylated lipid in the pharmaceutical composition is (19.7-80): (0.3-50), and may be (50-70): (20-40): (3-20), for example.

In some embodiments, the particles of the pharmaceutical composition formed from the double-stranded oligonucleotides of the present disclosure and the amine-containing transfection reagents described above have an average diameter of about 30nm to about 200nm, typically about 40nm to about 135nm, more typically the liposome particles have an average diameter of about 50nm to about 120nm, about 50nm to about 100nm, about 60nm to about 90nm, or about 70nm to about 90nm, e.g., the liposome particles have an average diameter of about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160nm.

In some embodiments, the weight ratio (weight/weight ratio) of the double-stranded oligonucleotide to total lipid (e.g., organic amine, helper lipid, and/or pegylated lipid) in the pharmaceutical composition formed by the double-stranded oligonucleotide of the present disclosure and the amine-containing transfection reagent described above is in the range from about 1:1 to about 1, from about 1:1 to about 130, from about 1:3 to about 120, from about 1:4 to about 118, from about 1:5 to about 1.

In some embodiments, the pharmaceutical compositions may be sold with the components present separately and may be in the form of a liquid formulation for use. In some embodiments, the pharmaceutical composition of the double-stranded oligonucleotide provided by the present disclosure and the above pharmaceutically acceptable carrier can be prepared according to various known methods, except that the double-stranded oligonucleotide provided by the present disclosure is used to replace the existing double-stranded oligonucleotide; in some embodiments, the following methods may be used:

suspending organic amine, auxiliary lipid and polyethylene glycol lipid in alcohol according to the molar ratio, and uniformly mixing to obtain a lipid solution; the amount of alcohol used is such that the total mass concentration of the resulting lipid solution is 2-25mg/mL, for example, 8-18mg/mL. The alcohol is selected from pharmaceutically acceptable alcohols such as alcohols that are liquid at about room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, which may be, for example, ethanol.

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

The lipid solution and the aqueous solution of the double-stranded oligonucleotide are mixed, and the resulting mixture is incubated at 40-60 ℃ for at least 2 minutes, for example, 5-30 minutes, to obtain a liposome preparation after incubation. The volume ratio of the lipid solution to the aqueous solution of the double-stranded oligonucleotide is 1 (2-5), and may be, for example, 1:4.

Concentrating or diluting the incubated liposome preparation, removing impurities, and sterilizing to obtain the pharmaceutical composition provided by the disclosure, wherein the physicochemical parameters are that the pH value is 6.5-8, the entrapment rate is not less than 80%, the particle size is 40-200nm, the polydispersity index is not higher than 0.30, and the osmotic pressure is 250-400mOsm/kg; for example, the physical and chemical parameters may be pH 7.2-7.6, encapsulation efficiency not less than 90%, particle size 60-100nm, polydispersity index not more than 0.20, and osmotic pressure 300-400mOsm/kg.

Wherein the concentration or dilution may be performed before, after or simultaneously with the removal of the impurities. The impurities can be removed by various methods, such as ultrafiltration using a cut-phase flow system and a hollow fiber column under 100K Da conditions, and the ultrafiltration exchange solution is Phosphate Buffered Saline (PBS) with pH 7.4. The sterilization can be carried out by various methods, for example, by filtration sterilization on a 0.22 μm filter.

Oligonucleotide conjugates

In a third aspect, the present disclosure provides an oligonucleotide conjugate comprising a double-stranded oligonucleotide provided by the present disclosure, and a conjugate group conjugated to the double-stranded oligonucleotide. In some embodiments, the conjugate group comprises a linker and a pharmaceutically acceptable targeting group and/or a delivery assisting group, and the double-stranded oligonucleotide, the linker and the targeting group or the delivery assisting group are covalently or non-covalently linked in that order, each targeting group is selected from a ligand capable of binding to a cell surface receptor, and each delivery assisting group is selected from a group capable of increasing biocompatibility of the oligonucleotide conjugate in delivering a target organ or tissue.

In the context of the present disclosure, "conjugated," means that two or more chemical moieties, each having a particular function, are linked to each other in a covalent linkage, unless otherwise indicated; accordingly, "conjugate" refers to a compound formed by covalent linkage between the various chemical moieties. Further, "oligonucleotide conjugate" means a compound formed by covalently attaching one or more chemical moieties having a specific function to an oligonucleotide. An oligonucleotide conjugate is to be understood as a generic term for a plurality of oligonucleotide conjugates or an oligonucleotide conjugate of a certain formula, depending on the context. In the context of the present disclosure, a "conjugate molecule" is understood to be a specific compound that can be conjugated to an oligonucleotide by a reaction, ultimately forming an oligonucleotide conjugate of the present disclosure.

Generally, the conjugate group comprises at least one targeting group and optionally a linker (linker) that are pharmaceutically acceptable, and the double-stranded oligonucleotide, the linker and the targeting group are linked in sequence. In one embodiment, the number of targeting groups is 1-6. In one embodiment, the targeting group is 2 to 4. The double-stranded oligonucleotide molecule may be non-covalently or covalently conjugated to the conjugate group, e.g. may be covalently conjugated to the conjugate group. The site of conjugation of the double-stranded oligonucleotide to the conjugate group may be at the 3' end or 5' end of the sense strand of the double-stranded oligonucleotide, at the 5' end of the antisense strand, or in the internal sequence of the double-stranded oligonucleotide. In some embodiments, the site of conjugation of the double-stranded oligonucleotide to the conjugate group is at the 3' end of the sense strand of the double-stranded oligonucleotide.

In some embodiments, the conjugate group may be attached to the phosphate group, the hydroxyl group at the 2' -position, or the base of the nucleotide. In some embodiments, the conjugate group may be attached to the hydroxyl group at the 3' -position, when 2' -5' phosphodiester linkages are used between nucleotides. When a conjugate group is attached to the terminus of a double-stranded oligonucleotide chain, the conjugate group is typically attached to the phosphate group of the nucleotide; when a conjugate group is attached to the internal sequence of a double-stranded oligonucleotide, the conjugate group is typically attached to a ribose sugar ring or base. Reference may be made to the following connection modes: siRNA conjugates and subsequent assembled tertiary N-acetyl amino acids in ACS Chemical biology 2015,10 (5) 1181-7.

In some embodiments, the double-stranded oligonucleotide and the conjugate group may be linked by acid-labile, or reducible chemical bonds that are degradable under the acidic environment of the cellular endosome, thereby leaving the double-stranded oligonucleotide free. For non-degradable conjugation, the conjugate group can be attached to the sense strand of the double-stranded oligonucleotide, thereby minimizing the effect of conjugation on the activity of the double-stranded oligonucleotide.

The targeting group may be attached to the double-stranded oligonucleotide molecule via a suitable linker, which may be selected by one skilled in the art according to the particular type of targeting group. These linkers, the type of targeting group, and the manner of attachment to the double-stranded oligonucleotide can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.

In some embodiments, the targeting moiety may be a ligand conventionally used in the art of double-stranded oligonucleotide administration, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.

In some embodiments, at least one or each of the targeting moieties is selected from a ligand capable of binding to a cell surface receptor expressing the target gene.

In some embodiments, at least one or each of said targeting moieties is selected from a ligand capable of binding to a mammalian hepatocyte surface receptor. In some embodiments, each of the targeting groups is independently a ligand that has affinity for asialoglycoprotein receptors on the surface of mammalian liver cells. In some embodiments, each of the targeting groups is independently an asialoglycoprotein or a sugar. <xnotran> , D- , L- , D- , D- , L- , D- , L- , D- , L- , α -D- , β -D- , α -D- , β -D- , α -D- , β -D- , α -D- , β -D- , α -D- , α -D- , α -D- , β -D- , α -D- , β -D- , , , , N- , N- , N- , N- , N- ,2- -3-O- [ (R) -1- ] -2- - β -D- ,2- -2- -L- , 3238 zxft 3238- -4- -3262 zxft 3262- -O- -D- ,2- -2- -D- , </xnotran> N-glycolyl-alpha-neuraminic acid, 5-thio-beta-D-glucopyranose, 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranoside methyl ester, 4-thio-beta-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-alpha-D-glucopyranoside ethyl ester, 2,5-anhydro-D-allose nitrile, ribose, D-4-thioribose, L-ribose, L-4-thioribose. In some embodiments, at least one or each of the targeting groups is galactose or N-acetylgalactosamine.

In some embodiments, at least one or each of the targeting moieties is selected from a ligand capable of binding to a lung epithelial cell surface receptor. In some embodiments, each of the targeting moieties is selected from a moiety targeting integrin α v β 6 or a moiety targeting integrin α v β 3. In some embodiments, each of the targeting moieties is independently a polypeptide or a small molecule ligand.

In some embodiments, at least one or each of the delivery assisting 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 assisting groups is selected from lipophilic molecules. In some embodiments, each of the delivery assisting groups is C ₅ -C ₁₈ Straight chain hydrocarbon groups or steroids.

In some embodiments, the linker in the oligonucleotide conjugates of the present disclosure has a structure as shown in formula (701):

wherein k is an integer of 1 to 3;

L ^A has a structure containing an amide bond as shown in formula (702), L ^B Has a structure shown as a formula (303) and comprises N-acyl pyrrolidine, and L contains carbonyl and oxygen atoms ^C Is a linker group based on hydroxymethylaminomethane, dimethylolaminomethane or trimethylaminomethane;

wherein n is ₃₀₂ 、q ₃₀₂ And p ₃₀₂ Each independently is an integer from 2 to 6, optionally n ₃₀₂ 、q ₃₀₂ And p ₃₀₂ Each independently is 2 or 3; n is ₃₀₃ Is an integer from 4 to 16, optionally n ₃₀₃ Is an integer of from 8 to 12, and,

indicates the site at which the group is covalently attached.

In the joint, each L ^A Each linked to one of said targeting groups by an ether bond and via L ^C Oxygen atoms of hydroxy groups in the moiety with L ^C Are linked in part by an ether linkage; l is ^B By reacting a carbonyl group of the formula (703) with L ^C The nitrogen atoms of the amino groups in the moiety form an amide bond and are linked to the double-stranded oligonucleotide via an oxygen atom to form a phosphate or phosphorothioate bond via an oxygen atom in formula (703).

In some embodiments, the oligonucleotide conjugates provided by the present disclosure have a structure as shown in formula (705):

wherein Nu denotes a double-stranded oligonucleotide provided by the present disclosure, or a double-stranded oligonucleotide obtained according to the method of the present disclosure.

In some embodiments, the linker in the oligonucleotide conjugates of the present disclosure has a structure represented by formula (706):

wherein n is ₃₀₆ Is an integer of 0 to 3, each p ₃₀₆ Independently an integer from 1 to 6,

represents the site of covalent attachment of a group; the linking group forms an ether linkage with the targeting group via an oxygen atom indicated by; the linking group is bonded to the double-stranded oligonucleotide by forming a phosphate bond or a phosphorothioate bond with at least one of the oxygen atoms indicated by #, and the remaining oxygen atoms indicated by #, are bonded to a hydrogen atom to form a hydroxyl group, or are bonded to C ₁ -C ₃ Alkyl groups being linked to form C ₁ -C ₃ An alkoxy group;

in some embodiments, the oligonucleotide conjugates of the present disclosure have a structure as shown in formula (707):

In some embodiments, the oligonucleotide conjugates of the present disclosure have a structure represented by formula (708):

wherein the content of the first and second substances,

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

each m1, m2 or m3 is independently an integer selected from 2 to 10;

R ₁₀ 、R ₁₁ 、R ₁₂ 、R ₁₃ 、R ₁₄ or R ₁₅ Each independently is H, or is selected from the group consisting of: c ₁ -C ₁₀ Alkyl radical, C ₁ -C ₁₀ Haloalkyl and C ₁ -C ₁₀ An alkoxy group;

R ₃ has a structure represented by formula a 59:

wherein E is ₁ Is OH, SH or BH ₂ Nu denotes a double-stranded oligonucleotide provided by the present disclosure;

R ₂ is a straight chain alkylene group of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH = N, S (O) ₂ 、C ₂ -C ₁₀ Alkenylene radical, C ₂ -C ₁₀ Alkynylene, C ₆ -C ₁₀ Arylene radical, C ₃ -C ₁₈ Heterocyclylene and C ₅ -C ₁₀ A heteroarylene group; and wherein R ₂ May optionally have a substituent of any one or more of the group consisting of: c ₁ -C ₁₀ Alkyl radical, C ₆ -C ₁₀ Aryl radical, C ₅ -C ₁₀ Heteroaryl group, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl, -C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkyl, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl, -C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ Haloalkyl, halogen substituents, -OH, -SH, -NH ₂ 、-C ₁ -C ₁₀ alkyl-NH ₂ 、-N(C ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkyl), -NH (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) (C ₁ -C ₁₀ Alkylphenyl), -NH (C) ₁ -C ₁₀ Alkylphenyl), cyano, nitro, -CO ₂ H、-C(O)O(C ₁ -C ₁₀ Alkyl), -CON (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkyl), -CONH (C) ₁ -C ₁₀ Alkyl), -CONH ₂ 、-NHC(O)(C ₁ -C ₁₀ Alkyl), -NHC (O) (phenyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ Alkyl, -C (O) C ₁ -C ₁₀ Alkylphenyl, -C (O) C ₁ -C ₁₀ Haloalkyl, -OC (O) C ₁ -C ₁₀ Alkyl, -SO ₂ (C ₁ -C ₁₀ Alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ Haloalkyl), -SO ₂ NH ₂ 、-SO ₂ NH(C ₁ -C ₁₀ Alkyl), -SO ₂ NH (phenyl), -NHSO ₂ (C ₁ -C ₁₀ Alkyl), -NHSO ₂ (phenyl) and-NHSO ₂ (C ₁ -C ₁₀ Haloalkyl);

each L ₁ Independently a linear alkylene group of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: c (O), NH, O, S, CH = N, S (O) ₂ 、C ₂ -C ₁₀ Alkenylene radical, C ₂ -C ₁₀ Alkynylene, C ₆ -C ₁₀ Arylene radical, C ₃ -C ₁₈ Heterocyclylene and C ₅ -C ₁₀ A heteroarylene group; and wherein L ₁ May optionally have a substituent of any one or more of the group consisting of: c ₁ -C ₁₀ Alkyl radical, C ₆ -C ₁₀ Aryl radical, C ₅ -C ₁₀ Heteroaryl group, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl, -C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkyl, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl, -C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ Haloalkyl, halogen substituents, -OH, -SH, -NH ₂ 、-C ₁ -C ₁₀ alkyl-NH ₂ 、-N(C ₁ -C ₁₀ Alkyl) (C ₁ -C ₁₀ Alkyl), -NH (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) (C) ₁ -C ₁₀ Alkylphenyl), -NH (C) ₁ -C ₁₀ Alkylphenyl), cyano, nitro, -CO ₂ H、-C(O)O(C ₁ -C ₁₀ Alkyl radicals),-CON(C ₁ -C ₁₀ Alkyl) (C ₁ -C ₁₀ Alkyl), -CONH (C) ₁ -C ₁₀ Alkyl), -CONH ₂ ，-NHC(O)(C ₁ -C ₁₀ Alkyl), -NHC (O) (phenyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (C) ₁ -C ₁₀ Alkyl), -N (C) ₁ -C ₁₀ Alkyl) C (O) (phenyl), -C (O) C ₁ -C ₁₀ Alkyl, -C (O) C ₁ -C ₁₀ Alkylphenyl, -C (O) C ₁ -C ₁₀ Haloalkyl, -OC (O) C ₁ -C ₁₀ Alkyl, -SO ₂ (C ₁ -C ₁₀ Alkyl), -SO ₂ (phenyl), -SO ₂ (C ₁ -C ₁₀ Haloalkyl), -SO ₂ NH ₂ 、-SO ₂ NH(C ₁ -C ₁₀ Alkyl), -SO ₂ NH (phenyl), -NHSO ₂ (C ₁ -C ₁₀ Alkyl), -NHSO ₂ (phenyl) and-NHSO ₂ (C ₁ -C ₁₀ Haloalkyl);

represents the site of covalent attachment of a group;

M ₁ refers to targeting groups, the definition and alternative ranges of which are the same as above. In some embodiments, each M is ₁ Independently selected from one of the ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells.

The skilled person will understand that although for convenience L is used ₁ Is defined as a linear alkyl group, but it may not be a linear group or differ in name, for example, by an amine or alkenyl group resulting from the above substitution and/or displacement. For purposes of this disclosure, L ₁ Is the number of atoms in the chain connecting the two attachment points. For this purpose, a ring (e.g., a heterocyclylene or heteroarylene) obtained by substituting a carbon atom of the linear alkylene group is counted as one atom. In some embodiments, each L is ₁ A linked combination of one or more independently selected from the groups of formulae A1-a 26:

wherein j1 is an integer of 1 to 20; j1 is an integer from 1 to 20; r' is C1-C10 alkyl; ra is selected from one of the groups of the formula A27-A45:

rb is C1-C10 alkyl;

when M is ₁ In the case of ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells, n1 may be an integer from 1 to 3 and n3 may be an integer from 0 to 4 in some embodiments, providing that M is present in the conjugate ₁ The number of ligands is at least 2; in some embodiments, n1+ n3 ≧ 2, which can result in M ₁ The number of ligands is at least 3, such that M ₁ The ligand binds more readily to the liver surface asialoglycoprotein receptor, thereby facilitating entry of the conjugate into cells by endocytosis. Experiments show that when M is used ₁ When the number of ligands is more than 3, M ₁ The increased ease of ligand binding to the hepatic surface asialoglycoprotein receptor is not significant, and thus, in some embodiments, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1+ n3=2-3, taken together in terms of ease of synthesis, structure/process cost, and delivery efficiency.

In some embodiments, when M1, M2 and M3 are independently selected from integers of 2 to 10, a plurality of M may be used ₁ Spatial position between ligands is adapted to M ₁ Binding of ligands to hepatic surface asialoglycoprotein receptorsIn order to make the conjugates provided by the present disclosure simpler, easier to synthesize, and/or lower cost, in some embodiments m1, m2, and m3 are each independently integers from 2 to 5, and in some embodiments, m1= m2= m3.

It will be understood by those skilled in the art that when R is present ₁₀ 、R ₁₁ 、R ₁₂ 、R ₁₃ 、R ₁₄ And R ₁₅ Each independently selected from H, C ₁ -C ₁₀ Alkyl radical, C ₁ -C ₁₀ Haloalkyl, and C ₁ -C ₁₀ One of the alkoxy groups, without altering the properties of the conjugates disclosed herein, can achieve the objectives of the present disclosure. In some embodiments, R ₁₀ 、R ₁₁ 、R ₁₂ 、R ₁₃ 、R ₁₄ And R ₁₅ Each independently selected from H, methyl and ethyl. In some embodiments, R ₁₀ 、R ₁₁ 、R ₁₂ 、R ₁₃ 、R ₁₄ And R ₁₅ Are all H.

Oligonucleotide conjugates provided according to the present disclosure, R ₃ A group of the structure represented by the formula A59, wherein E ₁ Is OH, SH or BH ₂ In some embodiments, E is based on considerations of ready availability of starting materials for preparation ₁ Is OH or SH.

In some embodiments, R ₂ Is selected to effect attachment to N and A59 on the nitrogen-containing backbone. In the context of the present disclosure, "nitrogen-containing backbone" means a linkage with R ₁₀ 、R ₁₁ 、R ₁₂ 、R ₁₃ 、R ₁₄ And R ₁₅ A chain structure in which the carbon atoms of (b) and N are linked to each other. Thus, R ₂ May be any linking group capable of linking the a59 group to N on the nitrogen-containing backbone in a suitable manner. In some embodiments, where the oligonucleotide conjugates of the present disclosure are prepared by a process of solid phase synthesis, R ₂ The group is required to contain a connecting site connected with N on the nitrogen-containing skeleton and R ₃ The attachment site to which P in (1) is attached. In some embodiments, R ₂ Wherein the site linked to N on the nitrogen-containing backbone forms an amide bond with N, said amide bond with R ₃ The site of attachment of P on (a) forms a phosphoester bond with P. In some embodiments, R ₂ Is B5, B6, B5 'or B6':

wherein the content of the first and second substances,

indicating the site at which the group is covalently attached.

q ₂ Can be an integer from 1 to 10, and in some embodiments, q is ₂ Is an integer of 1 to 5.

L ₁ Has the effect of mixing M ₁ Ligands are attached to the N on the nitrogen-containing backbone to provide targeting functions for the oligonucleotide conjugates of the present disclosure. In some embodiments, L ₁ One or more connecting combinations selected from the groups of the formulas A1 to A26. In some embodiments, L ₁ A combination of one or more selected from A1, A4, A5, A6, A8, A10, A11 and A13; in some embodiments, L ₁ A linkage combination of at least 2 selected from A1, A4, A8, a10 and a 11; in some embodiments, L ₁ At least 2 connection combinations selected from A1, A8 and A10.

In some embodiments, L ₁ Can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments, L is ₁ The length of (a) 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, 60 atoms.

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 implementationsWhere 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 C1-C5 alkyl, and in some embodiments Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', ra, rb are each selected in formulas A1-A26 to achieve M ₁ Ligands are attached to N on nitrogen-containing backbones and M is ₁ The spatial position between the ligands is more suitable for M ₁ The ligand binds to the hepatic surface asialoglycoprotein receptor.

In some embodiments, the oligonucleotide conjugates of the present disclosure have a structure represented by formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):

in some embodiments, P in formula a59 can be ligated to any possible position in the double-stranded oligonucleotide sequence, e.g., P in formula a59 can be ligated to either nucleotide of the sense or antisense strand of the double-stranded oligonucleotide; in some embodiments, P in formula a59 is attached to any one nucleotide of the sense strand of the double-stranded oligonucleotide. In some embodiments, P in formula a59 is ligated to the end of the sense or antisense strand of the double-stranded oligonucleotide; in some embodiments, P in formula a59 is ligated to the end of the sense strand of the double-stranded oligonucleotide. The end refers to the first 4 nucleotides of the sense strand or the antisense strand from one end thereof. In some embodiments, P in formula a59 is ligated to the end of the sense or antisense strand of the double-stranded oligonucleotide; in some embodiments, P in formula a59 is ligated to the 3' end of the sense strand of the double-stranded oligonucleotide. In the case of ligation to the sense strand of a double-stranded oligonucleotide at the above-described position, upon entry of the conjugate provided by the present disclosure into a cell, upon unwinding, the individual double-stranded oligonucleotide antisense strand may be released to modulate target gene expression.

P in formula A59 can be ligated to any possible position on a nucleotide in a double-stranded oligonucleotide, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide, or the base of the nucleotide. In some embodiments, P in formula a59 can be attached to the nucleotide in the double-stranded oligonucleotide at the 2' -position, the 3' -position, or the 5' -position by forming a phosphodiester bond. In some embodiments, P in formula a59 is attached to an oxygen atom formed after dehydrogenation of the 3' hydroxyl group of the 3' terminal nucleotide of the sense strand of the double-stranded oligonucleotide, or P in formula a59 is attached to a nucleotide by substitution of a hydrogen in the 2' -hydroxyl group of one nucleotide in the sense strand of the double-stranded oligonucleotide, or P in formula a59 is attached to a nucleotide by substitution of a hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide of the sense strand of the double-stranded oligonucleotide.

In some embodiments, the double-stranded oligonucleotide comprised by the oligonucleotide conjugates of the present disclosure may be an siRNA, when the oligonucleotide conjugates of the present disclosure are also referred to as siRNA conjugates. In some embodiments, the double-stranded oligonucleotide comprised by the oligonucleotide conjugates of the present disclosure may be, for example, an siRNA listed in table 1. Oligonucleotide conjugates comprising these sirnas exhibit low toxicity and high mRNA inhibitory activity of target gene expression.

Preparation of oligonucleotide conjugates of the disclosure

The above-described oligonucleotide conjugates can be synthesized by methods that have been described in detail in the prior art. For example, methods for the preparation of various siRNA conjugates are described in detail in WO2015006740 A2. In the case where the double-stranded oligonucleotide is an siRNA, the oligonucleotide conjugates of the present disclosure can also be obtained by means well known to those skilled in the art. For example, WO2014025805A1 describes a method for producing a structure of formula (705), and Rajeev et al in ChemBiochem 2015,16,903-908 describes a method for producing a structure of formula (707). Chinese patent application CN110959011a also discloses in detail a method for preparing an oligonucleotide conjugate represented by formula (708). The contents of the above documents are incorporated herein by reference in their entirety.

The oligonucleotide conjugates of the present disclosure may also be combined with other pharmaceutically acceptable excipients, which may be one or more of a variety of agents or compounds conventionally employed in the art, for details as described above with respect to the pharmaceutical compositions of the present disclosure.

Double-stranded oligonucleotides, pharmaceutical compositions, and uses of oligonucleotide conjugates of the disclosure

In some embodiments, the present disclosure provides use of a double-stranded oligonucleotide provided by the present disclosure, a double-stranded oligonucleotide obtained according to a method of the present disclosure, a pharmaceutical composition and/or an oligonucleotide conjugate in a medicament for treating and/or preventing a disease or a symptom associated with an mRNA level of target gene expression. In some embodiments, the specific gene is a gene that is abnormally expressed in a hepatocyte. 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 a tumor cell. In some embodiments, the mRNA expressed by the target gene is selected from one of 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, SOD, 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 mRNA expressed by a Hepatitis B Virus (HBV) gene, mRNA expressed by an angiopoietin-like protein 3 (ANGPTL 3) gene, or mRNA expressed by an apolipoprotein C3 (ApoC 3) gene. In some embodiments, the disease or condition associated with mRNA levels of target gene expression is chronic liver disease, hepatitis, liver fibrosis disease, liver proliferative disease, and/or dyslipidemia. In some embodiments, the disease or condition associated with mRNA levels of target gene expression is hepatitis b.

In some embodiments, the present disclosure provides a method of treating and/or preventing a disease or condition associated with mRNA levels of target gene expression, the method comprising administering to a subject in need thereof an effective amount of a double-stranded oligonucleotide, pharmaceutical composition, and/or oligonucleotide conjugate provided by the present disclosure. In some embodiments, the mRNA expressed by the target gene is selected from one of 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, HCVHSD17B13, p53, PCSK9, PNP, PLG, PKK, KNG, SARS-CoV-2, SCD1, SCNN1A, SOD, 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 mRNA expressed by a Hepatitis B Virus (HBV) gene, mRNA expressed by an angiopoietin-like protein 3 (ANGPTL 3) gene, or mRNA expressed by an apolipoprotein C3 (ApoC 3) gene. In some embodiments, the disease or condition associated with mRNA levels of target gene expression is chronic liver disease, hepatitis, liver fibrosis disease, liver proliferative disease, and/or dyslipidemia. In some embodiments, the disease or condition associated with mRNA levels of target gene expression is hepatitis b.

In some embodiments, the conjugates provided by the present disclosure may also be used to treat other liver diseases, including diseases characterized by unwanted cellular proliferation, hematologic diseases, metabolic diseases, and diseases characterized by inflammation. The proliferative disease of the liver may be a benign or malignant disease, such as cancer, hepatocellular carcinoma (HCC), liver metastasis or hepatoblastoma. The hematologic or inflammatory disease of the liver may be a disease involving coagulation factors, complement-mediated inflammation, or fibrosis. Metabolic disorders of the liver include dyslipidemia and irregularities in glucose regulation. In one embodiment, the disease is treated by administering one or more double-stranded oligonucleotides having high homology to the gene sequences involved in the disease.

In some embodiments, the present disclosure provides a method of modulating the level of expression of a target gene in a cell, the method comprising contacting an effective amount of a double-stranded oligonucleotide, pharmaceutical composition, and/or oligonucleotide conjugate provided by the present disclosure with the cell. In some embodiments, the mRNA expressed by the target gene is selected from one of 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, SOD, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT. In some embodiments, the modulation refers to inhibition of target gene expression in a cell, the target gene expressed mRNA is selected from the group consisting of Hepatitis B Virus (HBV) gene expressed mRNA, angiopoietin-like protein 3 (ANGPTL 3) gene expressed mRNA, or apolipoprotein C3 (ApoC 3) gene expressed mRNA.

By administering the double-stranded oligonucleotides, pharmaceutical compositions and/or oligonucleotide conjugates provided by the present disclosure to a subject in need thereof, prevention and/or treatment of pathological conditions or diseases caused by expression of a specific gene in a cell can be achieved through a mechanism of regulating gene expression. Thus, the double-stranded oligonucleotides provided by the present disclosure, the double-stranded oligonucleotides obtained according to the methods of the present disclosure, pharmaceutical compositions and/or oligonucleotide conjugates can be used for preventing and/or treating said pathological condition or disease, or for the preparation of a medicament for preventing and/or treating a pathological condition or disease as described herein.

The term "administering" as used herein refers to placing a double-stranded oligonucleotide, pharmaceutical composition, and/or oligonucleotide conjugate into a subject by a method or route that results in at least partially positioning the double-stranded oligonucleotide, pharmaceutical composition, and/or oligonucleotide conjugate at a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. In general, topical administration results in delivery of more double-stranded oligonucleotides, pharmaceutical compositions, and/or oligonucleotide conjugates to a particular site as compared to the entire body of the subject; whereas systemic administration results in delivery of the double-stranded oligonucleotide, pharmaceutical composition and/or oligonucleotide conjugate to substantially the entire body of the subject.

Administration to a subject can be by any suitable route known in the art, including but not limited to: oral or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal and topical (including buccal and sublingual) administration. The frequency of administration may be 1 or more times per day, week, month, or year.

The dosage of the double-stranded oligonucleotide, pharmaceutical composition and/or oligonucleotide conjugate described in the present disclosure may be a dosage that is conventional in the art, and the dosage may be determined according to various parameters, particularly age, weight and sex of the subject. Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose that gives rise to 50% of the maximal response intensity in a quantitative response, and in a qualitative response, the dose that gives rise to a positive response in 50% of the subjects). The range of human doses can be derived based on data obtained from cell culture analysis and animal studies.

In administering the double-stranded oligonucleotides, pharmaceutical compositions and/or oligonucleotide conjugates of the present disclosure, for example, for male or female, 6-12 weeks old, 18-25g weight of C57BL/6J or C3H/HeNCrlVr mice, based on the amount of double-stranded oligonucleotide in the double-stranded oligonucleotide, pharmaceutical composition and/or oligonucleotide conjugate: for oligonucleotide conjugates of a double-stranded oligonucleotide and a pharmaceutically acceptable conjugate molecule, the amount of double-stranded oligonucleotide may be from 0.001 to 100mg/kg body weight, in some embodiments from 0.01 to 50mg/kg body weight, in further embodiments from 0.05 to 20mg/kg body weight, in further embodiments from 0.1 to 15mg/kg body weight, and in yet further embodiments from 0.1 to 10mg/kg body weight. Such amounts may be preferred when administering the double-stranded oligonucleotides, pharmaceutical compositions and/or oligonucleotide conjugates described in the present disclosure.

In addition, by introducing the double-stranded oligonucleotide, pharmaceutical composition and/or oligonucleotide conjugate of the present disclosure into cells in which a specific gene is abnormally expressed, it is also possible to achieve the purpose of suppressing the expression of the specific gene in the cells by a mechanism of gene expression regulation. In some embodiments, the cell is a hepatocyte. In some embodiments, the hepatocytes may be cells or isolated primary hepatocytes, in some embodiments primary hepatocytes, selected from Hep3B, hepG, huh7, and like hepatoma cell lines.

The amount of double-stranded oligonucleotide in the provided double-stranded oligonucleotides, pharmaceutical compositions and/or oligonucleotide conjugates is readily determined by one skilled in the art based on the desired effect to be obtained using the methods provided by the present disclosure to inhibit the expression of a particular gene in a cell. For example, in some embodiments, the double-stranded oligonucleotide, pharmaceutical composition, and/or oligonucleotide conjugate is an siRNA conjugate, the amount of siRNA in the siRNA conjugate provided is an amount that: it is sufficient to reduce the expression of the target gene and result in an extracellular concentration at the surface of the target cell of 1pM to 1 μ M, or 0.01nM to 100nM, or 0.05nM to 50nM or to about 5 nM. The amount required to achieve this local concentration will vary depending on a variety of factors including the method of delivery, the site of delivery, the number of cell layers between the delivery site and the target cell or tissue, whether the delivery is local or systemic, and the like. The concentration at the delivery site may be significantly higher than the concentration at the surface of the target cell or tissue.

Reagent kit

The present disclosure provides a kit comprising a double-stranded oligonucleotide, a pharmaceutical composition, and/or an oligonucleotide conjugate provided by the present disclosure.

In some embodiments, the kits described herein can provide double-stranded oligonucleotides, pharmaceutical compositions, and/or conjugates in one container. In some embodiments, a kit described herein may comprise a container that provides a pharmaceutically acceptable excipient. In some embodiments, the kit may further comprise other ingredients, such as stabilizers or preservatives and the like. In some embodiments, the kits described herein may comprise at least one additional therapeutic agent in a container other than the container providing the double-stranded oligonucleotides, pharmaceutical compositions, and/or conjugates described herein. In some embodiments, the kit may comprise instructions for mixing the double-stranded oligonucleotide, pharmaceutical composition, and/or conjugate with a pharmaceutically acceptable carrier and/or adjuvant or other ingredient, if any.

In the kits of the present disclosure, the double-stranded oligonucleotide and the pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or conjugate, and/or pharmaceutically acceptable adjuvant may be provided in any form, such as a liquid form, a dried form, or a lyophilized form. In some embodiments, the double-stranded oligonucleotide and pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or conjugate and optionally pharmaceutically acceptable adjuvant are substantially pure and/or sterile. In some embodiments, sterile water may be provided in the kits of the present disclosure.

The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.

Without wishing to be bound, the present invention is described in further detail in the following embodiments and examples relating to exemplary embodiments in which the double stranded oligonucleotide in the pharmaceutical composition and/or oligonucleotide conjugate of the present disclosure is a small interfering RNA (siRNA). In this case, the double-stranded oligonucleotide, the pharmaceutical composition and the oligonucleotide conjugate of the present disclosure are siRNA, a pharmaceutical composition comprising siRNA and an siRNA conjugate, respectively. In the context of the present disclosure, the siRNA, the pharmaceutical composition comprising the siRNA, and the siRNA conjugate in these embodiments are also referred to as the siRNA of the present disclosure, the pharmaceutical composition of the present disclosure, and the siRNA conjugate of the present disclosure, for convenience of description. This does not mean that the double-stranded oligonucleotides of the present disclosure can only be sirnas, but rather, the double-stranded oligonucleotides can be other variants disclosed herein or known to those of skill in the art, such as small activating RNAs (sarnas), and the like. It is contemplated that other double-stranded oligonucleotides will similarly function when used alone, or in forming the pharmaceutical compositions and/or oligonucleotide conjugates described in the present disclosure, based on the detailed description of the siRNA, the pharmaceutical composition comprising the siRNA, and the siRNA conjugate.

Examples

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

Example 1 preparation of siRNA1

(1-1) Synthesis of Compound Z2

At 25 ℃, 25g of compound Z1 was dissolved in 100mL of anhydrous pyridine, placed under an argon atmosphere for sufficient protection, and then 32 was added to the reaction solution.8g of 4,4' -dimethoxytriphenylchloromethane were reacted for 3 hours. The anhydrous pyridine was evaporated under reduced pressure and the remaining mixture was dissolved in 500mL of dichloromethane and saturated NaHCO ₃ Washing 2 times, 300mL each time, and washing once with 350mL saturated saline, anhydrous Na ₂ SO ₄ Drying, filtering, concentrating the organic phase filtrate, and subjecting the obtained residue to silica gel column chromatography [ V (petroleum ether): V (ethyl acetate) =5:1-1:1]Gradient elution was carried out, and the eluate containing the reaction product was collected, and the solvent was evaporated to obtain 43.4g of compound Z2.M/z (ES +), theory, [ M + H ]]+,561.61, detect, [ M + H]+,561.23.

(1-2) Synthesis of Compound Z3

30g of the compound Z2 and 7.7g of imidazole were added to 150mL of N, N-dimethylformamide, and then 29.5g of t-butyldiphenylchlorosilane (TBDPSCl) was added to the reaction solution and reacted for 5 hours. Then, 50mL of a saturated sodium bicarbonate solution and 500mL of ethyl acetate were added to the reaction solution, and the obtained organic phase was washed once with 300mL of saturated saline, once with 300mL of a 5wt% citric acid aqueous solution, and dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 42.75g of compound Z3, which was used in the subsequent reaction without further purification. M/z (ES +), theory, [ M + H ] +,800.02, detect, [ M + H ] +,799.64

(1-3) Synthesis of Compound Z4

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

(1-4) Synthesis of Compound Z5

Adding 10.4g of compound Z4 into 120mL of dichloromethane under the protection of argon, then adding 34g of dess-martin periodinane (DMP), reacting for 30min, adding 300mL of saturated sodium thiosulfate solution and 500mL of ethyl acetate into the reaction solution, stirring for 30min, standing, separating, extracting, washing the organic phase once with 100mL of saturated sodium bicarbonate solution and once with 100mL of saline solution, washing the organic phase once with anhydrous Na at room temperature ₂ SO ₄ After drying and filtration, the solvent was distilled off from the obtained organic phase under reduced pressure to obtain 9.8g of compound Z5.M/z (ES +), theory, [ M + NH4]+,512.66, detect, [ M + NH4]+,512.80

(1-5) Synthesis of Compound Z6

13.9g of triphenyl phosphine and 38g of carbon tetrabromide are respectively added into 100mL of dichloromethane at 0 ℃ under the protection of argon, stirred for 30min, then added with 9.8g of compound Z5, and reacted for 2h. Then, 5mL of a saturated ammonium chloride solution and 100mL of dichloromethane were added to the reaction mixture, the mixture was stirred for 30min, liquid separation was performed, the obtained organic phase was concentrated under reduced pressure to remove the solvent, and column chromatography was performed [ V (petroleum ether) = V (ethyl acetate) = 20:1 ]]Purification, the eluate containing the reaction product was collected, and the solvent was evaporated to give 4.68g of Compound Z6.M/z (ES +), theory, [ M + H ]] ⁺ 651.44 assay, [ M + H] ⁺ 651.50

(1-6) Synthesis of Compound Z7

1.8g of dimethyl phosphate, 1.1g of triethylamine and 4.68g of the compound Z6 were added to 50mL of a solution of N, N-dimethylformamide, respectively, and reacted at room temperature for 2 hours. 150mL of ethyl acetate was added to the reaction mixture, which was then washed three times with 300mL of 5wt% sodium bicarbonate solution, twice with 100mL of saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography [ V (petroleum ether): V (ethyl acetate) =1:1 ]]Purification, collection of the eluate containing the reaction product and removal of the solvent by evaporation gave 2.4g of compound Z7.M/z (ES +), theory, [ M + H ]] ⁺ 572.54, detection, [ M + H] ⁺ ,572.7

(1-7) Synthesis of Compound Z8

Under an argon atmosphere, 2.4g of compound Z7 and 4.6mL of tetra-n-butylammonium fluoride (TBAF) were added to 25mL of tetrahydrofuran and reacted at room temperature for 2h. Concentrating the reaction solution, and performing column chromatography [ V (petroleum ether) = V (ethyl acetate) =1: 1%]Purification and removal of the solvent gave 1.0g of Compound Z8.M/z (ES +), theory, [ M + H ]] ⁺ 334.14, detection, [ M + H] ⁺ 334.80

(1-8) Synthesis of Compound S2

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

(1-9) Synthesis of Compound Z9

0.32g of the compound Z8, 0.1g of the compound S2, 0.068g of 1,1' -bis (diphenylphosphino) ferrocene, 800ul of propylene oxide and 0.017g of palladium acetate were added to 10mL of tetrahydrofuran and reacted at 70 ℃ for 1.5 hours under microwave. Then 300mL of saturated aqueous sodium bicarbonate solution and 200mL of ethyl acetate were added, the mixture was stirred for 5min, the mixture was allowed to stand for layering, the aqueous phase was extracted with 300mL of ethyl acetate, the organic phases were combined, the resulting organic phase was washed once with 300mL of saturated saline, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was subjected to gradient elution with a silica gel column (V (dichloromethane) = V (methanol) =50: 1-40: 1), purified, and dried under vacuum to give 92mg of Compound Z9.M/z (ES +): theory, [ M + H ]] ⁺ 1030.37, detection, [ M + H [ ]] ⁺ :1031.12。

(1-10) Synthesis of Compound Z10

0.65g of the compound Z9, 0.326g of N, N-diisopropylethylamine, 0.224g of 3- ((chloro (diisopropylamino) phosphono) oxy) propionitrile were added to 5mL of dichloromethane and reacted at room temperature for 3.0h. To the reaction solution, 300mL of a saturated aqueous sodium bicarbonate solution was added and stirred for 1 hour, the mixture was allowed to stand for separation, the aqueous phase was extracted with 300mL of dichloromethane, the organic phases were combined, the obtained organic phase was washed once with 100mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated, and the obtained residue was purified by silica gel column chromatography [ V (dichloromethane) = V (methanol) = 1-30]The mixed eluate was purified by column chromatography as a mobile phase, and the eluate containing the reaction product was collected and the solvent was removed by evaporation to obtain 300mg of compound Z10. The structure of the obtained Z10 compound is shown as a 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.

(1-11) Synthesis of sense Strand of siRNA1

Nucleoside monomers were linked one by one from the 3'-5' direction in accordance with the sense strand nucleotide arrangement order of siRNA1 in Table 1 by the solid phase phosphoramidite method. Each attachment of a nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, oxidation or sulfurization. When two nucleotides are connected by adopting phosphate ester and the next nucleoside monomer is connected, four-step reactions of deprotection, coupling, capping and oxidation are included. When two nucleotides are connected by phosphorothioate, and the latter nucleoside monomer is connected, the four-step reaction of protection, coupling, capping and sulfuration is included. The synthesis conditions are given as follows:

the nucleoside monomer is provided by acetonitrile solution with 0.1M concentration, the deprotection reaction conditions of each step are the same, namely the temperature is 25 ℃, the reaction time is 70 seconds, the deprotection reagent is dichloromethane solution of dichloroacetic acid (3%v/v), and the molar ratio of the dichloroacetic acid to 4,4' -dimethoxytrityl protecting group on the solid phase carrier is 5:1.

The coupling reaction conditions in each step are the same, and the coupling reaction conditions comprise that the temperature is 25 ℃, the molar ratio of the nucleic acid sequence connected on the solid phase carrier to the nucleoside monomer is 1.

The capping conditions were the same for each step, including a temperature of 25 ℃ and a reaction time of 15 seconds. The capping reagent solution is a mixed solution of CapA and CapB with the molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence connected to the solid phase carrier is acetic anhydride to N-methylimidazole to the nucleic acid sequence connected to the solid phase carrier = 1.

The oxidation reaction conditions in each step are the same, including the temperature of 25 ℃, the reaction time of 15 seconds, and the oxidizing agent of 0.05M iodine water. The molar ratio of iodine to nucleic acid sequence attached to the solid support in the coupling step is 30. The reaction was carried out in a mixed solvent of tetrahydrofuran, water, pyridine = 3.

The conditions of each step of sulfuration reaction are the same, including the temperature of 25 ℃, the reaction time of 300 seconds, and the sulfuration reagent of hydrogenated flavonol. The molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling step is 120. The reaction was carried out in a mixed solvent of acetonitrile pyridine = 1:1.

Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence with the attached carrier was added to 25wt% ammonia water in an amount of 0.5ml/μmol, reacted at 55 ℃ for 16 hours, the liquid was removed, and concentrated to dryness in vacuo.

Purification and desalting: purification of nucleic acids was accomplished by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the method comprises the following steps: eluent A:20mM sodium phosphate (pH 8.1) in water/acetonitrile =9:1 (volume ratio); eluent B:1.5M sodium chloride, 20mM sodium phosphate (pH 8.1), solvent water/acetonitrile =9:1 (volume ratio); elution gradient: eluent a eluent B =100, gradient elution. Collecting product eluates, mixing, desalting with reversed phase chromatography purification column, and eluting with deionized water, wherein the specific conditions include desalting with Sephadex column, and the filler is Sephadex G25.

And (3) detection: purity was checked using ion exchange chromatography (IEX-HPLC) and molecular weight was analyzed using liquid chromatography-mass spectrometry (LC-MS).

(1-12) Synthesis of antisense strand of siRNA1

By the solid phase phosphoramidite method, using a universal solid phase carrier (UnyLinker) ^TM loaded

HL Solid Supports, kinovate Life Sciences Co., ltd.) starting the cycle, synthesizing the antisense strand of siRNA1 according to the composition of the antisense strand of siRNA1 in Table 1, connecting nucleoside monomers one by one from the 3'-5' direction, and finally connectingWhen a nucleoside monomer is used, the compound represented by the formula Z10 prepared in the above step (1-10) is used. The conditions of deprotection, coupling, capping, oxidation or sulfuration reaction, cutting, deprotection, purification and desalination in the solid phase synthesis method are the same as those of the synthesis of a sense chain.

(1-13) annealing

Mixing the sense strand and the antisense strand in an equimolar ratio, dissolving in water for injection, heating to 95 ℃, and slowly cooling to room temperature to form a double-stranded structure by the action of hydrogen bonds on the two single strands. After the above synthesis was completed, the purity was checked using ion exchange chromatography (IEX-HPLC) and molecular weight detection was performed by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA is siRNA1 in table 1.

Example 2 preparation of sirnas 2

(2-1) Synthesis of Compound P2

Under the protection of argon, 11.9g of the compound P1 and 2.26g of tetrazole are dissolved in 110mL of anhydrous acetonitrile, the mixture is reacted for 10min at room temperature, 21.4mL of water is added, and the reaction is continued for 1.0h. 500mL of methylene chloride and 300mL of a saturated aqueous solution of sodium hydrogencarbonate were added to the reaction mixture, and the mixture was stirred and allowed to stand for separation. The resulting organic phase was washed once with 100mL of a saturated aqueous solution of sodium chloride, dried over anhydrous ammonium sulfate, filtered, concentrated, and dried under vacuum to give 9.3g of Compound P2.

(2-2) Synthesis of Compound P3

2.0g of compound Z8, 5.96g of compound P2, 1.33g of 1,1' -bis (diphenylphosphino) ferrocene (DPPF) and 0.37g of palladium acetate, obtained as described in steps 1 to 7 of example 1, are added to 20mL of tetrahydrofuran under argon, then 8mL of propylene oxide are added and the reaction is carried out at 70 ℃ for 5.0h. To the reaction solution was added 300mL of a saturated aqueous sodium bicarbonate solutionAnd 200mL of ethyl acetate, stirred for a while, and then allowed to stand for separation. The aqueous phase was extracted with 200mL of ethyl acetate, the combined organic phases were washed once with 100mL of a saturated aqueous salt solution, dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to silica gel column chromatography [ V (dichloromethane) = V (methanol) = 80: 1-20: 1 ]]The eluate containing the reaction product was purified, collected, evaporated to remove the solvent and dried in vacuo to give 4.2g of compound P3.M/z (ES +), theory, [ M + H ]] ⁺ 914.30, detection, [ M + H [ ]] ⁺ :914.88.

(2-3) Synthesis of Compound P4

4g of the compound P3 and 0.3g of tetrazole are added into 50mL of N, N-dimethylformamide under the protection of argon, and then 0.36g of N-methylimidazole and 1.98g of bis (diisopropylamino) (2-cyanoethoxy) phosphine are added into the reaction solution to react for 2.0h at room temperature. Adding 300mL saturated sodium bicarbonate aqueous solution into the reaction solution, stirring, standing for layering, extracting the water phase with 200mL dichloromethane, mixing the organic phases, washing the obtained organic phase with 100mL saturated saline solution, drying with anhydrous sodium sulfate, filtering, concentrating, and subjecting the residue to silica gel column chromatography [ V (dichloromethane) = V (methanol) =50:1-30: 1%]Purification, collection of the eluate containing the reaction product, removal of the solvent by evaporation and drying in vacuo gave 1.4g of compound P4. The structure of the obtained P4 compound is shown as a 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,2H),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.

(2-4) Synthesis of sense Strand of siRNA2

Nucleoside monomers were linked one by one from the 3'-5' direction in accordance with the sense strand nucleotide arrangement order of siRNA2 in Table 1 by the solid phase phosphoramidite method. Each attachment of a nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, oxidation or sulfurization. When two nucleotides are connected by adopting phosphate ester, and the next nucleoside monomer is connected, four-step reactions including deprotection, coupling, capping and oxidation are carried out. When two nucleotides are connected by phosphorothioate, and the latter nucleoside monomer is connected, the four-step reaction of protection, coupling, capping and sulfuration is included. The synthesis conditions are given as follows:

the nucleoside monomer is provided by acetonitrile solution with the concentration of 0.1M, the deprotection reaction conditions of each step are the same, namely the temperature is 25 ℃, the reaction time is 70 seconds, the deprotection reagent is dichloromethane solution (3%v/v) of dichloroacetic acid, and the molar ratio of the dichloroacetic acid to 4,4' -dimethoxytrityl protecting group on the solid phase carrier is 5:1.

The oxidation reaction conditions in each step are the same, including the temperature of 25 ℃, the reaction time of 15 seconds, and the oxidizing agent of 0.05M iodine water. The molar ratio of iodine to nucleic acid sequence attached to the solid support in the coupling step is 30. The reaction was carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3.

The conditions of each step of the sulfuration reaction are the same, including the temperature of 25 ℃, the reaction time of 300 seconds, and the sulfuration reagent is the hydrogenated flavonol. The molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling step is 120. The reaction was carried out in a mixed solvent of acetonitrile pyridine = 1:1.

Purification and desalting: purification of nucleic acids was accomplished by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the method comprises the following steps: eluent A:20mM sodium phosphate (pH 8.1) in water/acetonitrile =9:1 (volume ratio); eluent B:1.5M sodium chloride, 20mM sodium phosphate (pH 8.1), solvent water/acetonitrile =9:1 (volume ratio); elution gradient: eluent a eluent B =100, gradient elution. Collecting product eluates, mixing, desalting with reversed phase chromatography purification column, specifically desalting with Sephadex column as filler (Sephadex G25), and eluting with deionized water.

(2-5) Synthesis of antisense strand of siRNA2

HL Solid Supports, kinovate Life Sciences company), synthesizing the antisense strand of siRNA2 according to the composition of the antisense strand of siRNA2 in table 1, connecting nucleoside monomers one by one from 3'-5' direction, and using the compound represented by formula P4 prepared in the above step (2-3) when connecting the last nucleoside monomer. The conditions of deprotection, coupling, capping, oxidation or sulfuration reaction, cutting, deprotection, purification and desalination in the solid phase synthesis method are the same as those of the synthesis of a sense chain.

(2-6) annealing

Mixing the sense strand and the antisense strand in an equimolar ratio, dissolving in water for injection, heating to 95 ℃, and slowly cooling to room temperature to form a double-stranded structure by the action of hydrogen bonds on the two single strands. After the above synthesis was completed, the purity was checked using ion exchange chromatography (IEX-HPLC) and molecular weight detection was performed by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA is siRNA2 in table 1.

Example 3 preparation of sirnas 3

(3-1) Synthesis of Compound L2

Under the protection of argon, 3.0g of the compound L1 and 343mg of tetrazole are dissolved in 30ml of anhydrous acetonitrile, the mixture is reacted for 10min at room temperature, 5.2ml of water is added, and the reaction is continued for 1.0h. To the reaction solution were added 300ml of methylene chloride and 200ml of a saturated aqueous sodium bicarbonate solution, and the mixture was stirred for 5min and then allowed to stand for separation. The organic phase was washed once with 100mL of a saturated aqueous salt solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and dried under vacuum to give 2.5g of compound L2.M/z (ES +), theory, [ M + H ]] ⁺ 538.56, detection, [ M + H [ ]] ⁺ :538.62.

(3-2) Synthesis of Compound L3

2.7g of the compound Z8 obtained by the method described in steps 1 to 7 of example 1, 1.24g of the compound L2, 827mg of 1,1' -bis (diphenylphosphino) ferrocene, 154mg of palladium acetate and 4.4g of propylene oxide were added to 10mL of tetrahydrofuran, respectively, and reacted under an argon atmosphere for 3.0 hours. Adding 300mL saturated sodium bicarbonate water solution and 200mL ethyl acetate into the reaction solution, stirring for 5min, standing for layering, extracting the water phase with ethyl acetate, mixing the organic phases, washing with 100mL saturated sodium chloride water, drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and purifying the residue with silica gel chromatography column [ V (dichloromethane) = V (methanol) = 30: 1-20: 1 ]]Purification by gradient elution, collecting the eluate containing the reaction product, evaporating off the solvent and drying in vacuo, yield 840mg of compound L3. m/z: (Theory, [ M + H ]] ⁺ 790.78, detection, [ M + H [ ]] ⁺ :790.84.

(3-3) Synthesis of Compound L4

0.84g of the compound L3, 0.416g of bis (diisopropylamino) (2-cyanoethoxy) phosphine, 0.056g of tetrazole, and 0.022g of azomethylimidazole were added to 5mL of N, N-dimethylformamide at room temperature, and reacted for 1.0h. Adding 300mL saturated sodium bicarbonate water solution and 200mL ethyl acetate into the reaction solution, stirring for a while, standing for layering, extracting the water phase with ethyl acetate, mixing the organic phases, washing the obtained organic phase with 100mL saturated sodium chloride water, drying with anhydrous sodium sulfate, filtering, concentrating, and subjecting the obtained residue to silica gel column chromatography [ V (dichloromethane) = V (methanol) =50:1-30:1 ]]After gradient elution, the eluate containing the reaction product was collected and the solvent was evaporated off to give 400mg of compound L4. The structure of the obtained L4 compound is shown as a 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.

(3-4) Synthesis of sense Strand of siRNA3

Nucleoside monomers were linked one by one from the 3'-5' direction in accordance with the sense strand nucleotide arrangement order of siRNA3 in Table 1 by the solid phase phosphoramidite method. Each attachment of a nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, oxidation or sulfurization. When two nucleotides are connected by adopting phosphate ester and the next nucleoside monomer is connected, four-step reactions of deprotection, coupling, capping and oxidation are included. When two nucleotides are connected by phosphorothioate, and the latter nucleoside monomer is connected, the four-step reaction of protection, coupling, capping and sulfuration is included. The synthesis conditions are given as follows:

Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence with the connected carrier is added into ammonia water with the concentration of 25wt percent and the dosage of the ammonia water is 0.5 ml/mu mol, the reaction is carried out for 16h at 55 ℃, the liquid is removed, and the reaction is carried out until the solution is dry in vacuum.

(3-5) Synthesis of antisense strand of siRNA3

HL Solid Supports, kinovate Life Sciences company), synthesizing the antisense strand of siRNA1 according to the composition of the antisense strand of siRNA3 in table 1, connecting nucleoside monomers one by one from 3'-5' direction, and using the compound represented by formula L4 prepared in the above step (3-3) when connecting the last nucleoside monomer. The conditions of deprotection, coupling, capping, oxidation or sulfuration reaction, cutting, deprotection, purification and desalination in the solid phase synthesis method are the same as those of the synthesis of a sense chain.

(3-6) annealing

Mixing the sense strand and the antisense strand in an equimolar ratio, dissolving in water for injection, heating to 95 ℃, and slowly cooling to room temperature to form a double-stranded structure by the action of hydrogen bonds on the two single strands. After the above synthesis was completed, the purity was checked using ion exchange chromatography (IEX-HPLC) and molecular weight detection was performed by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA is siRNA3 in table 1.

Example 4 preparation of sirnas 4

(4-1) Synthesis of Compound Y2

Under the protection of argon2.61g of the compound Y1 and 490mg of tetrazole are dissolved in 25ml of anhydrous acetonitrile and reacted for 10min at room temperature, 4.5ml of water is added and the reaction is continued for 1.5h. To the reaction solution were added 200ml of ethyl acetate and 200ml of a saturated aqueous sodium bicarbonate solution, and the mixture was stirred for 5 minutes and then allowed to stand for separation. The organic phase was washed once with 100mL of a saturated aqueous salt solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and dried under vacuum to give 2.95g of Compound Y2.M/z (ES +), theory, [ M + H ]] ⁺ 775.58, detection, [ M + H [ ]] ⁺ :775.64.

(4-2) Synthesis of Compound Y3

0.84g of the compound Z8, 2.9g of the compound Y2, 560mg of 1,1' -bis (diphenylphosphino) ferrocene, 2.9g of propylene oxide and 113mg of palladium acetate, obtained by the method described in steps 1 to 7 of example 1, are added to 45ml of tetrahydrofuran at room temperature and reacted for 5.0h at 70 ℃. To the reaction mixture were added 200ml of a saturated aqueous sodium bicarbonate solution and 200ml of ethyl acetate, and the mixture was stirred for 5 minutes and allowed to stand for separation. The aqueous phase was extracted once with 100mL of ethyl acetate and the organic phases were combined, the resulting organic phase was washed once with 100mL of saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give a residue, which was subjected to silica gel column chromatography [ V (dichloromethane) = V (methanol) = 30: 1-20: 1 ]]Gradient elution, collecting the eluate containing the reaction product, and evaporation of the solvent gave 1.72g of compound Y3.M/z (ES +), theory, [ M + H ]] ⁺ 1028.00, detection, [ M + H [ ]] ⁺ :1028.06.

(4-3) Synthesis of Compound Y4

1.36g of the compound Y3, 0.6g of tetrazole, 27mg of N-methylimidazole and 600mg of bis (diisopropylamino) (2-cyanoethoxy) phosphine are added in succession to 15ml of dimethylformamide under argon protection at room temperatureReacting for 3.0h, adding 50mL saturated sodium bicarbonate water solution and 70mL ethyl acetate into the reaction solution, stirring for 5min, standing for layering, extracting the water phase with 70mL ethyl acetate, combining the organic phases, washing the obtained organic phase with 100mL saturated sodium chloride water, drying with anhydrous sodium sulfate, filtering, concentrating, and subjecting the obtained residue to silica gel column chromatography [ V (dichloromethane) = V (methanol) = 40: 1-20: 1%]Purification, the eluate containing the reaction product was collected, and the solvent was removed by evaporation to give 800mg of compound Y4. The structure of the obtained Y4 compound is shown as a 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),6.54(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＝11.1,5.7Hz,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.

(4-4) Synthesis of sense Strand of siRNA4

Nucleoside monomers were linked one by one from the 3'-5' direction in accordance with the sense strand nucleotide arrangement order of siRNA4 in Table 1 by the solid phase phosphoramidite method. Each attachment of a nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, oxidation or sulfurization. When two nucleotides are connected by adopting phosphate ester, and the next nucleoside monomer is connected, four-step reactions including deprotection, coupling, capping and oxidation are carried out. When two nucleotides are connected by phosphorothioate, and the latter nucleoside monomer is connected, the four-step reaction of protection, coupling, capping and sulfuration is included. The synthesis conditions are given as follows:

Purification and desalting: purification of nucleic acids was accomplished by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the method comprises the following steps: eluent A:20mM sodium phosphate (pH 8.1), solvent water/acetonitrile =9:1 (volume ratio); eluent B:1.5M sodium chloride, 20mM sodium phosphate (pH 8.1), solvent water/acetonitrile =9:1 (volume ratio); elution gradient: eluent a eluent B =100, gradient elution. Collecting product eluates, mixing, desalting with reversed phase chromatography purification column, specifically desalting with Sephadex column as filler (Sephadex G25), and eluting with deionized water.

(4-5) Synthesis of antisense strand of siRNA4

HL Solid Supports, kinovate Life Sciences company), synthesizing the antisense strand of siRNA1 according to the composition of the antisense strand of siRNA4 in table 1, connecting nucleoside monomers one by one from 3'-5' direction, and using the compound represented by formula Y4 prepared in the above step (5-3) when connecting the last nucleoside monomer. The conditions of deprotection, coupling, capping, oxidation or sulfuration reaction, cutting, deprotection, purification and desalination in the solid phase synthesis method are the same as those of the synthesis of a sense chain.

(4-6) annealing

Mixing the sense strand and the antisense strand in an equimolar ratio, dissolving in water for injection, heating to 95 ℃, and slowly cooling to room temperature to form a double-stranded structure by the action of hydrogen bonds on the two single strands. After the above synthesis was completed, the purity was checked using ion exchange chromatography (IEX-HPLC) and molecular weight detection was performed by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA is siRNA4 in table 1.

Example 5 preparation of siRNA5

(5-1) Synthesis of Compound K2

Under the protection of argon, 3.75g of the compound K1 and 0.63g of tetrazole are added into 28mL of anhydrous acetonitrile, the mixture reacts for 10min at room temperature, 5mL of water is added, and the reaction is continued for 2h. To the reaction solution were added 200ml of ethyl acetate and 200ml of ethyl acetateSaturated aqueous sodium bicarbonate solution, stirred for 5 minutes and then allowed to stand for delamination. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined, and the resulting organic phase was washed once with 100mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 3.45g of Compound K2.M/z (ES +), theory, [ M + H ]] ⁺ 751.75, detection, [ M + H [ ]] ⁺ :751.82.

(5-2) Synthesis of Compound K3

964mg of compound Z8, 3.26g of compound K2, 641mg of 1,1' -bis (diphenylphosphino) ferrocene, 3.36g of propylene oxide and 129mg of palladium acetate obtained by the method described in steps 1 to 7 of example 1 were added to 45ml of tetrahydrofuran under an argon atmosphere, and reacted at 70 ℃ for 5.0 hours, and 200ml of a saturated aqueous sodium bicarbonate solution and 200ml of ethyl acetate were added to the reaction solution, stirred for 5 minutes and left to separate into layers. After extraction of the aqueous phase with 100ml of ethyl acetate, the organic phases are combined. The organic phase was washed once with 100mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a residue, which was subjected to silica gel column chromatography [ V (dichloromethane) = V (methanol) =50: 1-20: 1 ]]Gradient elution, collecting the eluate containing the reaction product, and evaporation of the solvent gave 1.47g of compound K3.M/z (ES +), theory, [ M + H ]] ⁺ 1003.98, detection, [ M + H [ ]] ⁺ :1004.04.

(5-3) Synthesis of Compound K4

1.45g of the compound K3, 78.6mg of tetrazole, 30mg of N-methylimidazole and 655mg of bis (diisopropylamino) (2-cyanoethoxy) phosphine were added to 15mL of a methylformamide solution under an argon atmosphere, a reaction was carried out at room temperature for 3.0 hours, 50mL of a saturated aqueous sodium bicarbonate solution and 70mL of ethyl acetate were added to the reaction mixture, the mixture was stirred for 5 minutes and then allowed to stand for separation, and the aqueous phase was extracted with 70mL of ethyl acetateExtracting the ester, mixing the organic phases, washing the obtained organic phase with 100mL saturated saline solution, drying with anhydrous sodium sulfate, filtering, concentrating, and subjecting the obtained residue to silica gel column chromatography [ V (dichloromethane) = V (methanol) =50: 1-20: 1%]Gradient elution, collecting the eluate containing the reaction product, and evaporating off the solvent to obtain 700mg of compound K4. The structure of the obtained K4 compound is shown as a 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＝3.6Hz,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.

(5-4) Synthesis of sense Strand of siRNA5

Nucleoside monomers were linked one by one from the 3'-5' direction in accordance with the sense strand nucleotide arrangement order of siRNA5 in Table 1 by the solid phase phosphoramidite method. Each attachment of a nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, oxidation, or sulfurization. When two nucleotides are connected by adopting phosphate ester, and the next nucleoside monomer is connected, four-step reactions including deprotection, coupling, capping and oxidation are carried out. When two nucleotides are connected by phosphorothioate and the next nucleoside monomer is connected, four-step reaction including protection, coupling, capping and sulfuration is carried out. The synthesis conditions are given as follows:

The capping conditions were the same for each step, including a temperature of 25 ℃ and a reaction time of 15 seconds. The capping reagent solution is a mixture solution of CapA and CapB with a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence attached to the solid phase carrier is acetic anhydride to N-methylimidazole to the nucleic acid sequence attached to the solid phase carrier = 1.

(5-5) Synthesis of antisense strand of siRNA5

HL Solid Supports, kinovate Life Sciences company), synthesizing the antisense strand of siRNA1 according to the composition of the antisense strand of siRNA3 in table 1, connecting nucleoside monomers one by one from 3'-5' direction, and using the compound represented by formula K4 prepared in the above step (4-3) when connecting the last nucleoside monomer. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cutting and deprotection, purification and desalting conditions in the solid-phase synthesis method are the same as those of the synthesized sense chain.

(5-6) annealing

Mixing the sense strand and the antisense strand in an equimolar ratio, dissolving in water for injection, heating to 95 ℃, and slowly cooling to room temperature to form a double-stranded structure by the action of hydrogen bonds on the two single strands. After the above synthesis was completed, purity was checked using ion exchange chromatography (IEX-HPLC) and molecular weight detection was performed by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA is siRNA5 in table 1.

Example 6 preparation of sirnas 6

(6-1) Synthesis of Compound N2

2.08g of the compound N1 are added to 30mL of N, N-dimethylformamide under argon, and then 3.88g of di-tert-butylsilylbis (trifluoromethanesulfonic acid) are added and reacted at 0 ℃ for 40min. The obtained reaction mixture was used for the next reaction without purification.

(6-2) Synthesis of Compound N3

And (3) under the protection of argon, adding the reaction mixture obtained in the step (6-1) into 2.4g of imidazole in an ice bath, raising the temperature to room temperature for reaction for 30min, adding 3.26g of tert-butyldimethylsilyl chloride, and continuing the reaction for 2h. 50mL of sodium bicarbonate solution was added to the reaction mixture, the aqueous phase was extracted with 500mL of ethyl acetate, the organic phases were combined, and the resulting organic phase was washed once with 100mL of saturated brine and washed with anhydrous Na ₂ SO ₄ After drying, filtration and concentration, 4.3g of compound N3 was obtained. M/z (ES +), theory, [ M + H ]] ⁺ :515.87, detection, [ M + H] ⁺ :515.89

(6-3) Synthesis of Compound N4

Under the protection of argon and at the temperature of ice bath, 10mL of pyridine was added to 1.5mL of a hydrogen fluoride pyridine solution to form a mixed solution A, 7.5g of the compound N3 prepared according to the method in (6-2) was dissolved in 70mL of dichloromethane to form a mixed solution B, and the mixed solution A was added to the mixed solution B to react for 2 hours. Then, 50mL of sodium bicarbonate solution was added to the reaction mixture, the aqueous phase was extracted with 50mL of ethyl acetate, the organic phases were combined, the resulting organic phase was washed 5 times with 50mL of 5wt% aqueous citric acid solution and then once with 100mL of saturated sodium bicarbonate solution, and the organic phase was washed with anhydrous Na ₂ SO ₄ After drying, filtration and concentration of the organic phase, 5.77g of compound N4 are obtained. M/z (ES +), theory, [ M + Na +] ⁺ 397.49, detection, [ M + H] ⁺ :397.50.

(6-4) Synthesis of Compound N5

5.55g of Compound N4 was added to 50mL of anhydrous pyridine at 25 ℃ and 4.84g of 4,4' -dimethoxytriphenylchloromethane was added to the reaction mixture and reacted for 3 hours. The solvent was evaporated under reduced pressure and the residue was taken up in 100mLDissolving ethyl acetate and 50mL of 5wt% citric acid aqueous solution, eluting the organic phase obtained by separating with 5wt% citric acid aqueous solution for 5 times, each time 100mL, and finally 200mL saturated NaHCO ₃ And 100mL brine, and the organic phase was washed with anhydrous Na ₂ SO ₄ After drying and filtration, the residue obtained after concentration of the organic phase is purified by silica gel column chromatography [ V (petroleum ether) = V (ethyl acetate) =10:1-7:1]Purification by gradient elution and collection of the eluate containing the reaction product and removal of the solvent by evaporation afforded 6.23g of compound N5.M/z (ES +), theory, [ M + H ]] ⁺ 677.88, detection, [ M + H] ⁺ :677.90.

(6-5) Synthesis of Compound N6

5.4g of the compound N5, 432mg of tetrazole, 164mg of N-methylimidazole and 3.6g of bis (diisopropylamino) (2-cyanoethoxy) phosphine were added to 50ml of dimethylformamide under an argon atmosphere, and reacted at room temperature for 3.0 hours. To the reaction solution, 100mL of a saturated aqueous sodium bicarbonate solution and 70mL of ethyl acetate were added, the mixture was stirred for 5 minutes, and then allowed to stand for separation, the aqueous phase was extracted with 200mL of ethyl acetate, the organic phases were combined, the obtained organic phase was washed once with 100mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated, and the obtained residue was purified by a silica gel column [ V (petroleum ether): V (dichloromethane) = V (ethyl acetate) =10]Gradient elution, collecting the eluent containing the reaction product, evaporating to remove the solvent, and vacuum drying to obtain 6.2g of compound N6.M/z (ES +), theory, [ M + H ]] ⁺ 878.10, detection, [ M + H [ ]] ⁺ :878.15.

(6-6) Synthesis of Compound N7

Under the protection of argon, 6.2g of compound N6 and 996mg of tetrazole are added into 50ml of anhydrous acetonitrile to react for 10 minutes at room temperature, 9ml of water is added to continue the reaction for 2 hours. To the direction ofTo the reaction mixture were added 200ml of ethyl acetate and 200ml of a saturated aqueous sodium bicarbonate solution, and the mixture was stirred for 5 minutes and then allowed to stand for separation. The organic phase was washed once with 100mL of a saturated aqueous salt solution, dried over anhydrous ammonium sulfate, filtered, concentrated under reduced pressure, and dried under vacuum using an oil pump to obtain 5.38g of Compound N7.M/z (ES +), theory, [ M + Na +] ⁺ 816.91, detection, [ M + Na [ ]] ⁺ :816.96.

(6-7) Synthesis of Compound N8

1.38g of compound Z8, 4.94g of compound N7, 920mg of 1,1' -bis (diphenylphosphino) ferrocene, 4.8g of propylene oxide and 186mg of palladium acetate, obtained as described in steps 1 to 7 of example 1, are added to 45ml of tetrahydrofuran under argon protection and reacted for 5.0h at 70 ℃. 200ml of a saturated aqueous sodium bicarbonate solution and 200ml of ethyl acetate were added, and the mixture was stirred for 5 minutes and allowed to stand for separation. Extracting the water phase with 100mL ethyl acetate, mixing the organic phases, washing the obtained organic phase with 100mL saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, vacuum drying, and subjecting the obtained residue to silica gel column chromatography [ V (dichloromethane): V (dioxane = 17: 1-4:1) ]]Gradient elution purification, collecting the eluent containing the reaction product, evaporating to remove the solvent, and vacuum drying to obtain 2.6g of compound N8.M/z (ES +), theory, [ M + H ]] ⁺ 1047.15, detection, [ M + H [ ]] ⁺ :1047.21.

(6-8) Synthesis of Compound N9

1.3g of the compound N8, 67mg of tetrazole, 25mg of N-methylimidazole and 561mg of bis (diisopropylamino) (2-cyanoethoxy) phosphine were added in this order to 15ml of a dimethylformamide solution under an argon atmosphere, and reacted at room temperature for 3.0 hours. 50ml of a saturated aqueous sodium bicarbonate solution and 70ml of ethyl acetate were added to the reaction mixture,stirring for 5 minutes, standing for layering, extracting the aqueous phase with ethyl acetate, combining the organic phases, washing the obtained organic phase with 100mL of saturated saline solution, drying with anhydrous sodium sulfate, filtering, concentrating, performing gradient elution on the residue through a silica gel column (V (petroleum ether) = V (ethyl acetate) =1: 1-0:1), collecting eluent containing a reaction product, evaporating to remove the solvent, and performing vacuum drying to obtain 460mg of compound N9. The structure of the obtained N9 compound is shown as a 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),3.34(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.

(6-9) Synthesis of sense Strand of siRNA6

Nucleoside monomers were linked one by one from the 3'-5' direction in accordance with the sense strand nucleotide arrangement order of siRNA6 in Table 1 by the solid phase phosphoramidite method. Each attachment of a nucleoside monomer involves a four-step reaction of deprotection, coupling, capping, oxidation or sulfurization. When two nucleotides are connected by adopting phosphate ester, and the next nucleoside monomer is connected, four-step reactions including deprotection, coupling, capping and oxidation are carried out. When two nucleotides are connected by phosphorothioate, and the latter nucleoside monomer is connected, the four-step reaction of protection, coupling, capping and sulfuration is included. The synthesis conditions are given as follows:

(6-10) Synthesis of antisense strand of siRNA6

HL Solid Supports, kinovate Life Sciences company), synthesizing the antisense strand of siRNA1 according to the composition of the antisense strand of siRNA6 in table 1, connecting nucleoside monomers one by one from 3'-5' direction, and using the compound represented by formula N9 prepared in the above step (6-8) when connecting the last nucleoside monomer. The conditions of deprotection, coupling, capping, oxidation or sulfuration reaction, cutting, deprotection, purification and desalination in the solid phase synthesis method are the same as those of the synthesis of a sense chain.

(6-11) annealing

Mixing the sense strand and the antisense strand in an equimolar ratio, dissolving in water for injection, heating to 95 ℃, and slowly cooling to room temperature to form a double-stranded structure by the action of hydrogen bonds on the two single strands. After the above synthesis was completed, purity was checked using ion exchange chromatography (IEX-HPLC) and molecular weight detection was performed by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA is siRNA6 in table 1.

Example 7 preparation of siRNA8

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 in accordance with the nucleotide arrangement order shown for the sense strand and antisense strand of siRNA8 in Table 1, respectively. When the last nucleoside monomer of the antisense strand is linked, the compound represented by the formula Z10 prepared according to the procedure of (1-10) in example 1 is used.

According to the method described in step (1-13) of example 1, the sense strand and the antisense strand are mixed in an equimolar ratio, dissolved in water for injection and heated to 95 ℃ and slowly cooled to room temperature, and then the two single strands form a double-stranded structure by hydrogen bonding. After the above synthesis was completed, purity was checked using ion exchange chromatography (IEX-HPLC) and molecular weight detection was performed by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA is siRNA8 in table 1.

Example 8 preparation of siRNA9

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 order shown in the sense strand and the antisense strand of siRNA9 in table 1, respectively. When the last nucleoside monomer of the antisense strand is linked, the compound represented by the formula P4 prepared according to the step (2-3) in example 2 is used.

According to the method described in step (2-6) of example 2, the sense strand and the antisense strand are mixed in an equimolar ratio, dissolved in water for injection and heated to 95 ℃ and slowly cooled to room temperature, and then the two single strands form a double-stranded structure by hydrogen bonding. After the above synthesis was completed, purity was checked using ion exchange chromatography (IEX-HPLC) and molecular weight detection was performed by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA is siRNA9 in table 1.

Example 9 preparation of sirnas 15

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 in accordance with the nucleotide arrangement order shown for the sense strand and antisense strand of siRNA15 in Table 1, respectively. The compound represented by the formula Z10 prepared in the step (1-10) in example 1 was used in the ligation of the last nucleoside monomer of the antisense strand.

According to the method described in step (1-13) of example 1, the sense strand and the antisense strand are mixed in an equimolar ratio, dissolved in water for injection and heated to 95 ℃ and slowly cooled to room temperature, and then the two single strands form a double-stranded structure by hydrogen bonding. After the above synthesis was completed, purity was checked using ion exchange chromatography (IEX-HPLC) and molecular weight detection was performed by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA is siRNA15 in table 1.

Example 10 preparation of siRNA16

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 in accordance with the nucleotide arrangement order shown for the sense strand and antisense strand of siRNA16 in Table 1, respectively. The compound of formula P4 prepared in step (2-3) of example 2 was used in the ligation of the last nucleoside monomer of the antisense strand.

According to the method described in step (2-6) of example 2, the sense strand and the antisense strand are mixed in an equimolar ratio, dissolved in water for injection and heated to 95 ℃ and slowly cooled to room temperature, and then the two single strands form a double-stranded structure by hydrogen bonding. After the above synthesis was completed, purity was checked using ion exchange chromatography (IEX-HPLC) and molecular weight detection was performed by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA is siRNA16 in table 1.

Example 11 Synthesis of conjugate 1, conjugate 8 and conjugate 15

Following the procedure for the preparation of "conjugate 1" in preparation example 1 of WO2019/105437A1, conjugates 1, 8 and 15 of the present disclosure were prepared, respectively, with the difference that: nucleoside monomers are connected one by one from the 3'-5' direction according to the nucleotide arrangement sequence of the sense strand and the antisense strand of siRNA1, siRNA8 and siRNA15 in the table 1 respectively; when the last nucleotide (i.e., the 5' -terminal nucleotide) of the antisense strand is ligated, the nucleoside phosphoramidite monomer required for ligation of the nucleotide is replaced with Z10 prepared by the method of step (1-10) in example 1 of the present disclosure. Molecular weight detection was performed by liquid chromatography-mass spectrometry (LC-MS). As a result, the theoretical value of the sense strand of conjugate 1: 7516.5, found sense strand: 7515.3; theoretical value of antisense strand: 7287.8, found for antisense strand: 7286.0. the observed value is in agreement with the theoretical value, thereby confirming that the synthesized conjugate 1 is a target-designed double-stranded nucleic acid sequence containing a group represented by formula (501); theoretical value of sense strand for conjugate 8: 7605.4, found sense strand: 7604.3; theoretical value of antisense strand: 7229.8, found for antisense strand: 7228.7. the observed value is in agreement with the theoretical value, thereby confirming that the synthesized conjugate 8 is a target-designed double-stranded nucleic acid sequence containing a group represented by formula (501); theoretical value of sense strand for conjugate 15: 7584.5, found for sense strand: 7583.5; theoretical value of antisense strand: 7233.7, found for antisense strand: 7233.6. the observed value is in agreement with the theoretical value, thereby confirming that the synthesized conjugate 15 is a target-designed double-stranded nucleic acid sequence containing a group represented by formula (501). The structures of conjugate 1, conjugate 8 and conjugate 15 are shown in formula (801):

wherein Nu in formula (801) is siRNA1, siRNA8 or siRNA15 in table 1 of the present disclosure.

Example 12 Synthesis of conjugate 2, conjugate 9 and conjugate 16

Conjugate 2 of the present disclosure was obtained following the procedure of "conjugate 1" in preparation example 1 in WO2019/105437A1, with the difference that: nucleoside monomers are connected one by one from the 3'-5' direction according to the nucleotide arrangement sequence of the sense strand and the antisense strand of siRNA2, siRNA9 and siRNA16 in the table 1 respectively; when the last nucleotide (i.e., the 5' terminal nucleotide) of the antisense strand is ligated, this nucleotide is replaced with compound P4 prepared according to step (2-3) in example 2 of the present disclosure. Molecular weight detection was performed by liquid chromatography-mass spectrometry (LC-MS). As a result, the theoretical value of the sense strand of conjugate 2: 7516.5, found for sense strand: 7515.3; theoretical value of antisense strand: 7285.8, found for antisense strand: 7284.6. the observed value is in agreement with the theoretical value, thereby confirming that the synthesized conjugate 2 is a target-designed double-stranded nucleic acid sequence containing a group represented by the formula (502); theoretical value of sense chain for conjugate 2: 7605.4, found for sense strand: 7604.3; theoretical value of antisense strand: 7227.8, found for antisense strand: 7226.6. the observed value is in agreement with the theoretical value, thereby confirming that the synthesized conjugate 9 is a target designed double-stranded nucleic acid sequence containing a group represented by the formula (502); theoretical value of sense chain for conjugate 16: 7584.5, found for sense strand: 7583.5; theoretical value of antisense strand: 7233.7, found for antisense strand: 7232.6. the observed value is in agreement with the theoretical value, thereby confirming that the synthesized conjugate 16 is a target designed double-stranded nucleic acid sequence containing a group represented by the formula (502). The structures of conjugate 2, conjugate 9 and conjugate 16 are shown below (802):

wherein Nu in formula (802) is siRNA2, siRNA9 or siRNA16 in table 1 of the disclosure.

EXAMPLE 13 Synthesis of conjugates 3 to 6

Conjugates 3 to 6 of the present disclosure were obtained following the procedure of "conjugate 1" in preparation example 1 in WO2019/105437A1, the only difference being: when the last nucleotide (i.e., 5' -terminal nucleotide) of the antisense strand is ligated, the nucleotide is replaced with compound L4 prepared according to the method of step (3-3) in example 3 of the present disclosure, compound Y4 prepared according to the method of step (4-3) in example 4 of the present disclosure, compound K4 prepared according to the method of step (5-3) in example 5 of the present disclosure, or compound N4 prepared according to the method of step (6-3) in example 6 of the present disclosure, respectively, as a result, the sense strand theoretical value of conjugate 3: 7516.5, found for sense strand: 7515.3; theoretical value of antisense strand: 7161.7, found for antisense strand: 7160.7. the observed value is in agreement with the theoretical value, thereby confirming that the synthesized conjugate 3 is a target designed double-stranded nucleic acid sequence containing a group represented by the formula (503); theoretical value of sense strand for conjugate 4: 7516.5, found for sense strand: 7515.3; theoretical value of antisense strand: 7294.8, found for antisense strand: 7293.8. the observed value is in agreement with the theoretical value, thereby confirming that the synthesized conjugate 4 is a target-designed double-stranded nucleic acid sequence containing a group represented by formula (504); theoretical value of sense strand for conjugate 5: 7516.5, found sense strand: 7515.3; theoretical value of antisense strand: 7270.8, found for antisense strand: 7270.7. the observed value is in agreement with the theoretical value, thereby confirming that the synthesized conjugate 5 is a target-designed double-stranded nucleic acid sequence containing a group represented by formula (505); theoretical value of sense strand for conjugate 6: 7516.5, found for sense strand: 7515.3; theoretical value of antisense strand: 7303.8, found for antisense strand: 7302.8. the observed value is in agreement with the theoretical value, thereby confirming that the synthesized conjugate 6 is a target-designed double-stranded nucleic acid sequence containing a group represented by the formula (506). The structures of conjugate 3, conjugate 4, conjugate 5 and conjugate 6 are shown in formula (802).

Wherein Nu in formula (802) is siRNA3, siRNA4, siRNA5 or siRNA6 in table 1 of the present disclosure.

Comparative example 1 Synthesis of reference conjugates 1 to 3

Reference conjugate 1, reference conjugate 2, and reference conjugate 3 were obtained according to the method of "conjugate 1" in preparation example 1 in WO2019/105437A1, except that nucleoside monomers were linked one by one from the 3'-5' direction in the nucleotide arrangement order shown in the sense strand and antisense strand of siRNA22, siRNA23, and siRNA24 described below. The structures of reference conjugates 1 to 3 are shown below (803):

wherein, for reference conjugate 1, nu in formula (803) is siRNA22 having the following composition:

siRNA22

sense strand: cmsCmUmUmGmGfCfAmUmMemUmUmMemMemAm (SEQ ID NO: 25)

Antisense strand: VPUmsUfsUmGmAmAfGmUmUmGmCmCmUfCmAfAmGmGmUmsUm (SEQ ID NO: 26)

Theoretical value of sense strand: 7516.5, found for sense strand: 7515.3; theoretical value of antisense strand: 7061.7, found for antisense strand: 7060.7. the observed value is consistent with the theoretical value, thereby confirming that the synthesized reference conjugate 1 is the targeted designed double-stranded siRNA conjugate having the structure represented by formula (803).

For reference conjugate 2, nu in formula (803) is siRNA23 with the following composition:

siRNA23

sense strand: gmUmsAmCmGmUmGfGfAfCmUmGmGmAmUmGm (SEQ ID NO: 27)

Antisense strand: VPCmsafGmAmAmAmUfCmAmGmMemCMmAFCmUmAmmCumUmsUm (SEQ ID NO: 28)

The theoretical value of the sense strand is 7605.4, the measured value of the sense strand is 7604.3; theoretical value of antisense strand: 7002.6, found for antisense strand: 7001.5. the observed value is consistent with the theoretical value, thereby confirming that the synthesized reference conjugate 2 is the targeted designed double-stranded siRNA conjugate having the structure represented by formula (803).

For reference conjugate 3, nu in formula (803) is siRNA24 with the following composition:

siRNA24

sense strand: cmsCMAmmGmGfCfAfCmAmAmmAmmAmmAm (SEQ ID NO: 29)

Antisense strand: VPUmsAfs GmUmUmUmUmGmGmGmGmCmUfCmUfUmGmGmCmCmUm (SEQ ID NO: 30)

Theoretical value of sense chain: 7584.5, found for sense strand: 7583.5; theoretical value of antisense strand: 7007.4, found for antisense strand: 7006.4. the observed value is consistent with the theoretical value, thereby confirming that the synthesized reference conjugate 3 is the targeted designed double-stranded siRNA conjugate having the structure represented by formula (803).

After the preparation of the above siRNA or siRNA conjugate is complete, it is lyophilized to a solid powder using standard means for storage.

Experimental example 1 this experiment demonstrates the inhibitory efficiency of the siRNA conjugates of the present disclosure on HBV mRNA expression level in vitro (in vitro).

HBV transgenic mice C57BL/6J-Tg (A1 b1 HBV) 44Bri/J were purchased from the department of laboratory animals of the department of medicine of Beijing university, and mice with S/COV >10, hereinafter referred to as 44Bri mice, were selected for testing prior to the experiment.

Extracting fresh liver tissue of 44Bri mouse to obtain mouse liver primary cells, and adjusting the density of the mouse liver primary cells to 2 × 10 in Opti-MEM (1X) culture medium (GIBCO Co., ltd., cat. No. 31985-070) ⁵ cell/mL to obtain a mouse liver primary cell suspension. The obtained mouse liver primary cell suspensions were then added to different culture wells of a 12-well plate, and the mouse liver primary cells were inoculated into the culture wells. The volume of the added mouse liver primary cell suspension is 0.5 mL/hole, and the number of the mouse liver primary cells is 1 multiplied by 10 ⁵ Cells/well.

Each of the following siRNA conjugates, conjugate 1, conjugate 2 or reference conjugate 1, was formulated separately into 20 μ M (as siRNA) siRNA conjugate working solution using DEPC water.

In different culture wells added with the mouse liver primary cell suspension, the siRNA conjugate working solution of each conjugate was added respectively, and mixed uniformly, the addition amount was 2.5. Mu.L/well, each siRNA conjugate was added into 3 culture wells respectively, and a transfection mixture containing siRNA (calculated as siRNA, the final concentration was 10 nM) was obtained and recorded as a test group. The mixture in 3 additional culture wells to which the mouse liver primary cell suspension was added was designated as blank control.

Each siRNA-containing transfection mixture and blank control group was placed at 5% CO ₂ The incubator of (4) was further incubated at 37 ℃ for 24 hours.

Subsequently, total RNA in each well cell was extracted using TRIZOL (available from SIGMA, cat # T9424) according to the method described in the specification to obtain an aqueous total RNA solution.

For each well of cells, an aqueous total RNA solution containing 1. Mu.g of total RNA was taken separately, and a reverse transcription kit, golden star, was used ^TM RT6 cDNA Synthesis Kit (available from New Biotechnology, inc. of Beijing Okagaku, cat # TSK 301M) provided reagent, wherein Goldnstar was selected ^TM Oligo(dT) ₁₇ As a primer, 20. Mu.L of a reverse transcription reaction system is configured according to the reverse transcription operation steps in the kit specification, and the total RNA of each cell is subjected to reverse transcription. The reverse transcription conditions were: for each reverse transcription reaction system, the reverse transcription reaction system is incubated at 50 ℃ for 50min, then at 85 ℃ for 5min, and finally at 4 ℃ for 5min, and after the reaction is finished, 80 mu L of DEPC water is added into the reverse transcription reaction system to obtain a solution containing cDNA.

For each reverse transcription reaction system, 5. Mu.L of the above cDNA-containing solution was used as a template

15 μ L of qPCR reaction system was prepared using the reagents supplied by SYBR qPCR Supermix Plus kit (available from near shore protein science and technology Co., ltd., product No. E096-01B), wherein the sequences of PCR primers for amplifying the target gene HBV and the reference gene GAPDH are shown in Table 2, and the final concentration of each primer is 0.25 μ M. Each qPCR reaction system was placed on an ABI StepOneplus Real-Time PCR instrument using threeThe amplification is carried out by the step method, the amplification procedure is pre-denaturation at 95 ℃ for 10min, then denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 25s and extension at 72 ℃ for 25s, and the processes of denaturation, annealing and extension are repeated for 40 times to obtain a product W containing amplified target genes HBV and internal reference genes GAPDH. The product W is sequentially incubated at 95 ℃ for 1min,55 ℃ for 30s and 95 ℃ for 30s, and the real-time fluorescent quantitative PCR instrument respectively collects the dissolution curves of the target gene HBV and the internal reference gene GAPDH in the product W to obtain the Ct values of the target gene HBV and the internal reference gene GAPDH.

TABLE 2 sequence of detection primers

The relative expression level and the inhibition rate of the target gene HBV in each test group are calculated relatively and quantitatively by adopting a comparative Ct (delta Ct) method, and the calculation method is as follows:

delta Ct (test group) = Ct (test group target gene) -Ct (test group reference gene)

Δ Ct (control group) = Ct (control group target gene) -Ct (control group reference gene)

Δ Δ Ct (test group) = Δ Ct (test group) - Δ Ct (control group average)

Δ Δ Ct (control) = Δ Ct (control) - Δ Ct (control average)

Where Δ Ct (control mean) is the arithmetic mean of Δ Ct (control) for each of the three culture wells of the control. Thus, one Δ Δ Ct value was assigned to each culture well for the test and control groups.

Normalizing the expression level of HBV mRNA in the test group by taking the control group as a reference, defining the expression level of HBV mRNA in the blank control group as 100 percent,

test group HBV mRNA relative expression level =2 ^{Δ Δ Ct (test group)} ×100％

Test group HBV mRNA inhibition rate = (1-test group HBV mRNA relative expression level) × 100%

Table 3 below shows the results of the detection of HBV mRNA expression inhibitory activity of each conjugate of the present disclosure in hepatic primary cells.

Table 3 siRNA conjugates in vitro activity assay

siRNA conjugates	mRNA inhibition (%)
		Conjugate 1	81.63
Conjugate 2	84.04
		Reference conjugate 1	83.14

As can be seen from the results of table 3, both conjugate 1 and conjugate 2 of the present disclosure in table 3 showed excellent HBV gene expression inhibitory activity at a cellular level, maintaining the same level as that of reference conjugate 1.

Experimental example 2 this experiment demonstrates the inhibitory efficiency of the siRNA conjugates of the present disclosure on HBV mRNA expression level at day 8 in vivo (in vivo).

Using a hepatitis B virus surface antigen diagnostic kit (enzyme-linked immunosorbent assay) (Shanghai Kawawa) to detect the HbsAg content of 44Bri mice according to the method described in the specification, selecting mice with S/COV >10, randomly grouping the mice (all male), numbering 5 mice in each group, and subcutaneously injecting the

conjugates

1,2 or 1, respectively, in the form of 1 XPBS solution containing 0.02mg/ml (calculated as siRNA) of siRNA conjugate at the dose of 0.1mg/kg of mouse body weight (calculated as siRNA) to each mouse, wherein the administration volume is 5 ml/kg; each of the other 2 groups of mice was given 1 XPBS at a volume of 5ml/kg as a blank control group.

Animals were sacrificed on day 8, calculated as the administration time point as day 1, and liver tissue of each mouse was collected and preserved with RNA later (Sigma Aldrich company); 1mL of Trizol (Sigma) was added to each liver tissue, disrupted in a Tissuelyset II type fully automatic tissue homogenizer 3 times for 30 seconds to obtain a liver tissue homogenate, and 0.2mL of chloroform was added thereto and allowed to stand for 3min. After centrifugation at 12000rpm for 10min at 4 ℃ 0.4mL of the supernatant was collected. 0.5mL of isopropanol was added to the supernatant, and the mixture was allowed to stand at room temperature for 10min. The mixture was centrifuged at 12000rpm for 10min at 4 ℃ and the supernatant was discarded. The precipitate was washed with 1mL of ethanol, centrifuged at 12000rpm at 4 ℃ for 5min, and the supernatant was discarded. To the precipitate, 70. Mu.L of DEPC-treated water was added to obtain a total RNA solution.

For total RNA in liver tissue of each mouse, 10.5. Mu.L of a total RNA aqueous solution containing 1. Mu.g of total RNA was taken, and prepared into a Reverse Transcription reaction System of 20. Mu.L by using a Reverse Transcription kit Reverse Transcription System (purchased from Promega corporation, cat. No. A3500) according to the Reverse Transcription procedure in the kit instructions, and the total RNA was subjected to Reverse Transcription. The reverse transcription conditions were: for each reverse transcription reaction system, the reverse transcription reaction system is incubated at 42 ℃ for 30min, then incubated at 95 ℃ for 5min, and finally incubated at 4 ℃ for 5min, and after the reaction is finished, 80 μ L of DEPC water is added into the reverse transcription reaction system to obtain a solution containing cDNA.

For each reverse transcription reaction system, 5. Mu.L of the above cDNA-containing solution was used as a template, and 20. Mu.L of qPCR reaction system was prepared using a reagent provided by SYBR select Master Mix kit (Applied biosystems Co.), wherein the PCR primer sequences for amplifying the target gene HBV and the reference gene GAPDH are shown in Table 2, and the final concentration of each primer was 0.25. Mu.M. And (3) placing each qPCR reaction system on an ABI StepOnePlus Real-Time PCR instrument, amplifying by using a three-step method, wherein the amplification procedure is pre-denaturation at 95 ℃ for 10min, then denaturation at 95 ℃, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, and repeating the processes of denaturation, annealing and extension for 40 times to obtain a product W containing amplified target gene HBV and reference gene GAPDH. The product W is sequentially incubated at 95 ℃ for 1min,55 ℃ for 30s and 95 ℃ for 30s, and the real-time fluorescent quantitative PCR instrument respectively collects the dissolution curves of the target gene HBV and the internal reference gene GAPDH in the product W to obtain the Ct values of the target gene HBV and the internal reference gene GAPDH.

The relative expression level and inhibition rate of HBV mRNA in liver tissue of mice after administration of each siRNA conjugate were calculated from Ct values according to the method described in Experimental example 1.

Table 4 below shows the results of the detection of inhibitory activity of each conjugate of the present disclosure on HBV mRNA expression in mice.

TABLE 4 inhibition of mouse liver HBV mRNA by different conjugates

As can be seen from the results of table 4, both the conjugate 1 or the conjugate 2 of the present disclosure in table 4 showed excellent inhibitory activity of HBV gene expression in mice, having HBV mRNA inhibition rate of at least 67.54% at a lower dose of 0.1mg/kg, while the conjugate 1 showed more excellent inhibitory activity in vivo compared to the reference conjugate 1.

Experimental example 3 this experiment demonstrates the animal level toxicity of the siRNA conjugates of the invention.

On SD rats (purchased from si Bei Fu (beijing) biotechnology limited), 30mg/kg (in siRNA) of conjugate 1, conjugate 2 and reference conjugate 1 of the present disclosure, pbs, respectively, were subcutaneously and singly administered to each rat in a single dose for 14 days continuously during which the rats exhibited no death or abnormal behavior. The rat was dissected, liver tissue dissected, harvested, dehydrated, embedded, sectioned and stained to prepare pathological sections, which were 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 pathological liver sections of rats given

conjugates

1 and 2, the liver chordae structure of the tissues is clear, the arrangement of liver cells is tight, the boundary is clear, the cytoplasm is rich, the staining is uniform, the nucleus is circular, the size is normal, the vein endothelium is complete and normal, and no obvious abnormality is seen in the tissues, while in the pathological liver sections of rats given reference conjugate 1, extensive edema and degeneration of liver cells, cell swelling, cytopenia, mild cytopenia, a large number of liver cells accompanied with steatosis, circular vacuoles with different numbers in the cytoplasm and focal infiltration of inflammatory cells in lobules are seen. It can be seen that there is a significant improvement in toxicity for conjugate 1 and conjugate 2 of the present disclosure compared to reference conjugate 1.

Experimental example 4 this experiment demonstrates the inhibitory efficiency of the siRNA conjugates of the present disclosure on HBV mRNA expression level at day 8 in vivo (in vivo).

The conjugates 3,4, 5 or 6 were administered to mice, respectively, according to the method of experimental example 2, the siRNA conjugates were provided in the form of 1 XPBS solutions containing 0.02mg/ml (in terms of siRNA) of siRNA conjugates, each at a dose volume of 5ml/kg, as experimental groups; each of the 2 additional groups of mice was given 1 XPBS at a volume of 5ml/kg as a blank control group.

Relative expression level and inhibition rate of HBV mRNA in liver tissue of mice were obtained according to the method of Experimental example 2, with the administration time point as day 1, and the animals were sacrificed on day 8.

Table 5 below shows the results of the detection of inhibitory activity of each conjugate of the present disclosure on HBV mRNA expression in mice.

TABLE 5 inhibition of mouse liver HBV mRNA by different conjugates

As can be seen from the results of table 5, each of the conjugates 3 to 6 of the present disclosure showed excellent HBV gene expression inhibitory activity in mice, and the conjugate 3 reached an HBV mRNA inhibitory rate of 69.57% at a lower dose of 0.1 mg/kg.

Experimental example 5 this experiment demonstrates the inhibitory efficiency of the siRNA conjugates of the present disclosure on HBV mRNA expression level at day 15 in vivo (in vivo).

The inhibitory efficiency of the conjugate 2, the conjugate 3 and the reference conjugate 1 on the expression amount of HBV mRNA on day 15 in vivo (in vivo) was tested according to the method of Experimental example 2.

Table 6 below shows the results of the detection of inhibitory activity of each conjugate of the present disclosure on HBV mRNA expression in mice.

TABLE 6 inhibition of mouse liver HBV mRNA by different conjugates

As can be seen from the results of table 5, compared to reference conjugate 1, siRNA conjugate 2 or conjugate 3 of the present disclosure had comparable or even superior inhibitory activity against FXI mRNA expression at day 15 at siRNA administration dose of 0.1mg/kg to that of reference conjugate 1.

Experimental example 6 this experiment demonstrates the efficiency of inhibition of FXI mRNA expression levels in vivo (in vivo) by siRNA conjugates of the present disclosure.

C57BL/6N mice were randomly grouped (all female), and 5 mice per group were numbered separately. The test conjugate 8, conjugate 9 or reference conjugate 2 was administered to each group of mice as a subcutaneous injection at a dose of 3mg/kg (both expressed as siRNA). The siRNA conjugates were each provided as 1mg/ml of siRNA conjugate in PBS at a dose volume of 3ml/kg.

One group of mice was given 1 XPBS at a volume of 3ml/kg, and served as a control group.

Animals were sacrificed on day 8, calculated as the administration time point as day 1, and liver tissue of each mouse was collected and preserved with RNA later (Sigma Aldrich company); liver tissue was homogenized with a tissue homogenizer and total RNA was extracted using Trizol (Thermo Fisher Co.) according to the protocol described in the specification.

The C57BL/6N mice were randomly grouped (all female), and 5 mice per group were individually numbered and the test conjugate 8, conjugate 9, reference conjugate 2 and PBS (3 ml/kg) were administered at the same dose (3 mg/kg), concentration (1 mg/ml) and dosing volume (3 ml/kg) as described above. Animals were sacrificed on day 15, calculated as the administration time point as day 1, and liver tissue of each mouse was collected and preserved with RNA later (Sigma Aldrich company); liver tissue was homogenized with a tissue homogenizer and total RNA was extracted using Trizol (Thermo Fisher Co.) according to the protocol described in the specification.

The quantitative PCR detection of fluorescence and the calculation of the expression level and inhibition rate of FXI mRNA were carried out according to the method of Experimental example 1, specifically: the extracted total RNA was reverse-transcribed into cDNA using ImProm-IITM reverse transcription kit (Promega Co.) according to the instructions thereof to obtain a cDNA-containing solution, followed by detection of the expression level of FXI mRNA in liver tissue using a fluorescent quantitative PCR kit (Beijing kang, century Biotechnology Co., ltd.). In the fluorescent quantitative PCR method, murine GAPDH (mGAPDH) gene was used as an internal reference gene, and primers for FXI and murine GAPDH were used to detect FXI and murine GAPDH, respectively. The sequences of the detection primers are shown in Table 6. In the calculation of the expression level and inhibition rate of FXI mRNA, the control group was the control group mice administered with PBS in this experiment, and each test group was the administration group mice administered with different siRNA conjugates. The expression level of FXI mRNA in the control group was recorded as 100%, and the inhibition rate of the expression level of FXI mRNA was recorded as 0%, and the test results were normalized to the expression level of FXI mRNA in the control group, and the results are shown in Table 8.

TABLE 7 detection primer sequences

Table 8 below shows the results of the detection of FXI mRNA expression inhibitory activity of each conjugate of the present disclosure in mice. The relative expression levels of FXI mRNA in the liver of each group of mice are shown in FIG. 1.

TABLE 8 inhibition of FXI mRNA by siRNA conjugates

FIG. 1 is a line graph showing the relative expression levels of FXI mRNA at days 8, 15 and 29 after administration of 3mg/kg of different siRNA conjugates in liver tissues of mice compared to a blank control. As can be seen from the results of table 8 or fig. 1, the siRNA conjugates of the present disclosure showed superior inhibition effects on FXI mRNA expression at the siRNA administration dose of 3mg/kg on days 8, 15, and 29, compared to the reference conjugate 2, all over the FXI mRNA expression inhibition activity on days 8, 15, and 29.

Experimental example 7 this experiment demonstrates the efficiency of the inhibition of ANGPTL3mRNA expression levels by siRNA conjugates of the disclosure in vivo (in vivo).

C57BL/6N mice were randomly grouped into 5 mice per group, and each group was given conjugate 15, conjugate 16 and reference conjugate, and PBS, respectively. Each group of mice was administered test conjugate 15, conjugate 16 or reference conjugate 3 at a dose of 3mg/kg (both expressed as siRNA), respectively. The siRNA conjugates were each provided as 1mg/ml of siRNA conjugate in PBS at a dose volume of 3ml/kg.

The fluorescent quantitative PCR detection and the calculation of the expression level and the inhibition rate of ANGPTL3mRNA were carried out according to the method of Experimental example 1, specifically: the cDNA was obtained by reverse transcription using a reverse transcription kit (Promega corporation, cat. No. A3500) according to the protocol described in the specification. The amount of expression of ANGPTL3mRNA was measured using a 2X Ultra SYBR Mixture (with ROX) (Beijing Kan, a century Biotechnology Co., ltd., product number CW 0956) kit by the procedure described in the specification using cDNA as a template. Among them, PCR primers for amplifying ANGPTL3 and GAPDH as an internal reference gene are shown in Table 6.

Table 9: primer sequences

In the calculation of ANGPTL3mRNA expression levels and inhibition rates, the control group was the control group mice administered PBS in this experiment, and each test group was the group mice administered with a different siRNA conjugate. The ANGPTL3mRNA expression level of the control group was recorded as 100%, and accordingly, the inhibition ratio of the ANGPTL3mRNA expression level was recorded as 0%, and the test results were normalized to the ANGPTL3mRNA expression level of the control group, and the results are shown in table 10. The relative expression levels of ANGPTL3mRNA in the liver of each group of mice are shown in fig. 2.

TABLE 10 inhibition of ANGPTL3mRNA by siRNA conjugates

Figure 2 is a line graph showing the relative expression levels of ANGPTL3mRNA at day 8, day 15, and day 29 post-administration in liver tissue of mice after administration of 3mg/kg of different siRNA conjugates compared to a blank control. As can be seen from the results of table 10 or fig. 2, the siRNA conjugates of the present disclosure had comparable inhibitory activity against ANGPTL3mRNA expression at the administration dose of siRNA of 3mg/kg on days 8, 15, and 29 as the reference conjugate 3. Some embodiments of the present disclosure are described in detail above, however, the present disclosure is not limited to the specific details in the above embodiments, and many simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

It should be noted that, in some embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not further described.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Claims

1. A double-stranded oligonucleotide comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, each nucleotide in the double-stranded oligonucleotide being independently a modified or unmodified nucleotide, a portion of the sense strand and the antisense strand being reverse-complementary to form a double-stranded region, wherein the 5 'terminal nucleotide of the antisense strand is a nucleotide having a ribose 5' modification, the modified nucleotide having a structure according to formula (101), the group R being ₀ Constituting the 5' overhang of the antisense strand, R ₀ Has the structure shown as the formula (102),

wherein the content of the first and second substances,

R ₂₀₁ is hydroxyl or phosphate;

Bx ₁ is hydrogen, a heterocyclic base or a base substitution group, and the 3' end of the sense strand, if including an overhang, bx ₁ (ii) does not pair with a base of the overhang, and the base substitution group is phenyl or substituted phenyl; bx ₂ Is a heterocyclic base;

z is one of divalent linking groups represented by the structures represented by the formulae (Z1) to (Z5), or Z is 1,2-cycloalkylene or heterocyclylene having 3 to 6 carbon atoms, or substituted 1,2-cycloalkylene or heterocyclylene having 3 to 6 carbon atoms:

wherein, P ₁ And P ₂ Each independently is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, or substituted C2-C6 alkenyl; p ₃ Is O or S;

T ₂ is a phosphate subunit or a phosphorothioate subunit;

Y ₁ 、Y ₂ 、Y ₃ 、Y ₄ 、Y ₅ 、Y ₆ 、Y ₇ and Y ₈ Each independently is H, halogen, hydroxy, methyl, ethyl, n-propyl, or isopropyl;

indicates the site at which the group is covalently attached.

2. The double stranded oligonucleotide of claim 1, wherein the heterocyclic base is a pyrimidine, substituted pyrimidine, purine or substituted purine; optionally, the heterocyclic base is uracil, thymine, cytosine, 5-methylcytosine, adenine or guanine.

3. The double stranded oligonucleotide of claim 1, wherein the substituted phenyl is one or more hydrogens on the phenyl group independently substituted with F, cl, CH ₃ 、CH ₂ F or CF ₃ The resulting group is substituted.

4. The double-stranded oligonucleotide of claim 1, wherein G ₁ Is OH, T ₂ Are phosphorothioate subunits.

5. The double-stranded oligonucleotide of claim 1, wherein Z has a structure represented by formula (Z1) or (Z2), and P ₁ And P ₂ Are all H.

6. The double-stranded oligonucleotide of claim 1, wherein X ₂ Is OCH ₃ 。

7. The double-stranded oligonucleotide of claim 1, wherein Y is ₁ 、Y ₂ 、Y ₃ 、Y ₄ 、Y ₅ 、Y ₆ 、Y ₇ 、Y ₈ Are all H.

8. The double stranded oligonucleotide according to claim 1, wherein the group of formula (101) is selected from a group of formula (501), (502), (503), (504), (505), (506) or (507):

wherein Base is C, G, U, T or A.

9. The double-stranded oligonucleotide of any one of claims 1-8, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, wherein the 5 'terminal nucleotide of the nucleotide sequence II is a nucleotide having a ribose 5' modification, the modified nucleotide having the structure of formula (101); the nucleotide sequence I consists of 19 nucleotides, and a group shown as a formula (101) is taken as 2One nucleotide count, the nucleotide sequence II consisting of 20 nucleotides, the radical R ₀ An overhang forming the 5' end of the antisense strand, and the rest of the nucleotide sequence II forming a double-stranded region with the nucleotide sequence I, said nucleotide sequence II being at least partially reverse complementary to a first nucleotide sequence of 19 nucleotides in length in the mRNA expressed by the target gene; in the direction from the 5 'end to the 3' end, the 7 th to 9 th nucleotides of the nucleotide sequence I are fluoro-modified nucleotides; according to the direction from the 5 'end to the 3' end, the 3 rd, 7 th, 15 th and 17 th nucleotides of the nucleotide sequence II are fluorine modified nucleotides.

10. The double-stranded oligonucleotide of claim 9, wherein, in the 5 'to 3' terminal direction, the 7 th to 9 th nucleotides of the nucleotide sequence I are fluoro-modified nucleotides, and each nucleotide at other positions of the nucleotide sequence I is independently one of non-fluoro-modified nucleotides; according to the direction from 5 'end to 3' end, the 3 rd, 7 th, 15 th and 17 th nucleotides of the nucleotide sequence II are fluorine modified nucleotides, and each nucleotide at other positions of the nucleotide sequence II is independently one of non-fluorine modified nucleotides.

11. The double stranded oligonucleotide of claim 10, wherein nucleotides 2 to 20 of the nucleotide sequence II is substantially reverse complementary, or fully reverse complementary to the first stretch of nucleotide sequence in a5 'end to 3' end direction.

12. The double stranded oligonucleotide of claim 11, wherein the nucleotides at positions 3-20 of the nucleotide sequence II are fully reverse complementary to the nucleotides at positions 1-18 of the first stretch of nucleotide sequence in the 5 'to 3' direction.

13. The double stranded oligonucleotide of any one of claims 9-12, wherein nucleotides 2 to 20 of the nucleotide sequence II is substantially reverse complementary, or fully reverse complementary to the nucleotide sequence I in the 5 'end to 3' end direction.

14. The double-stranded oligonucleotide of claim 13, wherein the 2 nd to 20 th nucleotides of the nucleotide sequence II are completely reverse-complementary to the nucleotide sequence I in the 5 'to 3' terminal direction, or a base mismatch is present between the 3 rd nucleotide of the nucleotide sequence II and the 2 nd nucleotide of the nucleotide sequence I in the 3 'to 5' terminal direction in the 5 'to 3' terminal direction.

15. The double-stranded oligonucleotide of any one of claims 9-14, wherein the sense strand further comprises a nucleotide sequence III, the antisense strand further comprises a nucleotide sequence IV, each nucleotide of the nucleotide sequence III and the nucleotide sequence IV is independently one of non-fluorinated modified nucleotides, the nucleotide sequence III is 1,2, 3, or 4 nucleotides in length, the nucleotide sequence IV and the nucleotide sequence III are equal in length, and the nucleotide sequence IV and the nucleotide sequence III are substantially reverse-complementary or fully reverse-complementary, the nucleotide sequence III is linked to the 5 'end of the nucleotide sequence I, the nucleotide sequence IV is linked to the 3' end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse-complementary or fully reverse-complementary to a second nucleotide sequence that is adjacent to the first nucleotide sequence and has the same length as the nucleotide sequence IV in an mRNA expressed by a target gene.

16. The double stranded oligonucleotide of any one of claims 9-15, further comprising a nucleotide sequence V, each nucleotide of said nucleotide sequence V being independently one of the non-fluorinated modified nucleotides, said nucleotide sequence V being 1 to 3 nucleotides in length, attached to the 3 'terminus of the antisense strand, thereby constituting a 3' overhang of the antisense strand.

17. The double-stranded oligonucleotide according to claim 16, 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 2 consecutive thymine deoxyribonucleotides, 2 consecutive uracil ribonucleotides, or is completely reverse complementary to a third nucleotide sequence which is adjacent to the 5' end of the first nucleotide sequence or the second nucleotide sequence in the mRNA expressed by the target gene and has the same length as the nucleotide sequence V.

18. The double stranded oligonucleotide of any one of claims 9-17, wherein each non-fluorinated modified nucleotide is independently selected from one of a nucleotide or a nucleotide analogue in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group.

19. The double-stranded oligonucleotide of any one of claims 9 to 18, wherein each of the non-fluorinated modified nucleotides is a methoxy modified nucleotide, which is a nucleotide formed by substituting a 2' -hydroxyl group of a ribosyl group with a methoxy group.

20. The double stranded oligonucleotide of any one of claims 9-19, wherein at least 1 of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands is a phosphate group having a modifying group present at least one of the group consisting of:

the 5' terminal end of the sense strand is between the 2 nd nucleotide and the 3 rd nucleotide;

between the 1 st and 2 nd nucleotides at the 3' terminal end of the sense strand;

the 3' terminal end of the sense strand is between the 2 nd and 3 rd nucleotides;

21. The double stranded oligonucleotide of any one of claims 1-20, wherein the double stranded oligonucleotide is a saRNA or siRNA.

22. The double stranded oligonucleotide of any one of claims 1-21, 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.

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

24. An oligonucleotide conjugate comprising a double-stranded oligonucleotide of any one of claims 1 to 23 and a conjugate group conjugated to the double-stranded oligonucleotide, the conjugate group comprising a linker and a pharmaceutically acceptable targeting group and/or a delivery assisting group, and the double-stranded oligonucleotide, the linker and the targeting group or the delivery assisting group being covalently or non-covalently linked in that order, each targeting group being selected from a ligand capable of binding to a cell surface receptor and each delivery assisting group being selected from a group capable of increasing the biocompatibility of the oligonucleotide conjugate in delivering a target organ or tissue.

25. Use of the double-stranded oligonucleotide of any one of claims 1-22, and/or the pharmaceutical composition of claim 23, and/or the oligonucleotide conjugate of claim 24, in the manufacture of a medicament for treating and/or preventing a disease or condition associated with mRNA levels of target gene expression.

26. The use according to claim 25, wherein the mRNA expressed by the target gene is selected from one of the mrnas transcribed from: 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, SOD, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT.

27. The use of claim 26, wherein the mRNA expressed by the target gene is selected from mRNA expressed by a hepatitis b virus gene.

28. The use of any one of claims 25-27, wherein the disease or condition associated with mRNA levels of target gene expression is hepatitis b.

29. A method of modulating the level of expression of a target gene in a cell in vitro, the method comprising contacting the cell with an effective amount of the double-stranded oligonucleotide of any one of claims 1-22, and/or the pharmaceutical composition of claim 23 and/or the oligonucleotide conjugate of claim 24.

30. The method of claim 29, wherein the mRNA expressed by the target gene is selected from one of the mrnas transcribed from: 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, SOD, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT.

31. The method of claim 29 or 30, wherein the modulation is inhibition of target gene expression in the cell, and the mRNA expressed by the target gene is selected from the group consisting of mRNA expressed by hepatitis b virus genes.

32. A kit comprising a double stranded oligonucleotide according to any one of claims 1 to 22, and/or a pharmaceutical composition according to claim 23 and/or an oligonucleotide conjugate according to claim 24.