CN115677770A

CN115677770A - Modified nucleoside monomer, siRNA comprising modified nucleotide, pharmaceutical composition and conjugate

Info

Publication number: CN115677770A
Application number: CN202110836332.3A
Authority: CN
Inventors: 刘国成; 李海涛; 梁子才; 杨志伟
Original assignee: Suzhou Ruibo Biotechnology Co ltd; Beijing Ruibo Kaifa Pharmaceutical Technology Co ltd
Current assignee: Suzhou Ruibo Biotechnology Co ltd; Beijing Ruibo Kaifa Pharmaceutical Technology Co ltd
Priority date: 2021-07-23
Filing date: 2021-07-23
Publication date: 2023-02-03

Abstract

A modified nucleoside monomer, siRNA comprising a modified nucleotide, pharmaceutical composition and siRNA conjugate. The modified nucleoside monomer has a structure shown as a formula (1). The siRNA conjugate prepared by the modified nucleoside monomer has high bioavailability, better activity and lower toxicity.

Description

Modified nucleoside monomers, sirnas comprising modified nucleotides, pharmaceutical compositions and conjugates

Technical Field

The disclosure relates to a modified nucleoside monomer, and siRNA, pharmaceutical composition and siRNA conjugate containing modified nucleotide prepared from the nucleoside monomer. The disclosure also relates to methods of making and uses of the modified nucleosides, siRNA, pharmaceutical compositions, and siRNA conjugates.

Background

siRNA is known as a pharmaceutically active ingredient. In recent years, considerable progress has been made in the formulation of siRNA. As well known to those skilled in the art, the current siRNA preparation method is mainly performed by one ligation from different nucleoside monomers according to the phosphoramidite solid phase synthesis method. The nucleoside phosphoramidite monomer is used as an important reaction raw material of the synthesis method, is directly related to the structure and the performance of the prepared siRNA, and has very important function. In order to improve the activity, in vivo stability and pharmacokinetic properties of the prepared siRNA, one skilled in the art will often choose to chemically modify a specific nucleotide in the siRNA, and for this reason, a common means for achieving such chemical modification is to use a modified nucleoside phosphoramidite monomer in the corresponding step of the siRNA preparation process. However, another problem in the development of siRNA drugs is the safety of siRNA itself, i.e., how to minimize the toxicity of siRNA. However, how to obtain siRNA having both good pharmaceutical activity and low toxicity, and how to obtain modified nucleoside phosphoramidite monomers that can reduce the toxicity of the prepared siRNA, further intensive research is still needed in the art, and there is still an unresolved need in actual development.

Disclosure of Invention

The inventors of the present disclosure surprisingly found that modifying the 5' -position of ribose and the nucleobase of nucleoside monomer to obtain modified nucleoside monomer can be used for preparing siRNA. The siRNA, the pharmaceutical composition and the siRNA conjugate containing the modified nucleotide prepared by the modified nucleoside monomer have high bioavailability, better inhibitory activity on mRNA expressed by a target gene and lower toxicity.

In one aspect, the present disclosure provides a compound having a structure as shown in formula (1).

Wherein:

each J ₁ Independently is C1-C6 alkyl or substituted C1-C6 alkyl;

E ₁₀₁ is O or S;

R ₂₀₃ is a phosphoramidite functional group having the structure shown in formula (2):

wherein each B is ₁ Independently selected from substituted or unsubstituted C1-C5 hydrocarbyl; b ₂ One selected from C1-C5 alkyl, cyanoethyl, cyanopropyl and cyanobutyl;

D ₀ is one of divalent linking groups represented by formulae (D1) to (D4):

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;

bm is phenyl or substituted phenyl, the substituted phenyl refers to a group formed by substituting at least one hydrogen atom on the phenyl with a substituent, and each substituent is independently selected from F, cl and CH ₃ 、CH ₂ F or CF ₃ ；

Z ₀ Is H, protected hydroxy, OCH ₃ F or OCH ₂ CH ₂ OCH ₃ ；

Indicates the site at which the group is covalently attached.

In another aspect, the present disclosure provides an siRNA comprising a sense strand and an antisense strand, the sense strand and the antisense strand comprising 14 to 30 modified or unmodified nucleotides, 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 has a structure represented by formula (5):

wherein:

R ₂₀₁ and R ₂₀₂ Each independently is OH or OJ ₁ ，J ₁ Is C1-C6 alkyl or substituted C1-C6 alkyl;

each E ₁₀₁ Each independently is O or S;

D ₀ a group having one of divalent linking groups represented by the structures represented by the following formulae (D1) to (D4):

Z is H, OH, OCH ₃ F or OCH ₂ CH ₂ OCH ₃ ；

Represents the site of covalent attachment of a group;

the antisense strand is substantially reverse complementary or substantially reverse complementary to a nucleotide sequence in the same length as the antisense strand in an mRNA expressed by the target gene.

In yet another aspect, the present disclosure also provides a pharmaceutical composition comprising the siRNA of the present disclosure and a pharmaceutically acceptable carrier.

In yet another aspect, the present disclosure also provides an siRNA conjugate comprising an siRNA of the present disclosure and a conjugate group conjugated to the siRNA, the conjugate group comprising a linker and a pharmaceutically acceptable targeting group and/or a delivery assisting group, and the siRNA, the linker and the targeting group or the delivery assisting group are covalently or non-covalently linked in that order, each of the targeting groups 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 siRNA conjugate in delivering a target organ or tissue.

In yet another aspect, the present disclosure also provides use of the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure in the manufacture of a medicament for treating and/or preventing a disease or condition associated with mRNA levels of target gene expression.

In yet another 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 an siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure.

In yet another aspect, the present disclosure also provides a method of inhibiting the expression level of a target gene in a cell, the method comprising contacting the cell with an effective amount of an siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure.

In addition, the present disclosure also provides a kit comprising the siRNA, pharmaceutical composition and/or siRNA 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 siRNA, pharmaceutical composition and/or siRNA conjugate comprising modified nucleotide prepared from the modified nucleoside monomer of the present disclosure shows significantly lower toxicity while maintaining good stability, better target gene expression modulating activity. The concrete description is as follows:

first, the sirnas, pharmaceutical compositions, and/or siRNA conjugates provided by the present disclosure exhibit lower toxic responses in vivo. For example, the siRNA conjugates provided by the present disclosure are observed for 14 days under a single subcutaneous administration dose of 30mg/kg per rat, gross anatomy is performed, and liver histopathological sections are observed under an optical microscope, and it is found that the liver tissue of the rat given the conjugates of the present disclosure has clear liver chordae structure, close arrangement of liver cells, clear boundary, abundant cytoplasm, uniform staining, round nucleus, normal size, intact vein endothelium and no obvious abnormality of tissues, while the liver cells of the rat given the reference conjugates have extensive edema and degeneration, cell swelling, loose cytoplasm and light staining, a large number of liver cells are accompanied by steatosis, unequal numbers of round vacuoles in cytoplasm and multiple inflammatory focal infiltrations in lobules. Indicating a significant reduction in hepatotoxicity of siRNA conjugates of the present disclosure comprising specific modified nucleotides compared to reference siRNA conjugates.

Second, the siRNA, the pharmaceutical composition and/or the siRNA conjugate provided by the present disclosure show excellent target gene expression modulation activity in vitro cell experiments. For example, at an siRNA concentration of 10nM, the siRNA conjugates provided by the present disclosure show excellent target mRNA expression amount inhibition rates of 96.71% to 97.25% in HBV transgenic mouse liver primary cells.

In addition, the siRNA, pharmaceutical composition and/or siRNA conjugate provided by the present disclosure may also have higher stability and/or higher activity in vivo. For example, the siRNA conjugates provided by the present disclosure show high HBV mRNA inhibition rate of 74.5% to 77.2% in HBV transgenic mice even at a lower administration dose of 0.1 mg/kg.

Therefore, the siRNA, the pharmaceutical composition and/or the siRNA conjugate prepared from the modified nucleoside monomer provided by the disclosure can effectively inhibit the expression of the target gene in vitro and in vivo, so that the siRNA, the pharmaceutical composition and/or the siRNA conjugate can effectively treat and/or prevent the disease symptoms related to the mRNA level expressed by the target gene while having significantly higher safety, and have good application prospects.

Detailed Description

Specific embodiments of the present disclosure are described in detail below. 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 foregoing and following, nomenclature used in connection with, and procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those well known and commonly used in the art, unless a specific meaning is provided. 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 represent 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.

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 in the following, unless otherwise specified, "essentially 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 preparation method of the siRNA, the pharmaceutical composition or the siRNA conjugate of the present disclosure, unless otherwise specified, the Nucleoside monomer (Nucleoside monomer) means modified or unmodified Nucleoside phosphoramidite monomers (sometimes referred to as Nucleoside phosphoramidites) used in solid phase synthesis of phosphoramidites, depending on the kind and order of nucleotides in the siRNA or siRNA conjugate to be prepared. Solid phase phosphoramidite synthesis is a method known to those skilled in the art that is currently used primarily in the field of RNA synthesis. Unless otherwise mentioned, other nucleoside monomers useful in the present disclosure are commercially available in addition to the specific modified nucleoside monomers provided in the present disclosure.

In the above or below, unless otherwise specified, 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 group formed by replacing one or more hydrogen atoms in a C1-C5 hydrocarbyl group 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 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). 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 not 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, "hydrocarbyl" refers to a group formed after a corresponding hydrocarbon has lost one hydrogen atom. For example, a C4 hydrocarbyl group refers to a group formed by a hydrocarbon containing 4 carbon atoms having one hydrogen atom removed.

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-C5 alkyl groups include straight and branched chain alkyl groups of 1 to 5 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 molecule of hydrogen 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. Alkenylene is a subset of alkenyl and refers to the same residue as alkenyl, but having two points of attachment.

As used herein, "alkynyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond obtained by removing two hydrogen molecules from adjacent carbon atoms of the parent alkyl group. Typical alkynyl groups include, but are not limited to: 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 having two points of attachment.

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. Said aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, wherein at least one ring of said 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 groups include 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: azacyclotrienoyl, acridinyl, benzimidazolyl, benzindolyl, 1, 3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo [ d ] thiazolyl, benzothiadiazolyl, benzo [ b ] [1,4] dioxepinyl (benzo [ b ] [1,4] dioxapinyl), benzo [ b ] [1,4] oxazinyl (benzo [ b ] [1,4] oxazinyl), 1,4-benzodioxanyl (1, 4-benzodioxanyl), benzonaphthofuranyl, benzoxazolyl, benzodioxolyl (benzodioxolyl), benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzothiophenyl, benzothieno [3,2-d ] pyrimidinyl, benzotriazolyl, benzo [4,6] imidazo [1,2-a ] pyridinyl carbazolyl, cinnolinyl, cyclopenta [ d ] pyrimidinyl, 6, 7-dihydro-5H-cyclopenta [4,5] thieno [2,3-d ] pyrimidinyl, 5,6-dihydrobenzo [ H ] quinazolinyl (5, 6-dihydrobenzo [ H ] quinazolinyl), 5,6-dihydrobenzo [ H ] cinnolinyl (5, 6dihydrobenzo [ H ] cinnolinyl), 6, 7-dihydro-5H-benzo [6,7] cyclohepta [1,2-c ] pyridazinyl, and the like dibenzofuranyl, dibenzothienyl, furanyl, furanonyl, furo [3,2-c ] pyridyl, 5,6,7,8,9, 10-hexahydrocycloocta [ d ] pyrimidyl, 5,6,7,8,9, 10-hexahydrocycloocta [ d ] pyridazinyl, 5,6,7,8,9, 10-hexahydrocycloocta [ d ] pyridyl, isothiazolyl, imidazolyl, indazolyl, indolyl, isoindolyl, indolyl, etc, <xnotran> , , , (indolizinyl), ,5,8- -5,6,7,8- (5,8-methano-5,6,7,8-tetrahydroquinazolinyl), (naphthyridinyl), 1,6- (1,6-naphthyridinonyl), ,2- (2-oxoazepinyl), , (oxiranyl), 5,6,6a,7,8,9,10,10a- [ H ] ,1- -1H- , , , , (phthalazinyl), (pteridinyl), , , , [3,4-d ] , , [3,2-d ] , [3,4-d ] , , , , , (quinoxalinyl), , ,5,6,7,8- ,5,6,7,8- [4,5] [2,3-d ] ,6,7,8,9- -5H- [4,5] [2,3-d ] ,5,6,7,8- [4,5-c ] , , , , , , [2,3-d ] , [3,2-d ] , [2,3-c ] (thieno [2,3-c ] pridinyl) (thiophenyl/thienyl). </xnotran>

"heterocyclyl" refers to a stable 3-to 18-membered non-aromatic ring radical containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise stated in the specification, a heterocyclyl 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-diomorphinyl).

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 or substituted 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 or substituted pyrimidine. In some embodiments, the heterocyclic base is a non-naturally occurring pyrimidine or substituted pyrimidine.

Various protecting groups, such as hydroxyl protecting groups, may be used in the present disclosure. In general, protecting groups render chemical functional groups insensitive to particular reaction conditions, and can be added to and removed from the functional group in a molecule without substantially damaging the rest of the molecule. Representative hydroxyl protecting Groups are disclosed in Beaucage et al, tetrahedron 1992,48,2223-2311, and Greenea and 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," an siRNA, pharmaceutical composition, or siRNA conjugate can be administered to a subject at risk for 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.

Nucleoside monomer compound

In one aspect, the present disclosure provides a compound according to formula (1):

wherein: each J ₁ Independently is C1-C6 alkyl or substituted C1-C6 alkyl;

E ₁₀₁ is O or S;

Z ₀ Is H, protected hydroxy, OCH ₃ F or OCH ₂ CH ₂ OCH ₃ ；

Indicates the site at which the group is covalently attached.

Bm is phenyl or substituted phenyl. Without being bound by theory, when Bm is a substituted phenyl group, the choice of substituents in the substituted phenyl group can have an effect on the electron density on the phenyl ring, further enhancing or reducing the reactivity of the covalent bond between the carbon atom at the 1' position of the ribose ring and the adjacent oxygen atom in the ribose ring through a hyperconjugative effect. In some embodiments, each of saidEach substituent is independently an electron donating group. In some embodiments, it will be understood by those skilled in the art that when the substituents are each independently selected from F, cl, CH ₃ 、CH ₂ F or CF ₃ The objects of the present disclosure can be achieved without changing the properties of the compounds provided by the present disclosure. In some embodiments, each substituent in the substituted phenyl group is independently F, cl, or CH for simplicity of the compound structure ₃ . In some embodiments, the Bm group is selected from one of the following structures:

in some embodiments, G is a structure represented by formula (G1).

Without being limited by theory, D ₀ A divalent group capable of realizing the linkage of the five-membered ring ribose molecule in the formula (1) to the phosphoric acid structure, represented by any one of the formulae (D1) to (D4). In some embodiments, D ₀ Has a structure represented by the formula (D1) or (D2), and P ₁ And P ₂ Each independently is H. In some embodiments, D ₀ Has a structure represented by the formula (D4).

In the present disclosure, the modification of J is required depending on the structure and properties of the desired modified nucleoside represented by formula (1) ₁ And E ₁₀₁ A selection is made. . In some embodiments, J ₁ Is ethyl, E ₁₀₁ Is O.

In some embodiments, the protected hydroxyl group refers to a hydroxyl group protected by a TMS (trimethylsilyl), TES (triethylsilyl), TIPS (triisopropylsilyl), TBDPS (t-butyldiphenylsilyl), or TBDMS (t-butyldimethylsilyl) group.

In some embodiments, Z ₀ Is H, protected hydroxy, OCH ₃ F or OCH ₂ CH ₂ OCH ₃ ；。

In some embodiments, each B is for ease of synthesis of the compound ₁ Independently selected from isopropyl or isobutyl, B ₂ Selected from cyanoethyl or cyanopropyl.

In some embodiments, the phosphoramidite functionality of formula (2) has the structure shown below in formula (4):

in some embodiments, the compound of formula (1) has a structure of formula (101), (102), (103), (104), (105), (106), (107), (108), (109), (110), (111), (112), or (113):

the siRNA prepared by the modified nucleoside monomer represented by one of formulae (101) to (113) may have a further good balance of target gene expression inhibitory activity and low toxicity.

Preparation of modified nucleoside monomer Compound

One skilled in the art can prepare the modified nucleoside monomeric compounds of the present disclosure using any reasonable synthetic route.

For example, the compound represented by the formula (1) can be obtained by a method comprising the following steps: contacting a compound shown as a formula (401) with a compound shown as a formula (402) in an organic solvent under the condition of coupling reaction and in the presence of a coupling reagent and a reaction auxiliary agent, and separating to obtain a compound shown as a formula (1):

wherein, J ₁ 、E ₁₀₁ 、D ₀ 、Z ₀ 、Bm、B ₁ 、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 coupling reaction conditions and coupling reagents may employ any conditions and reagents capable of effecting the coupling reaction described above.

Generally, the coupling reaction conditions include a reaction temperature of from 0 ℃ to 50 ℃, and in some embodiments, from 15 ℃ to 35 ℃. The reaction time is from 0.5 to 5 hours, and in some embodiments from 1 to 3 hours. The reaction pressure may be normal pressure.

The molar ratio of coupling reagent to compound of formula (401) is 1 to 1, and in some embodiments 1 to 1. The coupling reagent is one or more selected from tetrazole, 5-ethylthio 1H-tetrazole, and 5-benzylthio 1H-tetrazole, and in some embodiments, tetrazole.

The organic solvent is one or more of epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N-dimethylformamide and N, N-diisopropylethylamine. The epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether in some embodiments, the haloalkane-based solvent is one or more of dichloromethane, trichloromethane and 1, 2-dichloroethane in some embodiments, and the organic solvent is N, N-dimethylformamide in some embodiments. 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 formula (401).

The reaction auxiliary agent is N-methylimidazole. The molar ratio of the reaction promoter to the compound represented by formula (401) is from 0.1 to 10, in some embodiments from 0.5.

The molar ratio of the compound represented by formula (402) to the compound represented by formula (401) is 1 to 20, and in some embodiments is 1 to 10. The compound represented by the formula (402) is a phosphoramidite compound which is commercially available or can be synthetically obtained by a method well known to those skilled in the art. In some embodiments, the compound of formula (402) is a readily commercially available bis (diisopropylamino) (2-cyanoethoxy) phosphine or 3- ((chloro (diisopropylamino) phosphono) oxy) propionitrile.

The compound of formula (1) 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 represented by formula (1) produced by the reaction is isolated using, for example, column chromatography, under conditions such as normal phase silica gel packing, with Petroleum Ether (PE) ethyl acetate (AcOEt) =2 (V: V) or a mixed eluent of petroleum ether: dichloromethane =1 (V: V) as a mobile phase.

The compounds of formula (401) are either commercially available or can be prepared by one skilled in the art by a reasonable synthetic route. In some embodiments, the compound of formula (401) may be prepared by the following method: the method comprises the following steps of contacting a compound shown as a formula (403) with a deprotection reagent in an organic solvent under a deprotection reaction condition, and separating to obtain a compound shown as a formula (401):

wherein, J ₁ 、E ₁₀₁ 、D ₀ 、Z ₀ The definitions and alternative ranges for Bm are as described above.

R _k Is a hydroxyl protecting group. In some embodiments, the protecting group R _k As the silane-based protecting group, TMS (trimethylsilyl), TES (triethylsilyl), TIPS (triisopropylsilyl), TBDPS (t-butyldiphenylsilyl) or TBDMS (t-butyldimethylsilyl) may, for example, be mentioned. In some embodiments, R _k May be TBDPS (tert-butyldiphenylsilyl).

The deprotection reaction conditions include a reaction temperature of 0 to 50 deg.C, and in some embodiments 15 to 35 deg.C. The reaction time is from 0.5 to 5 hours, and in some embodiments from 0.5 to 3 hours. 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. The epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether in some embodiments, the haloalkane-based solvent is one or more of dichloromethane, trichloromethane and 1, 2-dichloroethane in some embodiments, and the organic solvent is tetrahydrofuran in some embodiments. 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 (403).

The deprotection reagent is a fluorine ion-containing ammonium salt solution, and may be, for example, a solution containing pyridinium hydrogen fluoride, triethylamine hydrogen fluoride, tetrabutylammonium fluoride, tetraoctylammonium fluoride, tetramethylammonium fluoride, tetraethylammonium fluoride, or benzyltrimethylammonium fluoride; in some embodiments, the deprotection reagent is tetrabutylammonium fluoride in tetrahydrofuran. The molar ratio of the deprotecting reagent (as fluoride ion) to the compound represented by formula (403) is 1 to 1, and in some embodiments 1 to 5.

The compound of formula (401) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, the compound produced by the reaction as shown in formula (401) is separated by using, for example, column chromatography, and the separation conditions may be, for example, normal phase silica gel packing, and elution with a mixed eluent of Petroleum Ether (PE): ethyl acetate (AcOEt) =5 (V: V).

The compounds of formula (403) are commercially available or may be prepared by one skilled in the art by a reasonable synthetic route. In some embodiments, D is in a compound of formula (403) ₀ Is a divalent linking group represented by the formula (D4), in this case, the compound represented by the formula (403) can be obtained by the following production method: the method comprises the following steps of contacting a compound shown as a formula (404) with a methylenating reagent in an organic solvent under a methylenating reaction condition and in the presence of a catalyst, and separating to obtain a compound shown as a formula (403):

wherein, J ₁ 、E ₁₀₁ 、Z ₀ 、Bm、R _k The respective definitions and alternative ranges are as described above. D in formula (404) ₀ Is a group represented by the formula (D1) or (D2).

The methylenation reaction conditions include a reaction temperature of from 0 ℃ to 50 ℃, and in some embodiments, from 15 ℃ to 35 ℃. The reaction time is from 0.5 to 5 hours, and in some embodiments from 1 to 3 hours. The reaction pressure may be atmospheric 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, the haloalkane-based solvent is one or more of dichloromethane, chloroform and 1, 2-dichloroethane, and the organic solvent is dimethylsulfoxide. 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 (404).

The catalyst is sodium alkoxide or alkali metal hydride. In some embodiments, the catalyst is one of sodium hydride, potassium hydride, or lithium hydride. In some embodiments, the molar ratio of the catalyst to the compound of formula (404) is 10.

The methylating agent is any agent capable of causing the addition of a double bond to a methylene group to form a cyclopropane subunit. In some embodiments, the methyleneating agent is a carbene-based agent. In some embodiments, the methylenating agent is trimethyl sulfoxide iodide. In some embodiments, the molar ratio of the methylating agent to the compound of formula (404) is 1 to 1, in some embodiments 1 to 3.

The compound of formula (403) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, the reaction mixture is subjected to extraction, and after the organic phase is evaporated to remove the solvent, the compound produced by the reaction according to formula (403) is isolated using, for example, column chromatography, under conditions such as normal phase silica gel packing, elution with a gradient of Petroleum Ether (PE): ethyl acetate (AcOEt) = 30.

In some embodiments, D is in a compound of formula (403) ₀ Is a divalent linking group represented by the formula (D1) or (D2), in which case the compound represented by the formula (403) is the same as the compound represented by the formula (404), can be obtained by the following production method: the method comprises the following steps of contacting a compound shown as a formula (405) with a compound shown as a formula (406) in an organic solvent under condensation reaction conditions in the presence of a condensation reaction auxiliary agent, a catalyst and a base, and separating to obtain a compound shown as a formula (403):

wherein, E ₁₀₁ 、J ₁ 、Z ₀ 、Bm、R _k 、P ₁ The respective definitions and alternative ranges are as described above.

The condensation reaction conditions include a reaction temperature of from 0 ℃ to 50 ℃, and in some embodiments, from 15 ℃ to 35 ℃. The reaction time is from 0.5 to 30 hours, and in some embodiments from 2 to 20 hours. The reaction pressure may be atmospheric pressure.

The organic solvent is one or more of epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N-dimethylformamide and N, N-diisopropylethylamine. The epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether in some embodiments, the haloalkane-based solvent is one or more of dichloromethane, trichloromethane and 1, 2-dichloroethane in some embodiments, and the organic solvent is a mixed solvent of dimethyl sulfoxide and tetrahydrofuran in some embodiments. 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 (405).

The alkali is inorganic or organic strong alkali. In some embodiments, the base is an alkali metal hydroxide or an alkali metal alkoxide. In some embodiments, the base is potassium tert-butoxide. The molar ratio of the base to the compound of formula (405) is 1 to 10, in some embodiments 1.

The condensation reaction auxiliary is any agent capable of promoting condensation of the compound represented by the formula (406) and the compound represented by the formula (405). In some embodiments, the condensation reaction aid is Dicyclohexylcarbodiimide (DCC). In some embodiments, the molar ratio of the condensation reaction aid to the compound represented by formula (405) is 1 to 1, in some embodiments 1 to 5.

The catalyst is any agent that can increase the efficiency of the condensation reaction, but does not itself change in amount before and after the reaction. In some embodiments, the catalyst is a pyridinium trifluoroacetate. In some embodiments, the catalyst is added to the reaction mixture in the form of pyridine and trifluoroacetic acid, respectively, and reacts in the reaction solution to form a pyridinium trifluoroacetate, which acts as a catalyst. In some embodiments, the molar ratio of the catalyst to the compound of formula (405) is from 0.2 to 1, and in some embodiments from 0.8.

The compound represented by formula (406) is commercially available, or can be synthesized by a method known to those skilled in the art. On the other hand, the structure of the compound represented by the formula (406) determines E in the compound of the formula (403) or (404) ₁₀₁ 、J ₁ 、P ₁ Selection of (2). In some embodiments, the compound of formula (406) is a commercially available tetraethylmethylene diphosphate, when in compounds of formula (403) or (404), D ₀ Has a structure of formula (D1), and each E ₁₀₁ Are both O, or at least one E ₁₀₁ Is S, each J ₁ Are all ethyl radicals, and P ₁ Is hydrogen. The molar ratio of the compound represented by formula (406) to the compound represented by formula (405) is 1 to 10, and in some embodiments is 1. In some embodiments, the compound of formula (406) is added to the reaction mixture in the form of a base solution. In some embodiments, the compound of formula (406) is added to the reaction mixture comprising the compound of formula (405) as a solution with the base and organic solvent described above. In some embodiments, the organic solvent is tetrahydrofuran. In some embodiments, the organic solvent is used in an amount of 0.5 to 20L/mol, and in some embodiments, 1 to 10L/mol, relative to the compound of formula (406).

The compound of formula (403) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, after extraction and liquid separation, the organic phase is separated from the compound produced by the reaction according to formula (403) using, for example, column chromatography, and the separation conditions can be, for example, using normal phase silica gel packing, eluting with a gradient of Petroleum Ether (PE): ethyl acetate (AcOEt) = 30.

In some embodiments, D is in a compound of formula (403) ₀ Is a divalent linking group of formula (D3), in which case the compound of formula (403) can be prepared by following the procedure shown in example 25 in CN103154014B, with the only difference beingIn place of compound 15 in example 25 in CN103154014B, the preparation was carried out using a compound represented by formula (405).

The compound represented by formula (405) can be obtained commercially or can be synthesized by a method known to those skilled in the art. The compound represented by the formula (405) can be obtained by the following production method: the method comprises the following steps of contacting a compound shown as a formula (407) with acid in an organic solvent under deprotection conditions, and separating to obtain a compound shown as a formula (405):

wherein, Z ₀ 、Bm、R _k The respective definitions and alternative ranges are as described before.

R _j Is a hydroxyl protecting group and is selected from one of trityl, 4-methoxyl trityl, 4 '-double methoxyl trityl and 4,4' -trimethoxy benzyl; in some embodiments, R _j Is 4,4' -bis (methoxy) trityl.

The deprotection reaction conditions include a reaction temperature of 0-100 deg.C for 0.1-24 hours, in some embodiments 10-40 deg.C for 0.5-16 hours. The reaction pressure may be atmospheric pressure.

The organic solvent is one or more of epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N-dimethylformamide and N, N-diisopropylethylamine. The epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether in some embodiments, the haloalkane-based solvent is one or more of dichloromethane, trichloromethane and 1, 2-dichloroethane in some embodiments, and the organic solvent is dichloromethane in some embodiments. The organic solvent is used in an amount of 3 to 50L/mol, and in some embodiments 5 to 20L/mol, relative to the compound represented by formula (407).

The acid may be an acid commonly used in deprotection of a hydroxyl protecting group. In some embodiments, the acid is, for example, p-toluenesulfonic acid. In some embodiments, the acid is a solution obtained after TsOH (p-toluenesulfonic acid) is dissolved in dichloromethane and anhydrous ethanol, wherein the molar ratios of dichloromethane and anhydrous ethanol to p-toluenesulfonic acid are 1 to 1ml/g and 1 to 1. The molar ratio of the acid to the compound of formula (407) is 0.1 to 10, in some embodiments 0.3.

The compound of formula (405) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, after extraction, liquid separation, the organic phase is evaporated to remove the solvent and the compound produced by the reaction as shown in formula (405) is isolated using, for example, column chromatography, under conditions such as normal phase silica gel packing, eluting with a gradient of petroleum ether: ethyl acetate = 50. In some embodiments, the resulting crude compound of formula (405) is used in subsequent reactions without further isolation.

The compound represented by formula (407) 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 (407) may be obtained by the following preparation method: the method comprises the following steps of contacting a compound shown as a formula (408) with a silane hydroxyl protecting reagent in an organic solvent under the condition of hydroxyl protecting reaction and under the alkaline condition, and separating to obtain a compound shown as a formula (407):

wherein Z is ₀ 、Bm、R _j The respective definitions and alternative ranges are as described above.

The hydroxyl protection reaction conditions include a reaction temperature of 0-100 ℃ for 0.1-24 hours, in some embodiments 10-40 ℃ for 0.5-16 hours. 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. The epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether in some embodiments, the haloalkane-based solvent is one or more of dichloromethane, trichloromethane and 1, 2-dichloroethane in some embodiments, and the organic solvent is N, N-dimethylformamide in some embodiments. 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 of formula (408).

The alkaline condition means that an alkaline compound is contained in the reaction process. The molar ratio of the basic compound to the compound of formula (408) is 1 to 10, in some embodiments 1. In some embodiments, the basic compound is imidazole.

The silane hydroxyl protecting agent is TBDMSCl (tert-butyldimethylchlorosilane) or TBDPSCl (tert-butyldiphenylchlorosilane). In some embodiments, the silane-based hydroxyl protecting agent is TBDPSCl (t-butyldiphenylchlorosilane). The molar ratio of the silane-based hydroxyl protecting agent to the compound of formula (408) is 1 to 1, and in some embodiments is 2.

The compound of formula (407) may be isolated from the reaction mixture using any suitable separation method. In some embodiments, the reaction mixture is extracted, separated, and the organic phase is evaporated to remove the solvent to isolate the compound of formula (407) produced by the reaction. In some embodiments, the resulting crude compound of formula (407) is used in subsequent reactions without further isolation.

The compound represented by formula (408) can be obtained commercially, or can be synthesized by a method known to those skilled in the art. In some embodiments, the compound represented by formula (408) may be obtained by the following preparation method: the method comprises the following steps of contacting a compound shown as a formula (409) with a trityl hydroxyl protecting reagent in the presence of an organic solvent under hydroxyl protecting reaction conditions, and separating to obtain a compound shown as a formula (408):

wherein Z is ₀ The definitions and alternative ranges for Bm are as described above.

The organic solvent is one or more of epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N-dimethylformamide, N-diisopropylethylamine or anhydrous pyridine. 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 anhydrous pyridine. The organic solvent is used in an amount of 0.5 to 50L/mol, and in some embodiments 1 to 20L/mol, relative to the compound of formula (409).

The trityl hydroxyl protecting reagent contains R _j A hydroxyl protecting agent for the radical. In some embodiments, R _j One selected from trityl, 4-methoxytrityl, 4 '-bismethoxytrityl, and 4,4' -trimethoxytrityl. In some embodiments, the trityl-based hydroxyl protecting reagent is DMTrCl, i.e., 4' -bismethoxytrityl chloride. The molar ratio of the trityl-based hydroxyl protecting reagent to the compound of formula (409) is 1 to 1, and in some embodiments 1 to 1.

Any suitable separation method may be used to isolate the compound of formula (408) from the reaction mixture. In some embodiments, the reaction mixture is extracted, separated, and the organic phase is evaporated to remove the solvent to isolate the compound produced by the reaction, as shown in formula (408). In some embodiments, the resulting crude compound of formula (408) is used in subsequent reactions without further isolation. .

The compound represented by formula (409) may be commercially available, or may be synthetically obtained by a method known to those skilled in the art. In some embodiments, the compound of formula (409) may be prepared by the following method: the method comprises the steps of contacting a compound shown as a formula (410) with a deprotection reagent in the presence of an organic solvent under a hydrolysis reaction condition, and separating to obtain a compound shown as a formula (409).

Wherein Z is ₀ The definitions and alternative ranges for Bm are as described above. Each R ₄₁₀ Independently is a C1-C5 alkyl group. In some embodiments, each R is independently selected from R, and R ₄₀₉ Independently isopropyl or tert-butyl.

The hydrolysis reaction conditions include a reaction temperature of 0-100 ℃ and a reaction time of 0.1-24 hours, in some embodiments 10-40 ℃ and a reaction time of 0.5-16 hours. The reaction pressure may be atmospheric 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, the haloalkane-based solvent is one or more of dichloromethane, chloroform and 1, 2-dichloroethane, and the organic solvent is tetrahydrofuran. The amount of organic solvent used is 1 to 50L/mol, and in some embodiments 2 to 30L/mol, relative to the compound of formula (410).

The deprotecting agent is a fluoride ion-containing ammonium salt, and may be, for example, a pyridinium fluoride-containing salt, a triethylamine hydrogen fluoride salt, tetrabutylammonium fluoride, tetraoctylammonium fluoride, tetramethylammonium fluoride, tetraethylammonium fluoride, or benzyltrimethylammonium fluoride. In some embodiments, the deprotecting reagent is tetrabutylammonium fluoride. In some embodiments, the deprotecting reagent is added to the reaction mixture comprising the compound of formula (410) in an organic solvent to form a solution. The molar ratio of the deprotecting reagent to the compound represented by formula (410) is 1 to 10, in some embodiments 1 to 3.

The compound of formula (409) may be isolated from the reaction mixture using any suitable separation method. In some embodiments, the solvent is removed by evaporation, and the compound produced by the reaction as shown in formula (409) is separated using, for example, column chromatography, under conditions such as normal phase silica gel packing and elution with a mixed eluent of Petroleum Ether (PE) = ethyl acetate (AcOEt) =5 (V: V) as a mobile phase.

The compound represented by formula (410) can be obtained commercially, or can be synthesized by a method known to those skilled in the art. In some embodiments, Z in formula (410) ₀ Is OCH ₃ Or OCH ₂ CH ₂ OCH ₃ The compound represented by the formula (410) can be obtained by the following production method: the method comprises the steps of contacting a compound shown as a formula (411) with a reaction reagent under a substitution reaction condition in the presence of an organic solvent, and separating to obtain the compound shown as the formula (410).

Wherein, bm and R ₄₁₀ The definitions and alternative ranges of (a) are as previously described.

The substitution reaction conditions include a reaction temperature of-10 ℃ to 100 ℃ and a reaction time of 0.1 to 24 hours, in some embodiments a reaction temperature of-10 ℃ to 40 ℃ and a reaction time of 0.5 to 16 hours. The reaction pressure may be atmospheric 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, the haloalkane-based solvent is one or more of dichloromethane, chloroform and 1, 2-dichloroethane, and the organic solvent is N, N-dimethylformamide. The amount of organic solvent used is from 3 to 50L/mol, in some embodiments from 5 to 20L/mol, relative to the compound of formula (411).

The alkaline condition means that an alkaline compound is contained in the reaction process. The molar ratio of the basic compound to the compound of formula (411) is 1 to 10, in some embodiments 1. In some embodiments, the basic compound is a strong base. In some embodiments, the basic compound is NaH.

Z in formula (410) ₀ The difference in the structure of (a) determines the difference in the selected reagents. Z in formula (410) ₀ Is OCH ₃ When the reaction reagent is CH ₃ I、CH ₃ Cl、CH ₃ Br or CH ₃ F; z in formula (410) ₀ Is OCH ₂ CH ₂ OCH ₃ When the reaction reagent is CH ₃ OCH ₂ CH ₂ I、CH ₃ OCH ₂ CH ₂ F、CH ₃ OCH ₂ CH ₂ Br or CH ₃ OCH ₂ CH ₂ And (4) Cl. The molar ratio of the reactant to the compound represented by formula (411) is 1 to 20, in some embodiments 1 to 10.

The compound of formula (410) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, after extraction and liquid separation, the solvent is removed by concentration, and the compound produced by the reaction as shown in formula (410) is separated by column chromatography, for example, under the conditions of normal phase silica gel packing and elution with a mixed eluent of Petroleum Ether (PE): ethyl acetate (AcOEt) =20 (V: V).

The compound represented by formula (411) 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 (411) may be prepared by the following method: the method comprises the steps of contacting a compound shown as a formula (412) with a tetraalkyl disiloxane type hydroxyl protective agent under the condition of substitution reaction in the presence of an organic solvent, and separating to obtain a compound shown as a formula (411).

Where Bm is defined and optionally ranges are as previously described.

The substitution reaction conditions include a reaction temperature of 0-100 deg.C for 0.1-24 hours, and in some embodiments, a reaction temperature of 0-40 deg.C for 0.5-16 hours. The reaction pressure may be atmospheric pressure.

The organic solvent is one of anhydrous pyridine, 4-dimethylamino pyridine, 2-chloropyridine, 2, 6-dichloropyridine, 2-aminopyridine, anhydrous N, N-dimethyl amide and anhydrous N, N-dimethyl acetamide. In some embodiments, the organic solvent is anhydrous pyridine. The organic solvent is used in an amount of 1 to 50L/mol, and in some embodiments 5 to 20L/mol, relative to the compound of formula (412).

The tetraalkyldisiloxane-based protective agent may be, for example, TIPDSCl ₂ (1, 3-dichloro-1, 3-tetraisopropyldisiloxane). In some embodiments, TIPDSCl ₂ And a compound of formula (412) at a molar ratio of 1 to 1, in some embodiments 1 to 1.

The compound of formula (411) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, the solvent may be removed by evaporation and the compound produced by the reaction, as shown in formula (411), may be isolated using, for example, column chromatography, under conditions such as normal phase silica gel packing, elution with a gradient of Petroleum Ether (PE) = ethyl acetate (AcOEt) =5.

The compound represented by formula (412) 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 (412) may be prepared according to the methods disclosed in the document ACS chem.biol.2006,1,3, 176-183.

siRNA of the present disclosure

In another aspect, the present disclosure also discloses an siRNA comprising a sense strand and an antisense strand, each of the sense strand and the antisense strand comprising 14 to 30 modified or unmodified nucleotides, 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 has a structure represented by formula (5):

wherein, the first and the second end of the pipe are connected with each other,

each E ₁₀₁ Each independently is O or S;

Z is H, OH, OCH ₃ F or OCH ₂ CH ₂ OCH ₃ ；

Represents the site of covalent attachment of a group;

As previously described, in the sirnas 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 replaced with another group, or a nucleotide in which the base on the nucleotide is a modified base. Such modified nucleotides do not result in significant impairment or loss of function of the siRNA to regulate gene expression. For example, one can select modified nucleotides disclosed in J.K.Watts, G.F.Deleavey, and M.J.Damha, chemical modified siRNA: tools and applications, drug discovery Today,2008, 13 (19-20): 842-55.

In some embodiments, the siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises nucleotide sequence I and the antisense strand comprises nucleotide sequence II, and the 5' terminal nucleotide of the nucleotide sequence II has the structure represented by formula (5); the nucleotide sequence I and the nucleotide sequence II both consist of 19 nucleotides, the nucleotide sequence II and the nucleotide sequence I form a double-stranded region, the nucleotide sequence II is at least partially reversely complementary with 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; counting the group shown in the formula (5) as 1 nucleotide, wherein the 2 nd, 6 th, 14 th and 16 th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides according to the direction from the 5 'end to the 3' end.

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 2 nd, 6 th, 14 th and 16 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, the nucleotide sequence II is substantially reverse complementary or substantially reverse complementary to the first stretch of nucleotide sequence in the 5 'end to 3' end direction.

In some embodiments, the nucleotides at positions 2-19 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 nucleotide sequence II is substantially reverse complementary or substantially reverse complementary to the nucleotide sequence I.

In some embodiments, positions 2-19 of the nucleotide sequence II are fully reverse complementary to positions 1-18 of the nucleotide sequence I in the 5 'end to 3' end direction to form a double-stranded region.

In some embodiments, the nucleotide sequence II is substantially reverse complementary to the nucleotide sequence I in the 5 'end to 3' end direction, or a base mismatch exists between the 2 nd nucleotide in the nucleotide sequence II and the 2 nd nucleotide in 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 in mRNA expressed by the target gene and has the same length as the nucleotide sequence IV. Thus, the siRNA of the present disclosure can have a double-stranded complementary region of 19-23 nucleotides in length.

In some embodiments, the siRNA further comprises a nucleotide sequence V, each nucleotide of the nucleotide sequence V being independently one of a non-fluorinated modified nucleotide, the nucleotide sequence V being 1 to 3 nucleotides in length, linked at the 3 'end of the antisense strand, thereby constituting a 3' overhang of the antisense strand. Thus, the ratio of the lengths of the sense strand and the antisense strand of the siRNA provided by the present disclosure may be 19/19, 19/20, 19/21, 19/22, 20/20, 20/21, 20/22, 20/23, 21/21, 21/22, 21/23, 21/24, 22/22, 22/23, 22/24, 22/25, 23/23, 23/24, 23/25 or 23/26.

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 strand and the antisense strand of the siRNA of the present disclosure each have a length of 19/21 nucleotides or 21/23 nucleotides, and at this time, the siRNA of the present disclosure has better target gene expression modulation 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 (507). "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 (508). In some embodiments, 2 '-amino modified nucleotides (2' -NH) ₂ ) As shown in equation (509). In some embodiments, the 2' -Deoxynucleotide (DNA) is according to formula (510):

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. BNA can contain five-membered, six-membered, or seven-membered rings with "fixed" C3' -endo-sugar tapered bridging structures. 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 (512), ENA is as shown in equation (513), and cET BNA is as shown in equation (514):

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 according to formula (515) and GNA is according to formula (516):

in the above formulae (515) and (516), R is selected from H, OH or alkoxy (O-alkyl).

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 (517) or (518).

In the compounds of the above-mentioned formula (517) to formula (518), 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 the following, the terms "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" are the same, and all refer to a compound having a structure represented by formula (507) 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 (508) in which 2' -hydroxyl group of ribose group of nucleotide is substituted with methoxy group.

The modified siRNA is low in cost, and ribonuclease in blood is not easy to cut, so that the stability of the siRNA is improved, and the siRNA has stronger nuclease hydrolysis resistance. Meanwhile, the modified siRNA has higher activity of regulating the expression of target genes.

In some embodiments, at least 1 of the phosphate groups in the phosphate-sugar backbone of at least one single strand 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 in which at least one oxygen atom in a phosphodiester bond in the phosphate group is replaced 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 (521):

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

In some embodiments, in the siRNA, the phosphate group having the modifying group is present at least one position in 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;

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

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

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

between the 2 nd and 3 rd nucleotides at 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 sirnas of the present disclosure can be various sirnas that mediate gene expression. The use of the disclosed sirnas unexpectedly reduced toxicity, yet showed excellent target gene expression modulating activity.

The siRNA, pharmaceutical composition and siRNA conjugate provided by the present disclosure may be used to regulate various gene abnormal expression, and treat various pathological conditions or diseases caused by the gene abnormal expression. These genes may be various endogenous genes in the human or animal body, or genes of pathogens which propagate in the human or animal body. siRNA having a specific nucleotide sequence and the modification scheme can be designed and prepared based on mRNA expressed from a 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, HSD, p53, PCSK9, PNP, PLG, PKK, KNG, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3, TIMP-1, TMPRSS6, XO.

In some embodiments, the mRNA expressed by the target gene is selected from mRNA expressed by a hepatitis b virus gene.

In some embodiments, the compound of formula (5) has a structure of formula (201), (202), (203), (204), (205), (206), (207), (208), (209), (210), (211), (212), or (213):

in some embodiments, the siRNA is an siRNA having the structure shown in table 1 below.

TABLE 1 siRNA sequences

Among them, those in siRNA1-13Bxs represents a structure represented by formula (201) to formula (213). Wherein capital letters C, G, U and 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 a phosphorothioate linkage between the two nucleotides to the left of the letter.

Methods of making siRNA of the present disclosure

The preparation method of the siRNA nucleotide disclosed by the invention comprises the steps of respectively synthesizing a sense strand and an antisense strand of the siRNA according to an expected siRNA sequence by using a solid phase phosphoramidite method, connecting the last nucleotide at the 5' end of the antisense strand by using a structure shown in a formula (1) as a nucleoside phosphoramidite monomer, and carrying out a sulfuration reaction.

In some embodiments, the methods of making sirnas of the present disclosure comprise:

(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 from 2 to 1, and in some embodiments from 3 to 1.

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

The capping reaction conditions include a temperature of 0 to 50 deg.C, in some embodiments 15 to 35 deg.C, and a reaction time of 5 to 500 seconds, in some embodiments 10 to 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, iodine (provided in the form of iodine water in further embodiments). The molar ratio of oxidizing reagent to nucleic acid sequence attached to the solid support in the coupling step can be 1. 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 can be from 10. In some embodiments, the sulfurization reaction is carried out in a mixed solvent of acetonitrile pyridine = 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 checked at any time during the synthesis process to better control the quality of the synthesis, methods for 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 can be 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, etc., using methods such as liquid chromatography-mass spectrometry, etc., to determine sirnas designed for interest, e.g., corresponding to one of the sequences listed in table 1 above.

Pharmaceutical composition

In one aspect, the present disclosure provides a pharmaceutical composition comprising an siRNA provided by the present disclosure, and a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carrier can be a carrier conventionally used in the art of siRNA 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), chitosan (chitosan), 1,2-dioleoyl-3-trimethylammonium propane (1, 2-dioleoyl-3-trimethylammoniumpropane, DOTAP), poly (D-or L-lactic acid/glycolic acid copolymer)&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 siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly required, and in some embodiments, the weight ratio of siRNA to 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 between 200 and 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 may be selected from one or more of the amine-containing transfection compounds described in chinese patent application CN103380113A (incorporated herein by reference in its entirety), or pharmaceutically acceptable salts or derivatives thereof, helper lipid and pegylated lipid, respectively.

In some embodiments, the organic amine may 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 x 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) may 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 pharmaceutical composition particles formed from the sirnas of the present disclosure and the above-described amine-containing transfection reagents 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 siRNA to total lipid (e.g., organic amine, helper lipid, and/or pegylated lipid) is within the range of from 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 siRNA provided by the present disclosure and the above pharmaceutically acceptable carrier can be prepared according to various known methods except that the siRNA provided by the present disclosure is used to replace the existing siRNA; in some embodiments, the preparation may be as follows:

suspending organic amine, auxiliary lipid and pegylated 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 around 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 siRNA provided by the present disclosure is dissolved in a buffered salt solution to obtain an siRNA aqueous solution. The concentration of the buffered salt solution is 0.05-0.5M, such as 0.1-0.2M, the pH of the buffered salt solution is adjusted to 4.0-5.5, such as 5.0-5.2, and the amount of buffered salt solution is such that the concentration of siRNA does not exceed 0.6mg/mL, such as 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 siRNA solution are mixed, and the resulting mixture is incubated at 40-60 ℃ for at least 2 minutes, which may be, for example, 5-30 minutes, to obtain a post-incubation liposome preparation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1 (2-5), and may be, for example, 1.

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 can be pH 7.2-7.6, encapsulation efficiency not less than 90%, particle diameter 60-100nm, polydispersity index not higher 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.

siRNA conjugates

The present disclosure provides a siRNA conjugate containing the siRNA provided by the present disclosure, and a conjugate group conjugated to the siRNA. In some embodiments, the conjugate group comprises a linker and a pharmaceutically acceptable targeting group and/or a delivery assisting group, and the siRNA, the linker and the targeting group or the delivery assisting group are in turn covalently or non-covalently linked, each of the targeting groups is selected from a ligand capable of binding to a cell surface receptor, and each of the delivery assisting groups is selected from a group capable of increasing the biocompatibility of the siRNA conjugate in the delivery 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, "siRNA conjugate" means a compound formed by covalently attaching one or more chemical moieties having a specific function to siRNA. The siRNA conjugate is understood as a generic term of a plurality of siRNA conjugates or an siRNA conjugate represented by a certain chemical formula according to the context. In the context of the present disclosure, a "conjugate molecule" should be understood as a specific compound that can be conjugated to an siRNA by a reaction, ultimately forming an siRNA conjugate of the present disclosure.

Generally, the conjugate group comprises at least one targeting group that is pharmaceutically acceptable and optionally a linker (linker), and the siRNA, 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 siRNA molecule may be non-covalently or covalently conjugated to the conjugate group, e.g. may be covalently conjugated to the conjugate group. The conjugation site of the siRNA to the conjugate group may be at the 3' end or 5' end of the sense strand of the siRNA, or at the 5' end of the antisense strand, or within the internal sequence of the siRNA. In some embodiments, the site of conjugation of the siRNA to the conjugate group is at the 3' end of the sense strand of the siRNA.

In some embodiments, the conjugate group may be attached to the phosphate group, the hydroxyl group at the 2' -position, or the base of a 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 at the end of the siRNA strand, the conjugate group is typically attached to the phosphate group of the nucleotide; when a conjugate group is attached to the internal sequence of the siRNA, 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 vivo in contexts ACS Chemical biology 2015,10 (5): 1181-7.

In some embodiments, the siRNA may be attached to the conjugate group via acid labile, or reducible, chemical bonds that may degrade under the acidic environment of the cellular endosome, thereby leaving the siRNA in a free state. For non-degradable conjugation, the conjugation group can be attached to the sense strand of the siRNA, thereby minimizing the effect of conjugation on siRNA activity.

The targeting group can be attached to the siRNA molecule via a suitable linker, which one skilled in the art can select depending on the particular type of targeting group. The identity of these linkers, targeting groups and the manner of attachment to the siRNA can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.

In some embodiments, the targeting group may be a ligand conventionally used in the art of siRNA 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 hepatocytes. In some embodiments, each of the targeting groups is independently an asialoglycoprotein or a saccharide. <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- ,4,6- -4- -2,3- -O- -D- ,2- -2- -D- , </xnotran> One of 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-allositrile, ribose, D-4-thioribose, L-ribose and 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 siRNA 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 siRNA 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 via an ether linkage, 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 atom of the amino group in the moiety forms an amide bond to link and forms a phosphate bond or a phosphorothioate bond with the oxygen atom through the oxygen atom by the oxygen atom in formula (703).

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

wherein Nu represents an siRNA provided by the present disclosure, or an siRNA obtained according to the method of the present disclosure.

In some embodiments, the linker in the siRNA 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 linked by at least one of the oxygen atoms designated by #, forming a phosphoester bond or phosphorothioate bond with the siRNA, and the remaining oxygen atoms designated by #, forming a hydroxyl group, or being linked with a hydrogen atom to form a hydroxyl group ₁ -C ₃ Alkyl groups being linked to form C ₁ -C ₃ An alkoxy group;

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

In some embodiments, the siRNA conjugates of the present disclosure have the structure shown in formula (1) in chinese patent application CN110959011A, while this patent also discloses in detail the method of preparing the siRNA conjugates shown in formula (1), each of which is incorporated herein by reference in its entirety.

In some embodiments, the siRNA 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 siRNA 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 radical, 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), -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, an amine or an alkenyl group resulting from the above substitutions and/or replacements. 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 of 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 When the ligand has affinity for asialoglycoprotein receptors on the surface of mammalian liver cells, in some embodiments, n1 can be an integer from 1 to 3 and n3 can be an integer from 0 to 4, such 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 hepatic 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 present ₁ Spatial position between ligands is adapted to M ₁ Binding of a ligand to the liver surface asialoglycoprotein receptor, in 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 and C ₁ -C ₁₀ Alkyl radical, C ₁ -C ₁₀ Haloalkyl, and C ₁ -C ₁₀ One of the alkoxy groups does not alter the properties of the conjugates disclosed hereinAll can achieve the purpose 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.

siRNA 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 that an R group is attached ₁₀ 、R ₁₁ 、R ₁₂ 、R ₁₃ 、R ₁₄ And R ₁₅ A chain structure in which carbon atoms 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 siRNA conjugates of the present disclosure are prepared by a process of solid phase synthesis, R is ₂ The group is required to contain both a linking site to N on the nitrogen-containing skeleton and a linking site to 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':

indicating the site of covalent attachment of the group.

q ₂ Can be 1An integer of-10, in some embodiments, q ₂ Is an integer of 1 to 5.

L ₁ Has the function of mixing M ₁ Ligands are linked to the N on the nitrogen-containing backbone to provide targeting functions for the siRNA conjugates of the 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 embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3 to 5. R 'is a C1-C4 alkyl group, and in some embodiments, R' is one of methyl, ethyl, and isopropyl. Ra is one of a27, a28, a29, a30, and a31, and in some embodiments Ra is a27 or a28.Rb is 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 substituted 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 siRNA conjugates of the present disclosure have a structure represented by formula (903), (904), (905), (906), (907), (908), (909), (910), (911), (912), (913), (914), (915), (916), (917), (918), (919), (920), (921), or (922):

in some embodiments, P in formula a59 can be attached to any possible position in the siRNA sequence, e.g., P in formula a59 can be attached to any one nucleotide of the sense or antisense strand of the siRNA; in some embodiments, P in formula a59 is attached to any one nucleotide of the sense strand of the siRNA. In some embodiments, P in formula a59 is attached to the end of the sense or antisense strand of the siRNA; in some embodiments, P in formula a59 is attached to the end of the sense strand of the siRNA. 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 attached to the end of the sense or antisense strand of the siRNA; in some embodiments, P in formula a59 is attached to the 3' end of the sense strand of the siRNA. In the case of attachment to the above-described position of the sense strand of the siRNA, upon entry of the conjugate provided by the present disclosure into a cell, upon unwinding, the individual siRNA antisense strand may be released to modulate target gene expression.

P in formula A59 can be attached to any possible position on the nucleotide in the siRNA, 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 linked to the 2', 3', or 5' position of a nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments, P in formula a59 is attached to the oxygen atom formed after dehydrogenation of the 3' hydroxyl group of the 3' terminal nucleotide of the siRNA sense strand, 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 siRNA sense strand, 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 siRNA sense strand.

In some embodiments, the siRNA contained in the siRNA conjugates of the present disclosure may be, for example, an siRNA listed in table 1. siRNA conjugates comprising these sirnas exhibit low off-target effects and high mRNA inhibitory activity of target gene expression.

Preparation of siRNA conjugates of the disclosure

The above siRNA conjugates can be synthesized by methods that have been described in detail in the prior art. The siRNA conjugates of the present disclosure can be obtained by methods well known to those skilled in the art. As a method for preparing a structure represented by formula (705) is described in WO2014025805A1, rajeev et al in chem biochem 2015,16,903-908 describe a method for preparing a structure represented by formula (707). Chinese patent application CN110959011A also discloses in detail a method for preparing an oligonucleotide conjugate of formula (708). The contents of the above documents are incorporated herein in their entirety by this reference.

The siRNA conjugates of the present disclosure may also be combined with other pharmaceutically acceptable excipients, which may be one or more of a variety of formulations or compounds conventionally employed in the art, for details see the description above for the pharmaceutical compositions of the present disclosure.

siRNA and drug combination of the present disclosureSubstance and use of siRNA conjugate

In some embodiments, the present disclosure provides use of an siRNA, pharmaceutical composition and/or siRNA conjugate provided by the present disclosure in a medicament for treating and/or preventing a disease or condition associated with mRNA levels 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, SOD1, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT. In some embodiments, the mRNA expressed by the target gene is selected from the mRNA expressed by a hepatitis b virus gene (HBV), an angiopoietin-like protein 3 (ANGPTL 3) gene, or 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 an siRNA, pharmaceutical composition and/or siRNA 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, HCV, HSD17B13, p53, PCSK9, PNP, PLG, PKK, KNG, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT. In some embodiments, the mRNA expressed by the target gene is selected from the mRNA expressed by a hepatitis b virus gene (HBV), an angiopoietin-like protein 3 (ANGPTL 3) gene, or 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 siRNAs having a high degree of homology to a gene sequence involved in the disease.

In some embodiments, the present disclosure provides a method of inhibiting the expression level of a target gene in a cell, the method comprising contacting the cell with an effective amount of an siRNA, a pharmaceutical composition, and/or an siRNA 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, HCV, HSD17B13, p53, PCSK9, PNP, PLG, PKK, KNG, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT. In some embodiments, the mRNA expressed by the target gene is selected from the mRNA expressed by a hepatitis b virus gene (HBV), an angiopoietin-like protein 3 (ANGPTL 3) gene, or an apolipoprotein C3 (ApoC 3) gene.

By administering the sirnas, pharmaceutical compositions, and/or siRNA conjugates provided by the present disclosure to a subject in need thereof, the prevention and/or treatment of pathological conditions or diseases caused by the expression of specific genes in cells can be achieved through a mechanism that regulates gene expression. Accordingly, the sirnas provided by the present disclosure, sirnas obtained according to the methods of the present disclosure, pharmaceutical compositions and/or siRNA conjugates may be used for the prevention and/or treatment of said pathological conditions or diseases, or for the preparation of a medicament for the prevention and/or treatment of the pathological conditions or diseases described herein.

The term "administering" as used herein refers to placing an siRNA, pharmaceutical composition and/or siRNA conjugate into a subject by a method or route that results in at least partially positioning the siRNA, pharmaceutical composition and/or siRNA conjugate at a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include topical and systemic administration. In general, topical administration results in delivery of more siRNA, pharmaceutical composition, and/or siRNA conjugate to a particular site as compared to the subject's entire body; whereas systemic administration results in delivery of the siRNA, pharmaceutical composition and/or siRNA 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 siRNA, pharmaceutical composition and/or siRNA 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 a 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 siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure, for example, for a male or female, 6-12 weeks old, 18-25g body weight C57BL/6J or C3H/HeNCrlVr mouse, based on the amount of siRNA in the siRNA, pharmaceutical composition and/or siRNA conjugate: for siRNA conjugates formed by siRNA and a pharmaceutically acceptable conjugate molecule, the amount of siRNA 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 still further embodiments from 0.1 to 15mg/kg body weight, and in still further embodiments from 0.1 to 10mg/kg body weight. Such amounts may be preferred when administering the sirnas, pharmaceutical compositions, and/or siRNA conjugates described in the present disclosure.

In addition, by introducing the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure into cells in which a specific gene is abnormally expressed, the purpose of suppressing the expression of the specific gene in the cells can also be achieved by a mechanism of gene expression regulation. In some embodiments, the cell is a hepatocyte. In some embodiments, the hepatocyte may be a cell or isolated primary hepatocyte, in some embodiments a primary hepatocyte, selected from Hep3B, hepG2, huh7 and like hepatoma cell lines.

The amount of siRNA provided in the siRNA, pharmaceutical composition and/or siRNA conjugate can be readily determined by one skilled in the art based on the desired effect to be obtained by inhibiting the expression of a particular gene in a cell using the methods provided by the present disclosure. For example, in some embodiments, the siRNA, pharmaceutical composition and/or siRNA 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 an siRNA, a pharmaceutical composition and/or an siRNA conjugate provided by the present disclosure.

In some embodiments, the kits described herein can provide the siRNA, pharmaceutical composition, and/or conjugate 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 siRNA, pharmaceutical composition, and/or conjugate described herein. In some embodiments, the kit may comprise instructions for mixing the siRNA, pharmaceutical composition, and/or conjugate with a pharmaceutically acceptable carrier and/or adjuvant or other ingredients (if any).

In the kits of the present disclosure, the siRNA 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 siRNA 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.

Examples

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

EXAMPLE 1 preparation of Compound Z10

In this preparation example, the compound of example 1 was synthesized in the following manner.

(1-1) Synthesis of Z2

87.27g of TIPDSCl were added at 0 deg.C ₂ (1, 3-dichloro-1, 3-tetraisopropyl disiloxane, 276.67mmol, beijing coupling technology Co., ltd.) and 72g of compound Z1 (276.67 mmol) were added to 857.14mL of anhydrous pyridine (10.62 mol) solution, and the mixture was raised to 25 ℃ to react for 12 hours. 2000mL of ethyl acetate was added to the reaction solution, and the mixture was washed with 500mL of saturated brine 1 time, and the organic phase was washed with Na ₂ SO ₄ The residue obtained was purified by column chromatography (silica gel, V (petroleum ether): V (ethyl acetate) = 5) gradient elution, the eluate containing the reaction product was collected, and the solvent was evaporated to obtain 34g of compound Z2. ¹ H NMR:(400MHz,CDCl ₃ ) δ 7.25 (t, J =8.4hz, 1h), 6.68 (t, J =9.6hz, 1h), 4.98 (d, J =2.0hz, 1h), 4.27-4.26 (M, 1H), 4.06-4.00 (M, 1H), 3.97-3.94 (M, 3H), 2.80 (d, J =2.4hz, 1h), 2.14 (s, 3H), 1.03-0.89 (M, 3H) M/z (ES +): theory, [ M + H ] (M, 3H) ]] ⁺ 502.75, detection, [ M + H] ⁺ ,502.20。

(1-2) Synthesis of Z3

4.95g of sodium hydride (123.88 mmol) and 12.8g of sodium hydride are added at-10 deg.C5mL of iodomethane (12.85 mL) and 51.9g of Compound Z2 (103.23 mmol) obtained by the method described in step (1-1) were added to 520mL of anhydrous DMF and reacted for 3h. The reaction mixture was added to 2000mL of saturated ammonium chloride solution at 0 deg.C, extracted 1 time with 1000mL of ethyl acetate, the organic phases were combined, washed 3 times with 500mL each time of saturated brine, and then with anhydrous Na ₂ SO ₄ Drying, filtration and concentration under reduced pressure, the obtained residue was purified by column chromatography (silica gel, V (petroleum ether): V (ethyl acetate) = 20), the eluate containing the reaction product was collected, and the solvent was evaporated to obtain 36g of compound Z3.M/z (ES +), theory, [ M + H ]] ⁺ 517.77, detection, [ M + H] ⁺ ,517.89。

(1-3) Synthesis of Z4

27.58mL of a 1M solution of tetrabutylammonium fluoride (TBAF) in tetrahydrofuran and 5.70g of compound Z3 were added to 70mL of the tetrahydrofuran solution at room temperature and reacted for 12h. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in 1000mL of ethyl acetate, and the resulting organic phase was washed with 500mL of saturated saline 3 times and then with anhydrous Na ₂ SO ₄ After drying, filtration and concentration under reduced pressure, the obtained residue was purified by column chromatography (silica gel, V (petroleum ether): V (ethyl acetate) = 1), and the eluate containing the reaction product was collected, and the solvent was evaporated to obtain 21g of compound Z4.1H NMR (400MHz, CDCl) ₃ ): δ 7.25 (t, J =9.6hz, 1h), 6.78 (t, J =9.6hz, 1h), 5.06 (d, J =4.4hz, 1h), 4.19-4.17 (M, 1H), 3.98-3.95 (M, 2H), 3.87-3.82 (M, 1H), 3.76-3.74 (M, 1H), 3.48 (s, 3H), 2.75 (d, J =7.2hz, 1h), 2.24 (s, 3H), 2.01 (t, J =5.6hz, 1h) M/z (ES +): theory, [ M + H = 7.1h)] ⁺ 576.64, detection, [ M + H] ⁺ :576.10

(1-4) Synthesis of Z5

Under the protection of argonThen, 8.24g of compound Z4 (30.0 mmol) was dissolved in 60mL of anhydrous pyridine, the reaction mixture was cooled to 5 ℃, 12.8g of DMTrCl (36.06 mmol) was added to the reaction mixture, stirring was carried out for 20min, then the mixture was warmed to room temperature, the reaction was continued for 7 hours, 100mL of a saturated sodium bicarbonate solution and 100mL of ethyl acetate were added to the reaction mixture, the organic phase was washed six times with 5wt% aqueous citric acid solution, each 100mL, then with 100mL of a saturated aqueous sodium bicarbonate solution and 100mL of a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure and the mixture was dried by suction filtration with an oil pump to give 18.4g of compound Z5.M/z (ES +): theory, [ M + H ]] ⁺ 577.64, detection, [ M + H] ⁺ ,577.24。

(1-5) Synthesis of Z6

18.4g of compound Z5 (26.7 mmol) and 10.89g of imidazole (160.2 mmol) are dissolved in 200mL of dry DMF under the protection of argon, 22g of TBDPSCl (80.1 mmol) are then added and the reaction mixture is reacted at room temperature for 3 hours, then 200mL of water and 200mL of ethyl acetate are added to the reaction mixture for separation, the aqueous phase is extracted 3 times with ethyl acetate, 100mL each time, the organic phases are combined and rinsed twice with 5% by weight aqueous citric acid solution, 100mL each time, and then 100mL of saturated saline solution is washed once, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 21.76g of compound Z6.M/z (ES +), theory, [ M + H ]] ⁺ 816,04, detection, [ M + H] ⁺ ,815.68。

(1-6) Synthesis of Z7

21.76g of Compound Z6 (26.7 mmol) was dissolved in 100mL of DCM, and 100mL of a buffer (2.0 g of TsOH in 70mL of DCM and 30mL of anhydrous methanol) previously refrigerated at 4 ℃ was added for reaction for 30min. Saturated sodium bicarbonate solution was added dropwise to the reaction solution until the solution became colorless, 100mL of DCM solution was added, and the organic phase obtained by liquid separation was reused with 200mL of saturated sodium bicarbonate solutionAfter extraction until the aqueous phase was basic, the residue was washed with 100mL of a saturated saline solution, dried over anhydrous sodium sulfate and desolventized under reduced pressure, and the obtained residue was purified by a silica gel column (gradient elution was performed with V (petroleum ether): V (ethyl acetate) = 50. M/z (ES +): theory, [ M + H ]] ⁺ 513.67, [ M + H ] detection] ⁺ :513.57。

(1-7) Synthesis of Z8

12.82g of tetraethyl methylenediphosphonate (44.51 mmol) was dissolved in 90mL of tetrahydrofuran under the protection of argon, after cooling to 0 ℃, 6.79g of potassium tert-butoxide (60.54 mmol) was added slowly and stirred for 15min, then the mixture was allowed to warm to room temperature and reacted for 30min to obtain a mixed solution containing tetraethyl methylenediphosphonate.

12.25g of Compound Z7 (23.93 mmol) was dissolved in 100mL of a Dimethylsulfoxide (DMSO) solution under an argon atmosphere, and then 9.86g of dicyclohexylcarbodiimide (DCC, 47.86 mmol) was added to the reaction solution, followed by stirring, and then 2.46g of anhydrous pyridine and 2.73g of trifluoroacetic acid (TFA) were added to the reaction solution, followed by reaction at room temperature for 16 hours. Then, the reaction solution was cooled to 0 ℃ in an ice bath, and the obtained mixture was added to the reaction solution and reacted at room temperature for 3 hours. Water and ethyl acetate were added to the reaction mixture to separate layers, insoluble materials were filtered off, the aqueous phase was extracted with 300mL of ethyl acetate, the organic layers were combined, washed with 100mL of a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, and the solvent was removed by evaporation under reduced pressure, and the obtained residue was purified by silica gel column chromatography (gradient elution was performed with V (petroleum ether): V (ethyl acetate) = 30). M/z (ES +), theory, [ M + H ]] ⁺ 645.77, detection, [ M + H] ⁺ ,645.00.

(1-8) Synthesis of Z9

Under argon atmosphere, 10.24g of compound Z8 (15.88 mmol) was added to 100mL of a tetrahydrofuran solution, 4.77mL of a 4M tetrahydrofuran solution of tetrabutylammonium fluoride (19.06 mmol) was then added, the reaction was carried out at room temperature for 30min, the mixture was concentrated under reduced pressure and then subjected to column chromatography, elution was carried out using a mixed solvent of V (petroleum ether): V (ethyl acetate) =1 as a mobile phase, an eluate containing the reaction product was collected, and the solvent was evaporated to give 5.77g of compound Z9.M/z (ES +), theory, [ M + H ]] ⁺ 407.36, detection, [ M + H] ⁺ ,407.00

(1-9) Synthesis of Z10

After 2.14g of the compound Z9 (5.27 mmol) and 108mg of tetrazole (1.32 mmol) were dissolved in 20mL of a dry N, N-dimethylformamide solution at room temperature and air was replaced with argon three times, 285mg of N-methylimidazole (4.0 mmol) and 2.7g of bis (diisopropylamino) (2-cyanoethoxy) phosphine (9.0 mmol) were added to the reaction solution and air was replaced with argon three times, followed by reaction at room temperature for 3 hours. To the reaction mixture, 200mL of a saturated aqueous sodium bicarbonate solution and 200mL of ethyl acetate were added, liquid separation was performed, the obtained organic layer was washed three times with 100mL of a saturated aqueous sodium bicarbonate solution, then washed once with 100mL of a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the residue was purified by a silica gel column, eluted with a mixed eluent of V (petroleum ether):v (ethyl acetate) =2 as a mobile phase, and the eluent containing the reaction product was collected and the solvent was evaporated to obtain 1.63g of compound Z10. The structure of the obtained Z10 compound is shown as a formula (101). ¹ HNMR(300MHz,DMSO-d6)δ7.41-7.29(m,1H),7.20(t,J＝10.2Hz,1H),6.77(tdd,J＝21.8,17.0,5.5Hz,1H),6.19-6.00(m,1H),5.05(t,J＝4.9Hz,1H),4.59(s,1H),4.53(s,1H),4.33-4.12(m,1H),4.08–3.90(m,4H),3.89-3.52(m,4H),3.36(d,J＝11.7Hz,3H),2.86-2.73(m,2H),2.20(s,3H),1.30-1.18(m,6H),1.24-1.07(m,12H). ¹ P NMR(122MHz,DMSO)δ149.0148.8,17.10,16.70.M/z (ES +): theoretical, [ M + H ]] ⁺ 607.58, detection, [ M + H] ⁺ ,607.00。

Preparation example 2 preparation of P10 Compound

In this preparation example, the compound of example 2 was synthesized in accordance with the following method.

(2-1) according to the method described in preparation example 1, step (1-7), a Z8 compound was prepared.

(2-2) Synthesis of P9 Compound

Under argon atmosphere, 4.75g of TMSOI (trimethylsulfoxonium iodide, wherein Me3SI is used) was dissolved in 25mL of dimethyl sulfoxide, and 1.55g of sodium hydride having a concentration of 60wt% was added thereto, followed by reaction with stirring for 1 hour, and 5.0g of Compound Z8 was added to the reaction mixture and allowed to react at room temperature for 2 hours. Then cooling the reaction solution to 0 ℃ in an ice bath, adding a saturated ammonium chloride solution to quench the reaction, stirring the mixture for a while, adding ethyl acetate into the reaction solution to separate the solution, washing an organic phase twice by using saline solution, each time washing the organic phase by using 100mL, performing reduced pressure concentration and vacuum drying, dissolving the residue by using 50mL of tetrahydrofuran, adding 8.5mL of a 1M tetrahydrofuran solution of tetrabutylammonium fluoride (TBAF), reacting the mixture at room temperature for 30min, concentrating the reaction system to remove the solvent, performing column chromatography purification (performing gradient elution by using a ratio of V (petroleum ether): V (ethyl acetate) = 30. M/z (ES +), theory, [ M + H ]] ⁺ 421.39, [ M + H ] detection] ⁺ ,421.16。

(2-3) Synthesis of P10 Compound

400mg of the compound P9, 19.5mg of 1-methylimidazole and 50mg of tetrazole were added to 50mL of a dichloromethane solution at room temperature, argon gas was substituted three times, and 430mg of bis (diiso-methyl-triazole) was addedPropylamino) (2-cyanoethoxy) phosphine, reacting at room temperature for 2 hours, placing the reaction solution in an ice-water bath, cooling to 0 ℃, adding 10mL of saturated sodium bicarbonate solution to quench the reaction, separating with 50mL of dichloromethane, washing the obtained organic phase with 100mL of sodium bicarbonate solution, concentrating, purifying by column chromatography, and purifying by a method of mixing the components in a ratio of V (petroleum ether):V (dichloromethane) =1:1 as the mobile phase, collecting the eluate containing the reaction product, and evaporating off the solvent to obtain 150mg of compound P10. The structure of the obtained P10 compound is shown as a formula (102). ¹ H NMR(400MHz,DMSO-d6)δ7.36(td,J＝8.6,2.5Hz,1H),7.21(t,J＝10.2Hz,1H),6.78(dddd,J＝29.2,21.8,17.1,5.5Hz,1H),6.18–6.03(m,1H),5.06(t,J＝5.6Hz,1H),4.64–4.50(m,1H),4.24(ddt,J＝33.9,10.7,5.3Hz,1H),4.10–3.93(m,4H),3.89–3.73(m,3H),3.77–3.60(m,2H),3.63–3.54(m,2H),3.39(s,2H),2.88–2.71(m,2H),2.67–2.55(m,1H),2.21(d,J＝2.3Hz,3H),1.34–1.18(m,9H),1.22–1.06(m,15H). ¹ P NMR (202MHz, DMSO). Delta.149.3, 149.1,16.7,16.3.M/z (ES +): theoretical, [ M + H ]] ⁺ 621.67, detection, [ M + H] ⁺ ,621.00。

Preparation example 3 Synthesis of siRNA1-13

(3-1) Synthesis of sense Strand of siRNA

Nucleoside monomers are connected one by one from the 3'-5' direction according to the arrangement sequence of sense strand nucleotides of each siRNA in Table 1 by a 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 was supplied as a 0.1M solution in acetonitrile, under the same conditions for the deprotection reaction in each step, i.e., at a temperature of 25 ℃ for 70 seconds, the deprotection reagent was dichloroacetic acid in dichloromethane (3% v/v), and the molar ratio of dichloroacetic acid to 4,4' -dimethoxytrityl protecting group on the solid support was 5.

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 a molar ratio of 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.

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.

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), solvent water/acetonitrile =9 (volume ratio); eluent B:1.5M sodium chloride, 20mM sodium phosphate (pH 8.1), solvent water/acetonitrile =9 (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.

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

(3-2) antisense strand of synthetic siRNA

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

HL Solid Supports, kinovate Life Sciences) and synthesizing antisense strands of siRNA1-13 according to the composition of the antisense strand of siRNA in table 1. 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.

(3-3) 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 the molecular weight was checked by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA were siRNA1 to 13 in Table 1.

Preparation example 4 Synthesis of conjugate 1

Conjugate 1 of the present disclosure was obtained following the procedure of "conjugate 1" in preparation example 1 in WO2019/105437A1, the only difference being: when the last nucleotide (i.e., the 5' terminal nucleotide) of the antisense strand is ligated, this nucleotide is replaced with Z10 in example 1 of the present disclosure. The molecular weight measurement was carried out using a Liquid Chromatography Mass spectrometer (LC-MS, liquid Chromatography-Mass Spectrometry, available from Waters, inc., model: LCT Premier). As a result, the observed value agreed with the theoretical value, and it was confirmed that the synthesized conjugate 1 was the target designed double-stranded nucleic acid sequence containing the group represented by the formula (201). The structure of conjugate 1 is shown below (801):

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

Preparation example 5 Synthesis of conjugate 2

Conjugate 2 of the present disclosure was obtained following the procedure of "conjugate 1" in preparation example 1 in WO2019/105437A1, the only difference being: when the last nucleotide of the antisense strand (i.e., the 5' terminal nucleotide) is ligated, this nucleotide is replaced with compound P10 in example 2 of the disclosure. The molecular weight measurement was carried out using a Liquid Chromatography Mass spectrometer (LC-MS, liquid Chromatography-Mass Spectrometry, available from Waters, inc., model: LCT Premier). As a result, the observed value agreed with the theoretical value, and it was confirmed that the synthesized conjugate 2 was the target designed double-stranded nucleic acid sequence containing the group represented by the formula (202). The structure of conjugate 2 is shown below (802):

wherein Nu in formula (802) is siRNA2 in table 1 of the present disclosure.

Comparative preparation example 1 Synthesis of reference conjugate 1

Reference conjugate 1 was obtained according to the method of preparation example 1 "conjugate 1" in WO2019/105437 A1. The structure of reference conjugate 1 is shown below (803):

wherein Nu in formula (803) is siRNA14 having the following composition.

siRNA14

A sense strand: .

CmsCmsUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm

(SEQ ID NO:27)

Antisense strand:

VPUmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmsUmsUm

(SEQ ID NO:28)

wherein, the letter combination VP indicates that one nucleotide adjacent to the right side of the letter combination VP is a vinylphosphate (5' - (E) -vinylphosphonate, E-VP) modified nucleotide.

After the preparation of the siRNA or conjugate of the present disclosure described above 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).

In this experimental example, the inhibitory efficiency of conjugate 1, conjugate 2 and reference conjugate 1 on the expression level of HBV mRNA in hepatic primary cells of HBV transgenic mouse C57BL/6j-TgN (AlblHBV) 44Bri was examined.

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 prepared separately as 20 μ M (in terms of siRNA) siRNA conjugate working solution using DEPC-treated water.

In different culture wells added with the primary cell suspension of mouse liver, the working solution of siRNA conjugate of each conjugate was added separately and mixed evenly, the addition amount was 2.5. Mu.L/well, each siRNA conjugate was added to 3 culture wells separately, and the transfection mixture containing siRNA (final concentration is 10nM in terms of siRNA) was obtained and recorded as test group. The cell fluid in 3 additional culture wells to which the primary cell suspension of mouse liver 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, goldenstar, was used ^TM RT6 cDNA Synthesis Kit (purchased from Beijing Ongjingkou Biotechnology Co., ltd., cat. No. TSK 301M) provided reagent, wherein Goldenstar 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. And (2) 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 25s and extension at 72 ℃ for 25s, 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 reference gene GAPDH in the product W to obtain a target geneCt values due to HBV and the reference gene GAPDH.

TABLE 2 detection of primer sequences

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:

Δ 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 control culture wells. Thus, each culture well of the test and control groups corresponds to a Δ Δ Ct value.

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 inhibitory activity of each conjugate of the present disclosure on HBV mRNA expression in primary liver cells.

Table 3 siRNA conjugates in vitro activity assay

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 in vivo (in vivo).

The serum HbsAg content of 44Bri mice was measured using the hepatitis b virus surface antigen diagnostic kit (enzyme linked immunosorbent assay) (shanghai kawawa) according to the method described in the specification, mice with S/COV >10 were selected, randomly grouped (all male), 5 mice per group, numbered separately, and conjugate 1, conjugate 2 or reference conjugate 1, siRNA conjugate was administered to each mouse in the form of a1 × PBS solution containing 0.02mg/ml (calculated as siRNA) of siRNA conjugate in a dose of 0.1mg/kg mouse body weight (calculated as siRNA) by subcutaneous injection, and the administration volume was 5ml/kg.

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); to each liver tissue, 1mL of Trizol (Sigma Co.) was added, disrupted in a Tissuelyset type II fully automatic tissue homogenizer 3 times for 30s each to obtain a liver tissue homogenate, to which 0.2mL of chloroform was added, and allowed to stand for 3min. After centrifugation at 12000rpm for 10min at 4 ℃ 0.4mL of supernatant was collected. 0.5mL of isopropyl alcohol 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 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 was incubated at 42 ℃ for 30min, then at 95 ℃ for 5min, and finally at 4 ℃ for 5min, and after the reaction was completed, 80. Mu.L of DEPC water was added to the reverse transcription reaction system to obtain a cDNA-containing solution.

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 inhibitory Activity of siRNA conjugates on HBV mRNA expression in mice

As can be seen from the results of table 4, both the conjugate 1 and the conjugate 2 of the present disclosure in table 4 showed excellent HBV gene expression inhibitory activity in mice, while the conjugate 1 and the conjugate 2 showed comparable levels in vivo as 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 sbefu (beijing) biotechnology limited), 30mg/kg (in siRNA) of conjugate 1, conjugate 2 and reference conjugate 1, pbs of the present disclosure were administered to each rat subcutaneously in a single dose, respectively, for 14 days continuously during which the rats exhibited no death or abnormal behavior. The gross body was dissected, liver tissue was dissected, taken, dehydrated, embedded, tabletted 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.

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 gist of the present disclosure.

Sequence listing

<110> Beijing Ruibo pioneer medicine science and technology, inc.; suzhou Ruibo Biotechnology Ltd

<120> modified nucleoside monomer, siRNA comprising modified nucleotide, pharmaceutical composition and conjugate

<130> CP1210704/CB

<160> 32

<170> SIPOSequenceListing 1.0

<210> 1

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA1

<400> 1

ccuugaggca uacuucaaa 19

<210> 2

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is Bx, bx is the structure shown in formula (201) to formula (203)

<400> 2

nuugaaguau gccucaaggu u 21

<210> 3

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA2

<400> 3

ccuugaggca uacuucaaa 19

<210> 4

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is Bx, bx is the structure shown in formula (201) to formula (203)

<400> 4

nuugaaguau gccucaaggu u 21

<210> 5

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA3

<400> 5

ccuugaggca uacuucaaa 19

<210> 6

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is Bx, bx is the structure shown in formula (201) to formula (203)

<400> 6

nuugaaguau gccucaaggu u 21

<210> 7

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA4

<400> 7

ccuugaggca uacuucaaa 19

<210> 8

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n represents Bx, and Bx represents a structure represented by formula (201) to formula (203)

<400> 8

nuugaaguau gccucaaggu u 21

<210> 9

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA5

<400> 9

ccuugaggca uacuucaaa 19

<210> 10

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<400> 10

nuugaaguau gccucaaggu u 21

<210> 11

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA6

<400> 11

ccuugaggca uacuucaaa 19

<210> 12

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<400> 12

nuugaaguau gccucaaggu u 21

<210> 13

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA7

<400> 13

ccuugaggca uacuucaaa 19

<210> 14

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is Bx, bx is the structure shown in formula (201) to formula (203)

<400> 14

nuugaaguau gccucaaggu u 21

<210> 15

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA8

<400> 15

ccuugaggca uacuucaaa 19

<210> 16

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is Bx, bx is the structure shown in formula (201) to formula (203)

<400> 16

nuugaaguau gccucaaggu u 21

<210> 17

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA9

<400> 17

ccuugaggca uacuucaaa 19

<210> 18

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is Bx, bx is the structure shown in formula (201) to formula (203)

<400> 18

nuugaaguau gccucaaggu u 21

<210> 19

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA10

<400> 19

ccuugaggca uacuucaaa 19

<210> 20

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<400> 20

nuugaaguau gccucaaggu u 21

<210> 21

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA11

<400> 21

ccuugaggca uacuucaaa 19

<210> 22

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is Bx, bx is the structure shown in formula (201) to formula (203)

<400> 22

nuugaaguau gccucaaggu u 21

<210> 23

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA12

<400> 23

ccuugaggca uacuucaaa 19

<210> 24

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is Bx, bx is the structure shown in formula (201) to formula (203)

<400> 24

nuugaaguau gccucaaggu u 21

<210> 25

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA13

<400> 25

ccuugaggca uacuucaaa 19

<210> 26

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1)

<223> n is Bx, bx is the structure shown in formula (201) to formula (203)

<400> 26

nuugaaguau gccucaaggu u 21

<210> 27

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA14

<400> 27

ccuugaggca uacuucaaa 19

<210> 28

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> siRNA14

<400> 28

uuugaaguau gccucaaggu u 21

<210> 29

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer (primer)

<400> 29

ccgtctgtgc cttctcatct 20

<210> 30

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer (primer)

<400> 30

taatctcctc ccccaactcc 20

<210> 31

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer (primer)

<400> 31

tgcaccacca actgcttag 19

<210> 32

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer (primer)

<400> 32

ggatgcaggg atgatgttc 19

Claims

1. A compound having the structure shown in formula (1):

wherein:

each J ₁ Independently is C1-C6 alkyl or substituted C1-C6 alkyl;

E ₁₀₁ is O or S;

wherein each B is ₁ Independently selected from substituted or unsubstituted C1-C5 hydrocarbyl; b ₂ One member selected from the group consisting of C1-C5 alkyl, cyanoethyl, cyanopropyl and cyanobutylSeed growing;

Z ₀ Is H, protected hydroxy, OCH ₃ F or OCH ₂ CH ₂ OCH ₃ ；

Indicates the site at which the group is covalently attached.

2. The compound of claim 1, wherein each of said substituents is independently F, cl or CH ₃ 。

3. The compound of claim 2, wherein Bm is selected from the group consisting of those represented by any one of formulas (G1) to (G12):

4. the compound of claim 1, wherein D is ₀ Is a divalent linking group represented by the formula (D1) or (D2), and P ₁ And P ₂ Each independently is H, or D ₀ Is a divalent linking group represented by the formula (D4).

5. The compound of claim 1, wherein each J is ₁ Is ethyl, E ₁₀₁ Is O.

6. The compound of claim 1, wherein Z is OCH ₃ Or OCH ₂ CH ₂ OCH ₃ 。

7. The compound of claim 1, wherein each B is ₁ Independently selected from isopropyl or isobutyl, B ₂ Selected from cyanoethyl or cyanopropyl.

8. The compound of claim 7, wherein the phosphoramidite functionality has the structure of formula (4):

9. the compound of claim 1, having a structure according to any one of formulas (101) - (113):

10. an siRNA comprising a sense strand and an antisense strand, each of said sense strand and antisense strand comprising 14 to 30 modified or unmodified nucleotides, a portion of the sense strand and antisense strand being reverse-complementary to form a double-stranded region, wherein the 5' -terminal nucleotide of said antisense strand has the structure of formula (5):

wherein:

R ₂₀₁ and R ₂₀₂ Each independently is OH or OJ ₁ ；

J ₁ Is C1-C6 alkyl or substituted C1-C6 alkyl;

each E ₁₀₁ Each independently is O or S;

D ₀ a group which is one of divalent linking groups represented by the structures represented by the following formulae (D1) to (D4):

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

Z is H, OH, OCH ₃ F or OCH ₂ CH ₂ OCH ₃ ；

Represents the site of covalent attachment of a group;

the antisense strand is substantially reverse complementary or substantially reverse complementary to a nucleotide sequence in the mRNA expressed by the target gene having the same length as the antisense strand.

11. The siRNA of claim 10, wherein said substituted phenyl is where at least one hydrogen atom on the phenyl ring is independently replaced by F, cl or CH ₃ And (4) substitution.

12. The siRNA of claim 11, wherein Bm is selected from the group consisting of any one of formulae (G1) to (G12):

13. the siRNA of claim 10, wherein D is ₀ Is a divalent linking group represented by the formula (D1) or (D2), and P ₁ And P ₂ Each independently is H; or D ₀ Is a divalent linking group represented by the formula (D4).

14. The siRNA of claim 10, wherein R ₂₀₁ And R ₂₀₂ Are all OH.

15. The siRNA of claim 10, wherein each E is ₁₀₁ Are both O, or at least one E ₁₀₁ Is S.

16. The siRNA according to claim 10, wherein the group represented by formula (5) has a structure represented by any one of formulae (201) to (213):

17. the siRNA according to claim 10, wherein the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II, and the 5' -terminal nucleotide of the nucleotide sequence II has the structure represented by formula (5); the nucleotide sequence I and the nucleotide sequence II both consist of 19 nucleotides, the nucleotide sequence II and the part of the nucleotide sequence I are reversely complementary to form a double-stranded region, the part of the nucleotide sequence II is reversely complementary with 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; counting the group shown in the formula (5) as 1 nucleotide, wherein the 2 nd, 6 th, 14 th and 16 th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides according to the direction from the 5 'end to the 3' end.

18. The siRNA according to claim 17, wherein, in the 5 'end to 3' end 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 the 5 'end to the 3' end, the 2 nd, 6 th, 14 th and 16 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.

19. The siRNA of claim 17, wherein positions 2-19 of said nucleotide sequence II are fully reverse complementary to positions 1-18 of said first nucleotide sequence in the 5 'to 3' direction.

20. The siRNA of claim 17, wherein positions 2-19 of said nucleotide sequence II are fully reverse complementary to positions 1-18 of said nucleotide sequence I in the 5 'to 3' direction to form a double-stranded region.

21. The siRNA according to claims 17 to 20, 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 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, which is a nucleotide sequence adjacent to the first nucleotide sequence and has the same length as the nucleotide sequence IV in the mRNA expressed by the target gene.

22. The siRNA of claims 17 to 21, wherein the siRNA further comprises a nucleotide sequence V, each nucleotide of the nucleotide sequence V being independently one of the non-fluorinated modified nucleotides, the nucleotide sequence V being 1 to 3 nucleotides in length, attached to the 3 'end of the antisense strand, thereby constituting a 3' overhang of the antisense strand.

23. The siRNA according to claim 22, wherein the nucleotide sequence V has a length of 2 nucleotides, and is a sequence of 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 a length equal to the nucleotide sequence V, in the direction from the 5' end to the 3' end.

24. The siRNA of claims 17 to 23, wherein each of the non-fluorinated modified nucleotides is independently selected from one of a nucleotide or a nucleotide analog in which a hydroxyl group at the 2' -position of a ribosyl group of the nucleotide is substituted with a non-fluorine group.

25. The siRNA of any one of claims 17 to 24, wherein each of the non-fluorinated modified nucleotides is a methoxy modified nucleotide, which is a nucleotide in which a 2' -hydroxyl group of a ribosyl group is substituted with a methoxy group.

26. The siRNA of any of claims 17 to 25, wherein at least 1 of the phosphate groups in the phosphate-sugar backbone of at least one single strand of said sense strand and said antisense strand is a phosphate group having a modifying group, said phosphate group having a modifying group being present at least one position of the group consisting of:

the 5' terminal end of the sense strand is between the 1 st nucleotide and the 2 nd nucleotide;

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 5' terminal end of the antisense strand;

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

27. The siRNA of any of claims 10 to 26, wherein the siRNA is siRNA1, siRNA2, siRNA3, siRNA4, siRNA5, siRNA6, siRNA7, siRNA8, siRNA9, siRNA10, siRNA11, siRNA12 or siRNA13.

28. A pharmaceutical composition comprising the siRNA of any one of claims 10-27 and a pharmaceutically acceptable carrier.

29. An siRNA conjugate comprising an siRNA according to any one of claims 10 to 27 and a conjugate group conjugated to the siRNA, the conjugate group comprising a linker and a pharmaceutically acceptable targeting group and/or a delivery assisting group, and the siRNA, 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 siRNA conjugate in the delivery of a target organ or tissue.

30. Use of an siRNA of any one of claims 10 to 27, and/or a pharmaceutical composition of claim 28 and/or an siRNA conjugate of claim 29, for the manufacture of a medicament for the treatment and/or prevention of a disease or condition associated with mRNA levels of target gene expression.

31. The use of claim 30, 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, SOD1, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT.

32. The use of claim 31, wherein the mRNA expressed by the target gene is selected from mRNA expressed by a hepatitis b virus gene.

33. The use of any one of claims 30-32, wherein the disease or condition associated with mRNA levels of target gene expression is hepatitis b.

34. 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 siRNA of any one of claims 10-27, and/or a pharmaceutical composition of claim 28 and/or an siRNA conjugate of claim 29.

35. The method of claim 34, 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, SOD1, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT.

36. The method of claim 34 or 35, wherein the mRNA expressed by the target gene is selected from mRNA expressed by a hepatitis b virus gene.

37. The method of any one of claims 34-36, wherein the disease or condition associated with mRNA levels of target gene expression is hepatitis b.

38. A method of inhibiting the expression level of a target gene in a cell, the method comprising contacting the cell with an effective amount of the siRNA of any one of claims 10 to 27, and/or the pharmaceutical composition of claim 28, and/or the siRNA conjugate of claim 29.

39. The method of claim 38, 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, SOD1, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT.

40. The method of claim 38 or 39, wherein the modulation is inhibition of target gene expression in the cell, wherein the mRNA expressed by the target gene is selected from the group consisting of hepatitis B virus genes.

41. A kit comprising the siRNA of any one of claims 10 to 27, and/or the pharmaceutical composition of claim 28, and/or the siRNA conjugate of claim 29.