CN112876534B

CN112876534B - Liver targeting compounds and conjugates

Info

Publication number: CN112876534B
Application number: CN201911198284.9A
Authority: CN
Inventors: 张鸿雁; 杨志伟; 黄金宇; 王秀莲; 黄敏印; 何涛
Original assignee: Suzhou Ruibo Biotechnology Co ltd
Current assignee: Suzhou Ruibo Biotechnology Co ltd
Priority date: 2019-11-29
Filing date: 2019-11-29
Publication date: 2024-02-09
Anticipated expiration: 2039-11-29
Also published as: CN112876534A

Abstract

The present invention provides a compound for forming a conjugate with an active agent, such as an oligonucleotide, the compound having a structure as shown in formula (I). The disclosure also provides corresponding conjugates, as well as oligonucleotide conjugates for modulating gene expression in hepatocytes, and the use of the conjugates in the manufacture of a medicament for the prevention and/or treatment of related diseases. The conjugate of the present disclosure can specifically target hepatocytes, thereby effectively solving the problems associated with in vivo delivery of oligonucleotide drugs, has low toxicity, and has excellent delivery efficiency while maintaining the delivered oligonucleotides highly stable.

Description

Liver targeting compounds and conjugates

Technical Field

The present invention relates to the field of delivering active agents using targeting ligands. In particular, the present invention relates to a novel liver targeting compound and conjugates comprising the same, in particular oligonucleotide conjugates, and methods for their preparation and use.

Background

Oligonucleotide compounds have medical important therapeutic applications. Oligonucleotides can be used to regulate specific genes. Such oligonucleotide compounds include, but are not limited to, small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), small activating RNAs (sarnas), and microRNAs (microRNAs), among others. In particular therapeutic applications, oligonucleotides and analogs thereof that are expressed only in a particular tissue or location may be selected to treat a disease or condition of interest at a particular target site.

Although tissue-specific active agents, represented by specific oligonucleotides and oligonucleotide analogs, have made partial progress in their use as therapeutic agents, there remains a need for improvements in their pharmacological properties, such as targeting delivery to lesions to enhance the selectivity of the therapeutic agent, enhancing its biological activity and efficacy. Recently targeted conjugated delivery technology has become one of the most widely studied delivery systems, focusing mainly on liver targeted delivery.

Disclosure of Invention

In a first aspect, the present invention provides a novel liver targeting compound (hereinafter sometimes referred to as "conjugate molecule") having a structure represented by formula (I):

wherein, the liquid crystal display device comprises a liquid crystal display device,

m represents an integer of 1 to 6;

each R ₂ Independently selected from H, C ₁ -C ₁₀ Alkyl, C ₁ -C ₁₀ Haloalkyl or C ₁ -C ₁₀ An alkoxy group;

L ₁ represents carbonyl, carbonyl (C) ₁ -C ₁₀ Alkylene) or carbonyl- (oxyethyl) _k Wherein k represents an integer of 1 to 10;

g represents a first hydroxyl protecting group;

L ₂ represents an amino group or a hydroxyl group, or any group capable of forming a covalent bond with an amino group or a hydroxyl group; or alternatively

L ₂ Comprising a solid support linked by said covalent bond;

e represents ligand M having affinity for asialoglycoprotein receptor on the surface of mammalian liver cells ₁ Or (b)

Represents the ligand M ₁ A group formed by substitution of all of the active hydroxyl groups, if any, of the second hydroxyl protecting groups;

each L ₃ Is a linear alkylene group of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally replaced by one or more selected from the group consisting of: c (O), NH, O, S, CH = N, S (O) ₂ 、C ₂ -C ₁₀ Alkenylene, C ₂ -C ₁₀ Alkynylene, C ₆ -C ₁₀ Arylene group, C ₃ -C ₁₈ Heterocyclylene and C ₅ -C ₁₀ Heteroarylene; and, in addition, the processing unit,

each L ₃ Optionally having any one or more substituents selected from the group consisting of: c (C) ₁ -C ₁₀ Alkyl, C ₆ -C ₁₀ Aryl, C ₅ -C ₁₀ Heteroaryl, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkane, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ Haloalkyl, halogen substituent, -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 a second aspect, the present invention provides a novel oligonucleotide conjugate having a structure represented by formula (II):

E ₁ is OH, SH or BH ₂ Nu represents a functional oligonucleotide;

m represents an integer of 1 to 6;

E ₂ represents amino, hydroxy, -amino (C) ₁ -C ₁₀ Hydrocarbylene) amino, -amino (C) ₁ -C ₁₀ Hydrocarbylene) hydroxy, -hydroxy (C) ₁ -C ₁₀ Hydrocarbylene) amino or hydroxy (C ₁ -C ₁₀ Hydrocarbylene) hydroxy, the hydrocarbylene optionally having one or more substituentsThe substituents are selected from the group consisting of: c (C) ₁ -C ₁₀ Alkyl, C ₆ -C ₁₀ Aryl, C ₅ -C ₁₀ Heteroaryl, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkane, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ Haloalkyl, halogen substituent, -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), 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 A haloalkyl group);

M ₁ representing ligands having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells;

each L ₃ Is a straight chain alkyl group of 1 to 70 carbon atoms in length, wherein one or more carbonsThe atoms are optionally replaced by one or more selected from the group consisting of: c (O), NH, O, S, CH = N, S (O) ₂ 、C ₂ -C ₁₀ Alkenylene, C ₂ -C ₁₀ Alkynylene, C ₆ -C ₁₀ Arylene group, C ₃ -C ₁₈ Heterocyclylene and C ₅ -C ₁₀ Heteroarylene; and is also provided with

Each L ₃ Optionally having any one or more substituents selected from the group consisting of: c (C) ₁ -C ₁₀ Alkyl, C ₆ -C ₁₀ Aryl, C ₅ -C ₁₀ Heteroaryl, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkane, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ Haloalkyl, halogen substituent, -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 a third aspect, the present invention provides a liver targeting compound of formula (I) and a method of preparing an oligonucleotide conjugate of formula (II).

In a fourth aspect, the invention provides a method of modulating expression of a gene in a hepatocyte, said modulating comprising inhibiting or enhancing expression of said gene, the method comprising contacting said hepatocyte with an oligonucleotide conjugate of the invention.

The oligonucleotide conjugate of the invention shows higher gene expression inhibition activity in liver cells, and is expected to be capable of effectively improving the in vivo delivery efficiency of siRNA, has low toxicity and off-target effect and/or excellent stability, so that compared with the existing oligonucleotide preparation, the oligonucleotide conjugate of the invention is expected to achieve better treatment effect, thereby having wide industrial application prospect.

Other features and advantages of the present invention will be described in the detailed description section that follows.

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.

Detailed Description

The following describes specific embodiments of the present disclosure in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.

Definition of the definition

In the above and in the following, especially when describing the preparation method of the delivery compound of the present disclosure (hereinafter sometimes also referred to as "the conjugate molecule of the present disclosure" or simply as "conjugate molecule") or the preparation method of the oligonucleotide conjugate, the nucleoside monomer (nucleoside monomer) means, unless otherwise specified, that the "unmodified or modified RNA phosphoramidite (unmodified or modified RNA phosphoramidite)", respectively, is used for so-called solid phase phosphoramidite synthesis, which is a well-known method in the art for synthesizing RNA, depending on the RNA sequence to be prepared. RNA phosphoramidite is also referred to herein as nucleoside phosphoramidite (nucleoside phosphoramidites). Unless otherwise indicated, the nucleotide monomers used in the present disclosure are commercially available.

As used herein, a short dash ("-") that is not between two letters or between two symbols is used to indicate a position of a substituent point of attachment. For example: -C ₁ -C ₁₀ alkyl-NH ₂ Through C ₁ -C ₁₀ Alkyl groups.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" includes "alkyl" and "substituted alkyl" as defined below. Those skilled in the art will appreciate that for any group comprising one or more substituents, such groups are not intended to introduce any substitution or pattern of substitution that is sterically impractical, synthetically infeasible, and/or inherently unstable.

As used herein, "alkyl" refers to straight and branched chains having the indicated 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 6 carbon atoms. For example, C ₁ -C ₆ The alkyl groups comprise straight and branched alkyl groups of 1 to 6 carbon atoms. When referring to alkyl residues having a specific number of carbons, 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 groups, referring to residues identical to alkyl groups but having 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 loss of one hydrogen atom each from adjacent carbon atoms 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: vinyl; 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, such as but-1-en-1-yl, but-1-en-2-yl, 2-methylpropan-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 groups and refers to residues that are identical to alkenyl groups but have 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 the loss of two hydrogen atoms each from adjacent carbon atoms of the parent alkyl group. Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, and the like. In certain embodiments, alkynyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbons.

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

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

As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring, typically having 3 to 7 cyclic carbon atoms. The ring may be saturated or have one or more carbon-carbon double bonds. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl, as well as bridged and caged ring groups such as norbornane (norbornane).

As used herein, "halogen substituent" or "halo" refers to fluoro, chloro, bromo, and iodo, and the term "halo" includes fluoro, chloro, bromo, and iodo.

As used herein, "haloalkyl" refers to an alkyl group having a specified number of carbon atoms substituted with one or more up to the maximum allowable number of halogen atoms. Examples of haloalkyl include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl and pentafluoroethyl.

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

"heteroaryl" refers to groups derived from 3-to 18-membered aromatic ring radicals containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, heteroaryl groups may be monocyclic, bicyclic, tricyclic or tetracyclic systems, wherein at least one ring of the ring system is fully unsaturated, i.e. comprises a cyclic delocalized (4n+2) pi-electron system according to the huckel theory. Heteroaryl groups include fused or bridged ring systems. The heteroatoms in the heteroaryl group are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. Heteroaryl groups are attached through any atom in the ring. Examples of heteroaryl groups include, but are not limited to: azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1, 3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo [ d ] thiazolyl, benzothiadiazolyl, benzo [ b ] [1,4] dioxaheptyl (benzob ] [1,4] dioxazinyl), benzo [ b ] [1,4] oxazinyl (benzob ] [1,4] oxazinyl), 1,4-benzodioxanyl (1, 4-benzodioxanyl), benzonaphthalenyl, benzoxazolyl, benzodioxolyl (benzodioxanyl), benzodioxanyl (benzopyranyl, benzopyranonyl, benzofuranyl, benzothienyl, benzothiophenyl, benzotriazolyl, 6-imidazo [1,2 ] pyridyl; 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 ] cinnolinyl, 6, 7-dihydro-5H-benzo [6,7] cyclohepta [1,2-c ] pyridazinyl dibenzofuranyl, dibenzothienyl, furyl, furanonyl, furo [3,2-c ] pyridyl, 5,6,7,8,9, 10-hexahydrocyclooctano [ d ] pyrimidinyl, 5,6,7,8,9, 10-hexahydrocyclooctano [ d ] pyridazinyl, 5,6,7,8,9, 10-hexahydrocyclooctano [ d ] pyridyl, isothiazolyl, imidazolyl, indazolyl (indazolyl), indolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolinyl, indolizinyl (indoziyl), isoxazolyl, oxetanyl (oxalanyl), 5,6,7,8-tetrahydroquinazolinyl (5, 8-metheno-5, 6,7, 8-tetrahydroquinazolinyl), naphthyridinyl (naphthyridinyl), 1, 6-naphthyridinyl (1, 6-naphthyridinyl), oxadiazolyl, 2-oxaazepinyl (2-oxozepinyl), oxazolyl, oxetanyl (oxalanyl), 5, 6a,7,8,9,10 a-octahydrobenzo [ H ] quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phthalazinyl (phtalazinyl), pteridinyl (ptyleyl), purinyl, pyrazolo [3, 4-pyridinyl ] pyrazolo, 4-pyridinyl; pyrido [3,2-d ] pyrimidinyl, pyrido [3,4-d ] pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl (quinoxalinyl), quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7, 8-tetrahydrobenzo [4,5] thieno [2,3-d ] pyrimidinyl, 6,7,8, 9-tetrahydro-5H-cyclohepto [4,5] thieno [2,3-d ] pyrimidinyl, 5,6,7, 8-tetrahydropyrido [4,5-c ] pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno [2,3-d ] pyrimidinyl, thieno [3,2-d ] pyrimidinyl, thieno [2,3-c ] pyridinyl (thio) and thienyl (thio/thienyl).

Various hydroxyl protecting groups may be used in the present disclosure. In general, the protecting group renders the chemical functional group insensitive to specific reaction conditions and may be added and removed from the functional group in the molecule without substantially damaging the remainder of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al, tetrahedron 1992,48,2223-2311, and Greeneand Wuts, protective Groups in Organic Synthesis, chapter 2,2d ed,John Wiley&Sons,New York,1991, which are incorporated herein by reference in their entirety. In some embodiments, the protecting group is stable under alkaline conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthin-9-yl (Pixyl), and 9- (p-methoxyphenyl) xanthin-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) and t-butyldimethylsilyl (TBS or TBDMS). Non-exclusive examples of hydroxyl protecting groups that may be used herein include hydrocarboyl groups.

The term "subject" as used herein refers to any animal, such as a mammal or a pouched animal. 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, cattle, sheep, rats, and any variety of poultry.

As used herein, "treating," "alleviating," or "ameliorating" may be used interchangeably herein. These terms refer to methods of achieving a beneficial or desired result, including but not limited to therapeutic benefit. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is obtained by eradicating or ameliorating one or more of the 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, "prevent" and "prevent" are used interchangeably. These terms refer to methods of achieving a beneficial or desired result, including but not limited to prophylactic benefit. To obtain a "prophylactic benefit," the conjugate or composition may be administered to a subject at risk of suffering from a particular disease, or to a subject reporting one or more pathological symptoms of the disease, even though a diagnosis of the disease may not have been made.

Liver targeting compounds

In one aspect, the invention discloses a conjugate molecule for delivering an active agent or active drug. In some embodiments, the conjugate molecules of the present disclosure facilitate tissue-specific targeting. In some embodiments, the conjugate molecules of the present disclosure bind to a cell surface receptor. For this purpose, any cell surface receptor or biomarker or portion thereof is considered suitable. In some embodiments, the conjugate molecules of the present disclosure specifically bind to specific receptors of a particular tissue, thereby achieving tissue-specific targeting. In some embodiments, the conjugate molecules of the present disclosure specifically target to hepatocyte surface receptors, thereby specifically targeting to liver tissue. In some embodiments, the conjugate molecules of the present disclosure specifically target cell surface receptors specific for hepatocytes. In some embodiments, the conjugate molecules of the present disclosure specifically target asialoglycoprotein receptors (asialoglycoprotein receptors, ASGPR) to the surface of hepatocytes.

As used herein, "active agent" and "active drug" are used interchangeably, and refer to a molecule capable of being delivered by a conjugated molecule of the present disclosure. In some embodiments, the active agent is an agent capable of being delivered to hepatocytes. Such reagents are well known to those skilled in the art and include, but are not limited to, functional oligonucleotides, particularly those disclosed herein.

In some embodiments, the present invention provides a novel liver targeting compound having a structure represented by formula (I):

m represents an integer of 1 to 6;

g represents a first hydroxyl protecting group;

L ₂ Comprising a solid support linked by said covalent bond;

In some embodiments, m may be selected from an integer from 2 to 5, thereby ensuring that the number of E groups in the liver targeting compound (I) (also referred to as a "conjugate molecule") is at least 2; in some embodiments, m.gtoreq.3, such that in oligonucleotide conjugate (II) formed from the conjugate molecule, M ₁ The number of ligands may be at least 3, so that M ₁ The ligand binds more readily to hepatic surface asialoglycoprotein receptors, thereby facilitating entry of the oligonucleotide conjugate into the cell by endocytosis. Experiments show that when M ₁ When the number of the ligands is more than 3, M ₁ The increased ease of ligand binding to hepatic surface asialoglycoprotein receptors is not significant and thus, in some embodiments m is an integer from 2 to 4, from a combination of aspects such as ease of synthesis, structural/process costs, and delivery efficiency.

In some embodiments, each R ₂ Independently of one another, H, methyl or ethyl; in some embodiments, each R ₂ All are H;

L ₁ the function of (a) is to provide a suitable spatial position for the attachment of the liver targeting compound to the oligonucleotideAnd chemical environment. To this end, in some embodiments, L ₁ Represents carbonyl, carbonyl (C) ₁ -C ₁₀ Alkylene) or carbonyl- (oxyethyl) _k Wherein k represents an integer of 1 to 10. In some embodiments, L ₁ Is carbonyl (C) ₂ -C ₆ Alkylene) or carbonyl (oxyethyl) _k Wherein k is an integer from 2 to 7, preferably an integer from 2 to 4. In some embodiments, L ₁ Is carbonyl butylene.

G represents a first hydroxyl protecting group. In some embodiments, the first hydroxyl protecting group hydrolyzes under suitable conditions to release an active hydroxyl group for covalent attachment to the oligonucleotide. The hydroxyl protecting group G is selected according to the desired subsequent synthetic process. For example, G may be selected from any one of trityl, 4-methoxytrityl, 4 '-dimethoxytrityl (DMTr), 4',4 "-trimethoxybenzyl and t-butyldimethylsilyl (TBS or TBDMS); in some embodiments, G is 4,4' -dimethoxytrityl (DMTr).

In some embodiments, L ₂ Comprising groups capable of being attached to a solid support by covalent bonds. In some embodiments, L ₂ Comprising groups capable of bridging by hydrocarbyl diacids, thereby linking by amide or ester bonds with hydroxyl or amino groups on the solid support. In some embodiments, L ₂ Represents-amino (C) ₁ -C ₁₀ Alkyl alcohol) or-amino (ethoxy) _q2 Ethanol, wherein q2 represents an integer from 1 to 10, preferably an integer from 2 to 7. In further embodiments, L ₂ Having groups capable of forming an ester bond or an amide bond with a hydroxyl group or an amino group on the solid support; in some embodiments, L ₂ Comprising a carboxylic or cationic carboxylate, which may be, for example, a metal ion, an ammonium ion, a cation formed from an organic amine, or a quaternary ammonium cation. In some embodiments, L ₂ Comprising an oxyalkylenecarboxyl group (or carboxylate) or an aminoacylalkylenecarboxyl group (or carboxylate). In some embodiments, L ₂ Comprising solid phases linked by formation of ester or amide bonds with hydroxy or amino groupsA carrier. In some embodiments, L ₂ Containing amino or hydroxy groups, or L ₂ Comprising a structure represented by the formula (C1), (C2), (C3), (C4), (C1 '), (C3 ') or (C4 '):

Wherein each q ₁ Independently an integer of 1 to 5, each X is independently O or NH, M ⁺ Is a cation; in some embodiments, M ⁺ Selected from any one of hydrogen ion, ammonium ion, alkali metal ion or alkaline earth metal ion, SPS represents a solid phase carrier,indicating the site of covalent attachment of the group.

Represents the ligand M ₁ All of the active hydroxyl groups (if any) of the (a) are protected by a second hydroxyl protecting group. In some embodiments of the present disclosure, the hydroxyl group protected by the second hydroxyl protecting group has the form YCOO-, wherein each Y is independently selected from the group consisting of: c (C) ₁ -C ₁₀ Alkyl and C ₆ -C ₁₀ Aryl, said C ₁ -C ₁₀ Alkyl or C ₆ -C ₁₀ The aryl group optionally has one or more substituents selected from the group consisting of halogen substituents and C ₁ -C ₆ Alkyl groups. In some embodiments, each Y is independently selected from methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and C ₁ -C ₆ One of alkylphenyl groupsSeed;

the ligand M having affinity for asialoglycoprotein receptor on the surface of mammalian liver cells ₁ May be any ligand having an affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes, the class of which is well known to those skilled in the art. In some embodiments, at least one ligand M ₁ Is a saccharide. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand M ₁ Is monosaccharide, disaccharide, trisaccharide or polysaccharide. In some embodiments, each ligand M ₁ Is monosaccharide, disaccharide, trisaccharide or polysaccharide. In some embodiments, at least one ligand M ₁ Is a modified sugar. In some embodiments, each ligand M ₁ Is a modified sugar. In some embodiments, each ligand M ₁ Independently selected from the group consisting of polysaccharides, modified polysaccharides, monosaccharides, or monosaccharide derivatives. In some embodiments, each or at least one ligand M ₁ Possibly independently selected from the group consisting of: glucose and its derivatives, mannans and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.

In some embodiments, the ligand M ₁ May be independently selected from the group consisting of D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-glucofuranose, beta 0-D-mannopyranose, beta 1-D-mannopyranose, beta 2-D-glucopyranose, beta 3-D-glucopyranose, alpha-D-glucofuranose, beta-D-glucofuranose, alpha-D-fructofuranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetyl galactosamine, N-propionyl galactosamine, N-N-butyryl galactosamine, N-isobutyryl galactosamine, 2-amino-3-O1- [ (R) -1 ]Carboxyethyl group]-2-deoxy- β -D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-glucopyranose, 2-deoxy-2-sulphonamino-D-glucopyranose, N-glycolyl- α -neuraminic acid, 5-thio- β -D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl- α -D-glucopyranoside methyl ester, 4-thio- β -D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio- α -D-glucoheptopyranoside ethyl ester, 2, 5-anhydro-D-psicose, ribose, D-4-thioribose, L-ribose, L-4-thioribose. In some embodiments, at least one M ₁ Is N-acetylgalactosamine (GalNAc); in some embodiments, each M ₁ All are N-acetyl galactosamine. In some embodiments, ligand M ₁ See, for example, CN105378082a, the entire disclosure of which is incorporated herein by reference. CN105378082a discloses a compound comprising a modified oligonucleotide and a conjugate group comprising at least one phosphorus or neutral linking group and 1 or more ligands, each ligand is selected from the group consisting of polysaccharide, modified polysaccharide, mannose, galactose, mannose derivative, galactose derivative, D-glucopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-glucofuranose, beta 0-D-glucopyranose, beta 1-D-glucopyranose, beta 2-D-glucopyranose, beta 3-D-glucopyranose, beta 4-D-glucofuranose, beta-D-glucofuranose, alpha-D-fructofuranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactosamine, sialic acid, alpha-D-galactosamine, N-acetylgalactosamine, 2-amino-3-O- [ (R) -1-carboxyethyl ]-2-deoxy-beta-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamide-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulphonamino-D-glucopyranose, 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- β -D-galactopyranoside, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio- α -D-glucoheptopyranoside ethyl ester, 2, 5-anhydro-D-allose nitrile, ribose, D-4-thioribose, L-ribose or L-4-thioribose. The compounds are said to reduce the amount or activity of nucleic acid transcripts in cells.

WO2016077321A1 discloses numerous sirnas specifically targeting HBV genes and methods of delivering them and enhances their serum stability by modifying the nucleotides of the sirnas. The document also discloses siRNA conjugates, and further specifically discloses several siRNA conjugates having specific structures.

WO2016168286A1 discloses numerous sirnas specifically targeting the ANGPTL3 gene and methods of delivering them, and by modifying the nucleotides of the sirnas, the serum stability thereof is enhanced. The document also discloses siRNA conjugates.

N-acetylgalactosamine (GalNAc), a ligand that binds to hepatic surface asialoglycoprotein receptors. The asialoglycoprotein receptor (asialoglycoprotein receptor, ASGPR) is an endocytic receptor for hepatocyte-specific expression. In recent years, a high-affinity ligand N-acetylgalactosamine (GalNAc) of ASGPR is used as a targeting molecule, and a better effect is obtained in the aspect of liver targeting delivery of nucleic acid drugs. For example, siRNA based on GalNAc conjugation technology was first reported to exert interfering activity in mice by alnilla corporation (Alnylam pharmaceuticals, inc.) (Nair et al, j.am. Chem. Soc.,2014, 136, 16958-16961). The article reports that three clusters of GalNAc conjugated sirnas exhibit good delivery activity in both in vivo and in vitro experiments. Single dose ED by in vivo experiments in subcutaneously administered mice ₅₀ The single injection dosage is less than 1ml at 1 mg/kg. In long-term dosing experiments, once weekly subcutaneous injections, stable interfering activities of up to 9 months were obtained.

In some embodiments, each or at least one of the ligands is selected from any one of the following: alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionyl galactosamine, N-N-butyryl galactosamine, N-isobutyryl galactosamine, 4-thio-beta-D-galactopyranose.

In some preferred embodiments, each E is independently selected from one of the groups of formulas A46-A54:

in some embodiments, E is formula a49 or a50. In some embodiments, each Y is methyl.

L ₃ The function of (a) is to provide a linkage for the ligand to the N atom on the pyrrolidine ring in formula (I) of the present disclosure, thereby providing liver targeting function for the oligonucleotide conjugates of the present disclosure. In some embodiments, L ₃ Independently selected from the group consisting of groups of formulas A1-a26, and any combination thereof:

wherein each j1 is independently an integer from 1 to 20;

each j2 is independently an integer from 1 to 20;

each R' is independently C ₁ -C ₁₀ -an alkyl group;

each Ra is independently selected from the group consisting of groups of formulae a27-a45 and any combination thereof:

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each Rb is independently C ₁ -C ₁₀ -an alkyl group;

indicating the site of covalent attachment of the group.

In some embodiments, L ₃ A linked combination of one or more selected from A1, A4, A5, A6, A8, a10, a11 and a 13. In some embodiments, L ₃ A combination of linkages selected from at least 2 of A1, A4, A8, a10 and a 11. In some embodiments, L ₃ A combination of linkages selected from at least 2 of A1, A8, a 10.

In some embodiments, L ₃ May be 3-25, 3-20, 4-15 or 5-12 atoms in length. In some embodiments, L ₃ Is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, or 60 atoms in length. The L is ₃ The length of (a) refers to the number of chain forming atoms from the atom attached to the N atom on the pyrrolidine ring to the longest atom chain formed by the atom attached to E in the compound of formula (I).

According to some embodiments of the present disclosure, each j1 is independently an integer from 2 to 10, in some embodiments, each j1 is independently an integer from 3 to 5. In some embodiments, each j2 is independently an integer from 2 to 10; in some embodiments, each j2 is independently an integer from 3 to 5. Each R' is independently C ₁ -C ₄ In some embodiments, each R' is independently one of methyl, ethyl, and isopropyl. In some embodiments, each Ra is independently one of a27, a28, a29, a30, and a31, and in some embodiments, each Ra is independently a27 or a28. In some embodiments, each Rb is independently C ₁ -C ₅ In some embodiments, each Rb is independently one of methyl, ethyl, isopropyl, and butyl. In some embodiments, each pair j1, j2, R' in formulas A1-A26, Ra, rb are selected to achieve a stable linkage of the ligand to N on the pyrrolidine ring and to make the spatial position between the ligands more suitable for binding of the ligand to the hepatic surface asialoglycoprotein receptor.

In one embodiment, the liver targeting compound of the present invention has a structure represented by formula (I-1), (I-2), (I-3), (I-4), (I-5) or (I-6):

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wherein SPS represents a solid support;

the solid phase carrier may be a solid phase carrier known in the art as being useful for solid phase synthesis of nucleic acids, for example, a commercially available universal solid phase carrier [ ]HL UnyLinker ^TM 300Oligonucleotide Synthesis Support,Kinovate Life Sciences company, structure shown as formula B80):

wherein Resin represents a Resin.

In some embodiments, SPS represents a resin. In some embodiments, SPS may be a hydroxyl or amino resin, or SPS further comprises a linking group, in which case L ₁ Covalent bonds may be formed directly, or through the linking group, with hydroxyl or amino groups on the resin.

Preparation of Compounds of formula (I)

The compounds of formula (I) may be prepared by any reasonable synthetic route.

For example, in some embodiments, L in formula (I) ₂ The group has a hydroxyl group, in which case the compound of formula (I) can be obtained by the following preparation method: the method comprises contacting a compound of formula (X-11) with a deprotecting agent in an organic solvent to remove the hydroxy protecting group X in the compound of formula (X-11) ₁₁ And (5) separating.

Wherein L is ₁ 、L ₃ 、G、E、R ₂ M is as defined above;

L ₁₁ is a linking group which is obtained by linking a hydroxyl group to the above L ₂ A group; x is X ₁₁ Is a hydroxyl protecting group.

The organic solvent can be halogenated alkyl, ether or nitrile solvent; in some embodiments, the organic solvent is Tetrahydrofuran (THF). The amount of the organic solvent may be 5 to 50L/mol, for example 8 to 20L/mol, relative to the compound of formula (X-11). Depending on the hydroxy-protecting group X used ₁₁ The deprotecting agent is selected. For example, in some embodiments, the hydroxyl protecting group X ₁₁ Is Tetramethylsilane (TMS) or t-butyldimethylsilyl (TBDMS), in which case the deprotecting agent may be, for example, tetrabutylammonium fluoride (TBAF). The molar ratio of the amount of the deprotection agent to the compound of formula (X-11) may be from 2:1 to 10:1, for example from 4:1 to 6:1. The reaction may be carried out at a suitable temperature, for example 0-25 c, for 2-8 hours. In some embodiments, the reaction is performed at room temperature for 4 hours. In some embodiments, the reaction is performed in an ice bath for 8 hours. In some embodiments, the compounds of formula (I) produced by the reaction may be separated using, for example, column chromatography, and the separation conditions may be, for example, gradient elution using ethyl acetate: methanol=100:1-8:1 (V: V). In one embodiment of the present invention, the compound of formula (X-11) is, for example The compounds shown below as PNB-11.

The compounds of formula (X-11) may be obtained or prepared by any method known to those skilled in the art. In some embodiments, the compound of formula (X-11) is obtainable by the following preparation method: contacting a compound of formula (X-10) with a compound of formula (X-22) in an organic solvent in the presence of a condensation catalyst and an organic base to effect a condensation reaction, and isolating.

Wherein L is ₁ 、L ₃ 、G、E、m、R ₂ 、L ₁₁ 、X ₁₁ Is as defined above;

the organic solvent can be halogenated alkyl, ether or nitrile solvent; in some embodiments, the organic solvent is dichloromethane. The amount of the organic solvent may be 5 to 30L/mol, for example 8 to 20L/mol, relative to the compound of formula (X-10). The condensing agent may be selected from amide-forming condensing agents suitable in the art. In some embodiments may be, for example, 2- (7-oxo-benzotriazol) -N, N' -tetramethyluronium Hexafluorophosphate (HATU). The condensing agent may be used in a molar ratio of 2:1 to 10:1, for example 3:1 to 6:1, relative to the compound of formula (X-10). The organic base may be, for example, an amine organic base, and in some embodiments triethylamine or N, N-Diisopropylethylamine (DIPEA) may be used. The organic base may be used in a molar ratio to the compound of formula (X-10) of from 1.5:1 to 8:1, for example from 2:1 to 5:1. The reaction may be carried out at a suitable temperature, for example 0-25 c, for 2-8 hours. In some embodiments, the reaction is performed in an ice bath for 4 hours. In some embodiments, the end point of the reaction may be monitored, for example, by LC-MS. In some embodiments, the compound of formula (X-11) produced by the reaction may be separated using, for example, column chromatography, and the separation conditions may be, for example, gradient elution using ethyl acetate: methanol=100:1-12:1.

The compounds of formula (X-22) may be prepared by any method by a person skilled in the art or are commercially available. For example, the number of the cells to be processed,when L ₁ In the case of carbonylbutylene, the compound of formula (X-22) may be obtained by protecting the hydroxy group by a method known to those skilled in the art from commercially available hydroxyvaleric acid.

The compounds of formula (X-10) may be obtained or prepared by any method known to those skilled in the art. In some embodiments, the compound of formula (X-10) is obtainable by the following preparation method: the method comprises contacting a compound of formula (X-9) with an organic base in an organic solvent to deprotect the amino protecting group G in the compound of formula (X-9) _N The crude product is isolated or used directly in the subsequent reaction.

Wherein L is ₃ 、E、m、R ₂ 、L ₁₁ 、X ₁₁ Is as defined above;

G _N is the amino protecting group described above; in some embodiments, G _N May be, for example, fmoc protecting groups.

The organic solvent can be halogenated alkyl, ether or nitrile solvent; in some embodiments, the organic solvent is acetonitrile. The amount of the organic solvent may be 2 to 15L/mol, for example 3 to 8L/mol, relative to the compound of formula (X-9). The organic base may be, for example, various organic amine bases such as diethylamine, triethylamine or N, N-diisopropylethylamine; the organic base may be used in a molar ratio relative to the compound of formula (X-9) of from 5:1 to 30:1L/mol, for example from 6:1 to 20:1. The deprotection reaction may be carried out at 0-40℃for 1-4h, for example at room temperature for 2h, and/or the reaction endpoint may be determined by monitoring the extent of reaction progress by Thin Layer Chromatography (TLC). In some embodiments, the reaction product may be used directly in the subsequent reaction as a crude product, e.g., after evaporation of the solvent. In some embodiments, the compound of formula (X-10) produced by the reaction may be separated using, for example, column chromatography, and the separation conditions may be, for example, gradient elution using ethyl acetate: methanol=100:1-10:1.

The compounds of formula (X-9) may be obtained or prepared by any method known to those skilled in the art. In some embodiments, the compound of formula (X-9) is obtainable by the following preparation method: the method comprises contacting a compound of formula (X-8) with a compound of formula (X-21) in an organic solvent in the presence of a condensing agent and an organic base to effect a condensation reaction, and isolating.

Wherein L is ₃ 、E、m、R ₂ 、L ₁₁ 、G _N 、X ₁₁ Is defined as before.

In some embodiments, the compound of formula (X-21) is a carboxylic acid, i.e., L ₃ Has a carbonyl group bonded to a hydroxyl group represented by the formula (X-21). At this time, the compound of formula (X-21) may be prepared by a person skilled in the art by various methods using, for example, the compounds disclosed in j.am.chem.soc.2014,136,16958-16961, or, alternatively, certain compounds of formula (X-21) may be prepared by the method disclosed in example 1 of US 8,106,022B2, the entire contents of which are incorporated herein by reference.

The organic solvent may be one or more of acetonitrile, an epoxy-based solvent, an ether-based solvent, an alkyl halide-based solvent, one or more of methylene chloride, chloroform, and 1, 2-dichloroethane, N-dimethylformamide, and N, N-diisopropylethylamine, the epoxy-based solvent in some embodiments being dioxane and/or tetrahydrofuran, the ether-based solvent in some embodiments being diethyl ether and/or methyl tert-butyl ether, the alkyl halide-based solvent in one embodiment being one or more of methylene chloride, chloroform, and 1, 2-dichloroethane, and the organic solvent in some embodiments being N, N-Dimethylformamide (DMF). The amount of the organic solvent may be 3 to 15L/mol, for example 5 to 10L/mol, relative to the compound of formula (X-8). In some embodiments, the compound of formula (X-21) is a carboxylic acid, in which case the condensing agent is an amide forming reaction condensing agent. In some embodiments, the amide forming reaction condensing agent is benzotriazol-1-yl-oxy-tripyrrolidinylphosphine hexafluorophosphate, 3-diethoxyphosphoryl-1, 2, 3-benzozol 4 (3H) -one (DEPBT), O-benzotriazol-tetramethylurea hexafluorophosphate, or 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, in further embodiments 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine Hydrochloride (HATU). The molar ratio of the amide-forming reaction condensing agent to the compound of formula (X-8) is from 1:1 to 5:1, in some embodiments from 1.2:1 to 3:1. The organic base may be a tertiary amine organic base. In some embodiments, the tertiary amine organic base is N-methylmorpholine, triethylamine or N, N-diisopropylethylamine, in some embodiments N, N-Diisopropylethylamine (DIPEA); the molar ratio of the tertiary amine organic base to the compound of formula (X-8) is from 2:1 to 20:1, in some embodiments from 3:1 to 10:1. The reaction may be carried out for 2 to 6 hours under ice bath conditions, for example. The compound of formula (X-9) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, the compound of formula (X-9) may be isolated by water and haloalkane solvent extraction, washing with a salt solvent after combining the organic phases, drying over sulfate, followed by chromatographic methods, e.g., separation may be performed using the following chromatographic conditions: (1) normal phase purification silica gel: 200-300 mesh silica gel packing, gradient elution with ethyl acetate: methanol = 100:1-20:1; or (2) reverse phase purification: c18, C8 reversed phase packing, eluting with methanol: acetonitrile=0.1:1-1:0.1 gradient. In some embodiments, the solvent may be directly removed to provide a crude compound of formula (X-9), which may be directly used in subsequent reactions.

In some embodiments, a compound of formula (X-8) is reacted with a sufficient amount of a compound of formula (X-21) in one step to form the desired compound of formula (X-9), in which case each E-L ₃ The parts are identical to each other. In some embodiments, the compound of formula (X-8) may be prepared by batchwise reacting a compound of formula (X-21), i.e., L, with a different compound of formula (X-21) ₃ And/or E is different, so that the compound of formula (X-9) contains more than two E and/or L ₃ . For example, for 1eq of a compound of formula (X-8), it may be contacted with 2eq of a first compound of formula (X-21), two amino groups in the compound of formula (X-8)To which the first E-L is attached ₃ Part, then, it is contacted with (m-2) eq of a second compound of formula (X-21) (m is defined and ranges of values as described above) to attach a second E-L to (m-2) amino groups in the compound of formula (7) ₃ Part(s).

The compounds of formula (X-8) may be obtained or prepared by any method known to those skilled in the art. In some embodiments, the compound of formula (X-8) is obtainable by the following preparation method: the process comprises contacting a compound of formula (X-7) with a hydroxy protecting agent in an organic solvent in the presence of an organic base to convert the hydroxy group in formula (X-7) to a protected hydroxy-OX ₁₁ And (5) separating.

Wherein m, L ₁₁ 、R ₂ 、G _N Is defined as before.

The organic solvent may be one or more of acetonitrile, an epoxy-based solvent, an ether-based solvent, an alkyl halide-based solvent, one or more of methylene chloride, chloroform, and 1, 2-dichloroethane, N-dimethylformamide, and N, N-diisopropylethylamine, the epoxy-based solvent in some embodiments being dioxane and/or tetrahydrofuran, the ether-based solvent in some embodiments being diethyl ether and/or methyl tert-butyl ether, the alkyl halide-based solvent in one embodiment being one or more of methylene chloride, chloroform, and 1, 2-dichloroethane, and the organic solvent in some embodiments being N, N-Dimethylformamide (DMF). The amount of the organic solvent may be 2 to 15L/mol, for example 3 to 8L/mol, relative to the compound of formula (X-7). In some embodiments, the hydroxyl protecting agent provides a hydroxyl protecting group X ₁₁ In some embodiments may be, for example, t-butyldimethylchlorosilane (TBSCl), in which case X ₁₁ Is tert-butyldimethylsilyl (-TBS). The molar ratio of the hydroxyl protecting agent to the compound of formula (X-7) is from 2:1 to 20:1, in some embodiments from 3:1 to 10:1. The organic base may be a tertiary amine organic base. In some embodiments, the tertiary amine organic base N-methylmorpholine, triethylamine or N, N-diisopropylethylamine, in some embodiments N, N-diisopropylethylamine; the molar ratio of the tertiary amine organic base to the compound of formula (X-7) is from 2:1 to 20:1, in some embodiments from 5:1 to 15:1. The compound of formula (X-8) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, the compound of formula (X-8) may be isolated by extraction with water and haloalkane solvents, combining the organic phases, washing with saline solvents, drying with sulfate, and then separating by chromatographic methods. In some embodiments, the solvent may be directly removed to provide a crude compound of formula (X-8), which may be directly used in subsequent reactions.

The compounds of formula (X-7) may be obtained or prepared by any method known to those skilled in the art. In some embodiments, the compound of formula (X-7) is obtainable by the following preparation method: the process comprises contacting a compound of formula (X-6) with an acid in a solvent to remove the amino protecting group G 'in formula (X-6)' _N And a hydroxyl protecting group X ₁₁ And (5) separating.

/>

Wherein m, L ₁₁ 、R ₂ 、G _N 、X ₁₁ Is defined as before.

G’ _N Is with G _N A second amino protecting group which is more reactive with acid than it is to be removed, e.g. when G _N When Fmoc protecting group, G' _N May be, for example, t-butoxycarbonyl (Boc). The solvent may be one or more of an alcohol solvent, an ether solvent, an ester solvent, and in some embodiments, the organic solvent is an alcohol solvent, such as ethanol. The organic solvent is used in an amount of 1 to 10L/mol, and in some embodiments 1.5 to 5L/mol, relative to the compound represented by the formula (X-6). In some embodiments, the acid may be an organic acid or an inorganic acid, such as hydrochloric acid (HCl). To ensure the reaction is complete, the acid should be in a significant excess and be related to the value of m. In the case where m is 2 to 5, the acid is of the formulaThe molar ratio of the compounds shown in (X-6) may be, for example, from 50:1 to 400:1, and in some embodiments, from 100:1 to 250:1. The reaction may be carried out, for example, at room temperature for 3 to 6 hours as long as the reaction is sufficient, and/or the reaction end point may be monitored using TLC. The compound of formula (X-7) may be isolated from the reaction mixture using any suitable isolation method. In some embodiments, the solvent may be directly removed to provide a crude compound of formula (X-7), which may be directly used in subsequent reactions.

According to the value of m, the compound of the formula (X-6) can be prepared by selecting any reasonable route from the compound of the formula (X-1).

For example, when m is 1, the compound of formula (X-6) (m=1) (hereinafter, also referred to as a compound of formula (X-6-1)) can be obtained by contacting the compound of formula (X-1) with a compound of formula (X-20) in an organic solvent in the presence of a condensing agent to undergo a condensation reaction:

X ₁ -L ₂₀ -OX ₁₁

(X-20)

Wherein m, L ₁₁ 、G _N 、G’ _N 、X ₁₁ Is defined as before.

L ₂₀ To correspond to L ₁₁ A moiety of a group; x is X ₁ Is amino or hydroxy, which is condensed with the carboxyl group of the compound of formula (X-1), together with L ₂₀ Part of which together obtain a linking group L ₁₁ . The organic solvent and the condensing agent may be selected according to amide or ester forming methods well known in the art. For example, in some embodiments, the organic solvent may be methylene chloride, which may be used in an amount of 5 to 20Lmol, e.g., 8 to 15L/mol, relative to the compound of formula (X-1), and the condensing agent may be an organic base such as Diisopropylethylamine (DIPEA), in a molar ratio of 1:1 to 10:1, e.g., 1.5:1 to 5:1, relative to the compound of formula (X-1). The condensation reaction may be carried out, for example, at room temperature for 3 hours, and/or the reaction endpoint monitored by TLC. In some embodimentsThe compound of formula (X-6) can be isolated by column chromatography, for example, using 200-300 mesh normal phase silica gel under normal phase column chromatography conditions, eluting with petroleum ether: ethyl acetate=20:1-1:1 gradient, collecting the eluted product and removing the solvent to obtain the compound of formula (X-6) (m=1).

The compounds of formula (X-1) can be readily prepared by the person skilled in the art according to known preparation methods or are commercially available. For example, when each R ₂ Are all hydrogen, G _N Is Fmoc protecting group, G' _N In the case of Boc protecting groups, the compounds of formula (X-1) are commercially available from Anavia Ji.

In some embodiments, m is an integer greater than or equal to 2. At this time, on the basis of the above reaction, it is required to specifically remove the amino protecting group G in the previous intermediate (for example, in the case of preparing the X-6 compound (m=2) (similarly, also referred to as the compound of formula (X-6-2)) with the aforementioned X-6-1 compound as the "previous intermediate";inthe case of preparing the X-6 compound (m=3) (hereinafter, also referred to as the compound of formula (X-6-3)) with the X-6-2 compound as the "previous intermediate", and so on) _N And subjecting the obtained compound to the aforementioned condensation reaction with a compound of the formula (X-1). The solvents, reagents, conditions and isolation methods of the condensation reaction are the same as or different from those described above, and the reagents, reaction conditions and methods may be appropriately selected depending on the structure of the desired product.

In some embodiments, where m is 3, the compound of formula (X-6) (compound of formula (X-6-3)) may be prepared by:

(i) Preparing a compound of formula (X-6-1) as described previously;

(ii) Contacting a compound of formula (X-6-1) with a base in an organic solvent to remove the amino protecting group G _N Separating; wherein the organic solvent may be, for example, acetonitrile, the base may be an organic base, and further may be an amine-based organic base such as diethylamine or triethylamine; the reaction may be carried out, for example, at room temperature for 2h, followed by separation by, for example, ethyl acetate: methanol=100:1-10:1 (V: V) gradient elution column chromatography, to finally obtain the compound of formula (X-6-1 a);

(iii) Contacting a compound of formula (X-1) with a compound of formula (X-6-1 a) in an organic solvent in the presence of a condensing agent to effect a condensation reaction, and isolating to obtain a compound of formula (X-6-2); wherein the solvent, condensing agent and reaction conditions are selected the same as in step (i) and separated using, for example, ethyl acetate: methanol = 100:1-15:1 gradient elution column chromatography:

(iv) Contacting a compound of formula (X-6-2) with a base in an organic solvent to remove the amino protecting group G _N Isolating to obtain a compound of formula (X-6-2 a); the solvents, reagents and reaction conditions are the same as in step (ii) and are separated using, for example, ethyl acetate: methanol = 100:1-10:1 gradient eluent column chromatography;

(v) Contacting a compound of formula (X-1) with a compound of formula (X-6-2 a) in an organic solvent in the presence of a condensing agent to effect a condensation reaction, and isolating to obtain a compound of formula (X-6-3); wherein the solvent, condensing agent and reaction conditions are selected the same as in step (i) and separated using, for example, ethyl acetate: methanol = 100:1-8:1 gradient elution column chromatography:

oligonucleotide conjugates

In one aspect, the present disclosure provides an oligonucleotide conjugate having a structure represented by formula (II):

E ₁ is OH, SH or BH ₂ ；

Nu represents a functional oligonucleotide;

m、L ₁ 、L ₃ 、R ₂ the definition and optional ranges of (a) are the same as those described above;

M ₁ represents a ligand having an affinity for asialoglycoprotein receptors on the surface of mammalian liver cells, said ligand being defined and optionally ranging as in E above;

E ₂ represents amino, hydroxy, -amino (C) ₁ -C ₁₀ Hydrocarbylene) amino, -amino (C) ₁ -C ₁₀ Hydrocarbylene) hydroxy, -hydroxy (C) ₁ -C ₁₀ Hydrocarbylene) amino or hydroxy (C ₁ -C ₁₀ Hydrocarbylene) hydroxy, the hydrocarbylene optionally having one or more substituents selected from the group consisting of: c (C) ₁ -C ₁₀ Alkyl, C ₆ -C ₁₀ Aryl, C ₅ -C ₁₀ Heteroaryl, C ₁ -C ₁₀ Haloalkyl, -OC ₁ -C ₁₀ Alkyl, -OC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-OH, -OC ₁ -C ₁₀ Haloalkane, -SC ₁ -C ₁₀ Alkyl, -SC ₁ -C ₁₀ Alkylphenyl radicals C ₁ -C ₁₀ alkyl-SH, -SC ₁ -C ₁₀ Haloalkyl, halogen substituent, -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), 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 A haloalkyl group); in some embodiments, E ₂ Represents-amino (C) ₁ -C ₁₀ Alkylene) hydroxy; in some embodiments, E ₂ Is hydroxyethylamino (-NHCH) ₂ CH ₂ OH)。

By forming an oligonucleotide conjugate of formula (II) wherein the functional oligonucleotide is covalently linked to one or more M's having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells ₁ Ligands, which are thus readily enriched to the surface of hepatocytes and further into hepatocytes, to achieve specific targeted delivery of functional oligonucleotides.

In the present disclosure, preferably oligonucleotide conjugates of formula (II), wherein

E ₁ OH, m is an integer of 2 to 4;

each R ₂ Independently H, methyl or ethyl; or each R ₂ All are H;

L ₁ is carbonyl (C) ₂ -C ₆ Alkylene) or carbonyl (oxyethyl) _k Wherein k is an integer of 2 to 7;

E ₂ represents amino or-amino (C) ₂ -C ₆ Alkylene) hydroxy; and/or

Nu、M ₁ 、L ₃ The definition and optional ranges of (a) are the same as those described above.

In a specific embodiment, the oligonucleotide conjugate of the present invention has a structure represented by formula (II-1), (II-2) or (II-3):

/>

wherein Nu represents a functional oligonucleotide.

In the context of the present disclosure, unless otherwise indicated, "conjugated" means that two or more chemical moieties each having a particular function are linked to each other by covalent linkage; accordingly, "conjugate" refers to a compound formed by covalent linkage between the chemical moieties. Further, "oligonucleotide conjugate" refers to a compound formed by covalently attaching one or more chemical moieties having a specific function to an oligonucleotide. Hereinafter, the oligonucleotide conjugates of the present disclosure are also sometimes referred to simply as "conjugates". More specifically, in the context of the present disclosure, a "conjugate molecule" is understood to be a specific compound that can be conjugated to an oligonucleotide by reaction, ultimately forming an oligonucleotide conjugate of the present disclosure. In some embodiments, the oligonucleotide is an siRNA, in which case the conjugate of the present disclosure is an siRNA conjugate. More specifically, for purposes of this disclosure, a "conjugate molecule" may refer to a compound represented by formula (I), and correspondingly, an "oligonucleotide conjugate" may refer to a compound represented by formula (II).

In some embodiments, the oligonucleotides in the oligonucleotide conjugates of the present disclosure are functional oligonucleotides. Functional oligonucleotides refer to such oligonucleotides: the oligonucleotides are capable of up-regulating or down-regulating the expression of a target gene or causing alternative splicing of mRNA by creating stable and specific hybridization with the target sequence using principles such as RNA activation (RNAa), RNA interference (RNAi), antisense nucleic acid technology, exon skipping (exon skip) technology, etc. In some aspects, a functional oligonucleotide may also be a nucleic acid structure that produces stable and specific binding to a target protein. Furthermore, it will be readily appreciated by those skilled in the art that polynucleotides (e.g., mRNA itself or fragments thereof) are equally suitable for conjugation with the conjugate molecules provided by the present disclosure to form conjugates for targeted delivery, such as liver targeted delivery, to modulate expression of proteins transcribed from mRNA. Thus, in this context, the concept of "functional oligonucleotide" may also encompass mRNA or fragments thereof.

In some embodiments, the functional oligonucleotide is capable of interacting with the target sequence, thereby affecting the normal function of the target sequence molecule, such as causing mRNA cleavage or translational repression or exon skipping to initiate alternative splicing of the mRNA, etc. In some embodiments, the functional oligonucleotide may be substantially complementary to a base of the target sequence. In some embodiments, the functional oligonucleotide may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more base complementary to the target sequence, or fully complementary to the target sequence. In some embodiments, the functional oligonucleotide may contain 1, 2, or 3 bases that are not complementary to the target sequence. In some embodiments, the functional oligonucleotides include deoxyribonucleotides or ribonucleotides, as well as nucleotides with modifications. In some embodiments, the functional oligonucleotide may be single-stranded DNA, RNA, or DNA-RNA chimeras (chimers), or double-stranded DNA, RNA, or DNA-RNA hybrids.

Thus, in some embodiments, a functional oligonucleotide suitable for inclusion in an oligonucleotide conjugate of the present disclosure may be one of a small interfering RNA (siRNA), a microRNA (microRNA), an anti-microRNA (anti-micror), a microRNA antagonist (anti-microRNA), a microRNA mimetic (microRNA), a decoy oligonucleotide (decoy), an immunostimulatory substance (immune stimulatory), a G-quadrupole (G-quadraplex), an alternative splice (splice), a single-stranded RNA (ssRNA), an antisense nucleic acid (antisense), a nucleic acid aptamer (Nucleic Acid Aptamer), a small activating RNA (small activating RNA, saRNA), a stem-loop RNA (stem-loop RNA), or DNA. WO2015/006740A2 discloses a conjugate of a different ligand conjugated to an oligonucleotide, wherein the ligand is linked to the oligonucleotide by a linker (linker) selected from one of small interfering RNAs (siRNA), micrornas (micrornas), anti-micrornas (anti-micrornas), microRNA antagonists (anti-micrornas), microRNA mimics (microRNA mimics), decoy oligonucleotides (decoy), immunostimulants (immune stimulatory), G-quadrupoles (G-quales), alternative spliceosomes (splice), single-stranded RNAs (ssrnas), antisense nucleic acids (anti-sense), aptamers (stem-loop RNAs) or DNA. These conjugates exhibit good stability in the in vivo delivery of oligonucleotides. In a further embodiment, the functional oligonucleotides suitable for inclusion in the oligonucleotide conjugates of the present disclosure may be the oligonucleotides disclosed in WO2009082607A2, WO2009073809A2 or WO2015006740A2, the entire contents of which are incorporated herein by reference.

The oligonucleotide conjugates of the present disclosure can modulate abnormal expression of a particular gene in a particular cell, such as a hepatocyte, by increasing the liver targeted delivery efficiency of an active agent, such as a functional oligonucleotide, thereby enhancing the interaction between the functional oligonucleotide and the targeting sequence in the cell. In some embodiments, the specific gene may be an endogenous gene expressed in the liver, or may be a pathogen gene propagated in the liver. The genes that are abnormally expressed in hepatocytes may be, for example, apoB, apoC, ANGPTL3, PCSK9, SCD1, TIMP-1, col1A1, FVII, STAT3, p53, HBV, HCV, etc. In some embodiments, the gene that is abnormally expressed in hepatocytes is an HBV gene, an ANGPTL3 gene, or an APOC3 gene. In the context of the present disclosure, HBV gene refers to a gene whose sequence is shown as Genbank accession number nc_ 003977.1; ANGPTL3 gene refers to an mRNA sequence such as that shown in Genbank accession No. nm_ 014495.3; the APOC3 gene refers to a gene whose mRNA sequence is shown in Genbank accession No. nm_ 000040.1.

In some embodiments, a "target sequence" is a target mRNA. In the context of the present disclosure, "target mRNA" refers to mRNA corresponding to a gene that is abnormally expressed in hepatocytes, either as mRNA corresponding to an overexpressed gene or as mRNA corresponding to an underexpressed gene. Since most diseases result from overexpression of mRNA, in the present disclosure, target mRNA refers in particular to mRNA corresponding to the overexpressed gene. In some embodiments of the present disclosure, the target mRNA may be mRNA corresponding to the genes abnormally expressed as described above, apoB, apoC, ANGPTL, PCSK9, SCD1, TIMP-1, col1A1, FVII, STAT3, p53, HBV, HCV, etc. In some embodiments, the target mRNA may be mRNA transcribed from the corresponding HBV gene, or mRNA corresponding to the ANGPTL3 gene, or mRNA corresponding to the APOC3 gene.

The P (phosphorus atom) in formula (II) may be attached to any possible position in the oligonucleotide sequence, for example, to any one of the nucleotides of the oligonucleotide. In some embodiments, the linkage includes, but is not limited to, the P being linked to the nucleotide by formation of a phosphodiester linkage (sometimes also referred to simply as a phosphoester linkage). In some embodiments, the functional oligonucleotide in the oligonucleotide conjugates of the present disclosure is a single stranded oligonucleotide (e.g., a single stranded RNA or an aptamer), at which point P in formula (II) may be attached to the end of the single stranded oligonucleotide, which refers to the first 4 nucleotides of the single stranded oligonucleotide from one end. In some embodiments, P in formula (II) is attached to the end of the single stranded oligonucleotide.

The P in formula (II) may be attached to any possible position on the nucleotide in the oligonucleotide sequence, 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 (II) may be attached to the 2', 3', or 5' position of a nucleotide in the oligonucleotide sequence by formation of a phosphodiester bond. In some embodiments, P in formula (II) is attached to an oxygen atom formed after dehydrogenation of the 3' hydroxyl group of the 3' terminal nucleotide of the sense strand in the double-stranded oligonucleotide sequence (in which case the P and corresponding phosphate groups can be considered as being attributed to P and phosphate groups in the double-stranded oligonucleotide), or P in formula (II) is attached to the nucleotide by substitution of hydrogen in the 2' -hydroxyl group of one nucleotide in the sense strand in the double-stranded oligonucleotide sequence, or P in formula (II) is attached to the nucleotide by substitution of hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide of the sense strand in the double-stranded oligonucleotide sequence.

Without wishing to be limited, in the following embodiments and examples, the case where the functional oligonucleotides in the oligonucleotide conjugates of the present disclosure are small interfering RNAs (sirnas) is described in detail. At this time, the oligonucleotide conjugate of the present disclosure is an siRNA conjugate. In the context of this document, for convenience of description, the siRNA conjugates in these embodiments are also referred to as the siRNA conjugates of the present disclosure. This does not represent that the oligonucleotides in the oligonucleotide conjugates of the present disclosure may only be siRNA, but rather that the oligonucleotides may be other alternative drugs of the present disclosure or well known to those skilled in the art. From the detailed description of siRNA conjugates, it is contemplated that other functional oligonucleotides will also have similar effects when conjugated to the conjugate molecules provided by the present disclosure.

It is well known to those skilled in the art that siRNA contains a nucleotide group as a basic structural unit, the nucleotide group containing a phosphate group, a ribose group, and a base. Typically active, i.e., functional, siRNAs are about 12-40 nucleotides in length, in some embodiments about 15-30 nucleotides in length, each of which can independently be a modified or unmodified nucleotide, and at least one of which is a modified nucleotide for increased stability.

The inventors of the present disclosure found that the sirnas described in the following embodiments have higher activity and/or stability, and thus can be the object of the invention of the sirnas in the present disclosure.

In some embodiments, each nucleotide in an siRNA conjugate of the present disclosure (hereinafter also referred to as an siRNA of the present disclosure) is independently a modified or unmodified nucleotide, the siRNA comprising a sense strand and an antisense strand, wherein the sense strand comprises nucleotide sequence 1 and the antisense strand comprises nucleotide sequence 2, the nucleotide sequence 1 and the nucleotide sequence 2 are each 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides in length, and are at least partially reverse-complementary to form a complementary duplex region, at least a portion of the nucleotide sequence 2 is complementary to a first stretch of nucleotide sequence that is a stretch of nucleotide sequence in a target mRNA.

In some embodiments, the siRNA of the present disclosure refers to an siRNA capable of inhibiting at least 50% hepatitis b virus gene expression, at least 50% angiopoietin-like protein 3 gene expression, or at least 50% apolipoprotein C3 gene expression at a concentration of 3 mg/kg. In some embodiments, the siRNA of the present disclosure refers to an siRNA capable of inhibiting at least 55%, 60%, 65%, 70%, 75% or 80% hbv gene expression at a concentration of 3 mg/kg.

In some embodiments, the nucleotide sequence 1 is equal in length to the first stretch of nucleotide sequences and does not differ by more than 3 nucleotides; the nucleotide sequence 2 is equal to the nucleotide sequence B in length and does not differ by more than 3 nucleotides; the nucleotide sequence B is a nucleotide sequence which is completely reverse complementary to the first nucleotide sequence. Without wishing to be limited, these specific nucleotide differences do not significantly reduce the target gene inhibition capacity of the siRNA conjugates, and these siRNA conjugates comprising the specific nucleotide differences are also within the scope of the present disclosure.

In some embodiments, the nucleotide sequence 1 and the nucleotide sequence 2 are substantially reverse complementary, substantially fully reverse complementary, or fully reverse complementary.

In some embodiments, the nucleotide sequence 1 differs from the first stretch of nucleotides by no more than 1 nucleotide, and/or the nucleotide sequence 2 differs from the nucleotide sequence B by no more than 1 nucleotide. In some embodiments, the nucleotide difference between the nucleotide sequence 2 and the nucleotide sequence B comprises a difference in the Z ' position of the first nucleotide on the nucleotide sequence 2 in a 5' end to 3' end direction. In some embodiments, the last nucleotide Z on the nucleotide sequence 1 is a nucleotide complementary to Z ' in a 5' end to 3' end orientation.

In some embodiments, the sense strand further comprises nucleotide sequence 3, the antisense strand further comprises nucleotide sequence 4, the nucleotide sequence 3 and the nucleotide sequence 4 are equal in length and each is 1-4 nucleotides, the nucleotide sequence 3 is linked at the 5 'end of the nucleotide sequence 1, and the nucleotide sequence 4 is linked at the 3' end of the nucleotide sequence 2, the nucleotide sequence 4 is complementary to a second nucleotide sequence that is adjacent to the first nucleotide sequence and is identical in length to the nucleotide sequence 4 in the target mRNA. In some embodiments, the nucleotide sequence 3 and the nucleotide sequence 4 are substantially fully reverse complementary or fully reverse complementary. Thus, the sense and antisense strands may be 19-23 nucleotides in length.

In some embodiments, the siRNA of the present disclosure further comprises a nucleotide sequence 5, the nucleotide sequence 5 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; in some embodiments, the nucleotide sequence 5 is 1 or 2 nucleotides in length. Thus, in some embodiments, the ratio of the lengths of the sense strand and the antisense strand of the siRNAs of the present disclosure can be 19/20, 19/21, 20/22, 21/23, 22/24, 23/24, or 23/25.

In one embodiment, the nucleotide sequence 5 is 2 nucleotides in length and in the 5 'to 3' end direction, the nucleotide sequence 5 is a contiguous 2 deoxythymidine nucleotides, a contiguous 2 uracil nucleotides, or is complementary to a third nucleotide sequence that is adjacent to the first nucleotide sequence or adjacent to the second nucleotide sequence in the target mRNA and is equal in length to the nucleotide sequence 5. In one embodiment, the ratio of the length of the sense strand to the length of the antisense strand of the siRNA of the present disclosure is 19/21 or 21/23, at which time the siRNA of the present disclosure has better hepatocyte mRNA silencing activity.

In some embodiments, the nucleotides in the siRNA of the present disclosure are each independently modified or unmodified nucleotides. In some embodiments, the siRNA of the present disclosure does not contain modified nucleotide groups; in some embodiments, the siRNA of the present disclosure contains modified nucleotide groups.

Currently, there are a variety of ways available in the art for modifying siRNA, including backbone modifications (also known as internucleotide linkage modifications, such as phosphate group modifications), ribose group modifications, and base modifications, among others (see, e.g., watts, J.K., G.F.Deleavey and M.J.damha, chemically modified siRNA: tools and applications. Drug discovery Today,2008.13 (19-20): p.842-55, the entire contents of which are incorporated herein by reference).

In the context of the present disclosure, the term "modified nucleotide" is used to refer to a nucleotide or nucleotide analogue in which the ribosyl group of the nucleotide is modified, such as where the 2' -hydroxyl group is replaced with another group, or where the base on the nucleotide is a modified base.

In some embodiments of the disclosure, at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and/or at least one phosphate group is a phosphate group having a modification group, in other words, at least a portion of the phosphate groups and/or ribose groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand are phosphate groups having a modification group and/or ribose groups having a modification group (or modified phosphate groups and/or modified ribose groups). In some embodiments of the disclosure, all of the nucleotides in the sense strand and/or the antisense strand are modified nucleotides.

In some embodiments, each nucleotide in the sense strand and the antisense strand is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

The fluoro-modified 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 has a structure represented by the following formula (207).

Non-fluoro modified nucleotide refers to a nucleotide or nucleotide analogue formed by substituting the hydroxyl group at the 2' -position of the ribosyl of the nucleotide with a non-fluoro group. In some embodiments, each non-fluoro modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by substitution of the hydroxyl group at the 2' position of the ribosyl of the nucleotide with a non-fluoro group.

Nucleotides in which the hydroxyl group at the 2 '-position of the ribosyl group is substituted with a non-fluorine group are well known to those skilled in the art and may be selected from one of 2' -alkoxy-modified nucleotides, 2 '-substituted alkoxy-modified nucleotides, 2' -alkyl-modified nucleotides, 2 '-substituted alkyl-modified nucleotides, 2' -amino-modified nucleotides, 2 '-substituted amino-modified nucleotides, 2' -deoxynucleotides.

In some embodiments, the 2 '-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2' -OMe), as shown in formula (208). The 2' -substituted alkoxy-modified nucleotide may be, for example, a 2' -O-methoxyethyl-modified nucleotide (2 ' -MOE), as shown in formula (209). In some embodiments, 2 '-amino modified nucleotides (2' -NH) ₂ ) As shown in equation (210). In some embodiments, the 2' -Deoxynucleotide (DNA) is as shown in formula (211).

Nucleotide analogs refer to groups that are capable of replacing nucleotides in a nucleic acid, but that differ in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine. In some embodiments, the nucleotide analog may be, for example, an isonucleotide, a bridged nucleic acid (bridged nucleic acid, abbreviated BNA) nucleotide, or an acyclic nucleotide.

BNA nucleotides refer to constrained or inaccessible nucleotides. BNA may contain a five-, six-, or seven-membered ring bridging structure with "fixed" C3' -endo-saccharides tucked. The bridge is typically incorporated at the 2'-, 4' -position of the ribose ring to provide a 2',4' -BNA nucleotide, such as LNA, ENA, cret BNA, etc., where LNA is shown as formula (212), ENA is shown as formula (213), cret BNA is shown as formula (214).

Acyclic nucleotides are a class of nucleotides in which the sugar ring of the nucleotide is opened, such as Unlocking Nucleic Acid (UNA) nucleotides or Glycerol Nucleic Acid (GNA) nucleotides, where UNA is represented by formula (215) and GNA is represented by formula (216).

Wherein R is selected from H, OH or alkoxy (O-alkyl).

An isopucleotide refers to a compound in which the position of a base on the ribose ring is changed in a nucleotide, for example, a compound in which the base is shifted from the 1' -position to the 2' -position or the 3' -position of the ribose ring, as shown in formula (217) or (218).

Wherein Base represents a Base, such as 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 an iso-nucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluoro modified nucleotide is a methoxy modified nucleotide, which refers to a nucleotide formed by substitution of the 2' -hydroxy group of the ribosyl group with a methoxy group.

In the above and in the following, the meaning of "fluoro modified nucleotide", "2 '-fluoro modified nucleotide", "nucleotide in which the 2' -hydroxyl group of the ribose group is replaced with fluorine" and "2 '-fluoro ribose group" are the same, and refer to a compound having a structure as shown in formula (207) formed by replacing the 2' -hydroxyl group of the nucleotide with fluorine; "methoxy modified nucleotide", "2 '-methoxy modified nucleotide", "nucleotide in which the 2' -hydroxy group of the ribose group is replaced by methoxy" and "2 '-methoxyribosyl" are the same meaning, and each refers to a nucleotide in which the 2' -hydroxy group of the ribose group is replaced by methoxy to form a structure shown in formula (208).

In some embodiments, the siRNA of the present disclosure is an siRNA with modifications of: both the sense strand and the antisense strand comprise fluoro-modified nucleotides and non-fluoro-modified nucleotides, the fluoro-modified nucleotides being located in the aforementioned nucleotide sequence 1 and nucleotide sequence 2, the number of fluoro-modified nucleotides in the nucleotide sequence 1 being not more than 5, and the nucleotides at positions 7, 8, 9 of the nucleotide sequence 1 being fluoro-modified nucleotides in the direction from the 5 'end to the 3' end; the number of the fluoro-modified nucleotides in the nucleotide sequence 2 is not more than 7, and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence 2 are fluoro-modified nucleotides according to the direction from the 5 'end to the 3' end. In some embodiments, the siRNA of the present disclosure is an siRNA with modifications of: the nucleotides at positions 7, 8 and 9 of the nucleotide sequence 1 in the sense strand of the siRNA are-fluoro modified nucleotides according to the direction from the 5 'end to the 3' end, and the nucleotides at the rest positions in the sense strand are methoxy modified nucleotides; in the antisense strand, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence 2 are fluoro modified nucleotides, and the nucleotides at the rest positions in the antisense strand are methoxy modified nucleotides; in some embodiments, the siRNA of the present disclosure is an siRNA with modifications of: or according to the direction from the 5 'end to the 3' end, the nucleotides at the 5 th, 7 th, 8 th and 9 th positions of the nucleotide sequence 1 in the sense strand of the siRNA are fluoro modified nucleotides, and the nucleotides at the rest positions in the sense strand are methoxy modified nucleotides; in the antisense strand, the nucleotides at positions 2, 6, 8, 9, 14 and 16 of the nucleotide sequence 2 are fluoro-modified nucleotides, and the nucleotides at the rest positions in the antisense strand are methoxy-modified nucleotides; in some embodiments, the siRNA of the present disclosure is an siRNA with modifications of: the nucleotides at positions 7, 8 and 9 of the nucleotide sequence 1 in the sense strand of the siRNA are fluoro-modified nucleotides, the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides, and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence 2 in the antisense strand of the siRNA are fluoro-modified nucleotides, in the 5 'to 3' direction.

In some embodiments of the siRNA described in the present disclosure, the nucleotide contains a phosphate group modification. In the context of the present disclosure, the phosphate group modification is in one embodiment a phosphorothioate modification as shown in formula (201) below, i.e., substitution of one sulfur atom for a non-bridging oxygen atom in the phosphodiester linkage, thereby replacing the phosphodiester linkage with a phosphorothioate linkage. In some embodiments, the modification stabilizes the structure of the siRNA, maintaining high specificity and high affinity for base pairing.

According to some embodiments of the disclosure, in the siRNA, the phosphorothioate linkage is present at least one of the group consisting of: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above. In some embodiments, phosphorothioate linkages are present at all of the above positions except the 5' end of the sense strand. In some embodiments, phosphorothioate linkages are present at all of the above positions except the 3' end of the sense strand. In some embodiments, the phosphorothioate linkage is present in at least one of the following positions:

A linkage between nucleotide 1 and nucleotide 2 of the 5' terminal end of the sense strand;

a linkage between nucleotide 2 and nucleotide 3 of the 5' terminal end of the sense strand;

a linkage between nucleotide 1 and nucleotide 2 of the 3' -terminal end of the sense strand;

a linkage between nucleotide 2 and nucleotide 3 of the 3' -terminal end of the sense strand;

a linkage between nucleotide 1 and nucleotide 2 of the 5' terminal end of the antisense strand;

a linkage between nucleotide 2 and nucleotide 3 of the 5' terminal end of the antisense strand;

a linkage between nucleotide 1 and nucleotide 2 of the 3' -terminal end of the antisense strand; and

the 3' -terminal end of the antisense strand is linked between nucleotide 2 and nucleotide 3.

According to some embodiments of the disclosure, the 5' -terminal nucleotide of the antisense strand sequence of the siRNA molecule is a 5' -phosphonucleotide or a 5' -phosphoanalog modified nucleotide.

In some embodiments, the 5' -phosphate nucleotide may have a structure represented by formula (202):

meanwhile, the kinds of commonly used 5' -phosphate analogue-modified nucleotides are well known to those skilled in the art, for example, anastasia Khvorova and Jonathan K.Watts, the chemical evolution of oligonucleotide therapies of clinical availability.Nature Biotechnology,2017,35 (3): 4 nucleotides as shown in the following formulas (203) to (206) are disclosed in 238-48:

Wherein R represents a group selected from the group consisting of H, OH, F, and methoxy;

base represents a Base selected from A, U, C, G or T.

In some embodiments, the 5 '-phosphonucleotide or 5' -phosphoanalog modified nucleotide is a vinyl phosphate (E-vinylphosphonate, E-VP) containing nucleotide represented by formula (203), a 5 '-phospho modified nucleotide represented by formula (202), or a 5' -phosphorothioate modified nucleotide represented by formula (205).

The inventors of the present disclosure unexpectedly found that the siRNA conjugates of the present disclosure, while having significantly improved serum stability, also exhibit target mRNA silencing activity that is not significantly reduced and excellent gene expression inhibition effect; thus, the siRNA conjugates of the present disclosure are shown to have higher in vivo delivery efficiency. According to some embodiments of the present disclosure, the oligonucleotide conjugates of the present disclosure are siRNA conjugates comprising, for example, the sirnas shown in tables 1A-1F:

TABLE 1 siRNA sequences in some embodiments

TABLE 1A

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TABLE 1B

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TABLE 1C

TABLE 1D

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TABLE 1E

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TABLE 1F

* S: a sense strand; AS: antisense strand

Wherein capital C, G, U, A indicates the base composition of the nucleotide; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a 2' -methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a 2' -fluoro modified nucleotide; the lower case letter s indicates that the linkage between two nucleotides adjacent to the letter s is a phosphorothioate linkage; p1 represents that the one nucleotide adjacent to the right of P1 is a 5' -phosphonucleotide or a 5' -phosphoanalog modified nucleotide, in some embodiments a vinyl phosphate modified nucleotide (denoted as VP in the examples below), a 5' -phospho modified nucleotide (denoted as P in the examples below), or a phosphorothioate modified nucleotide (denoted as Ps in the examples below).

It will be apparent to those skilled in the art that the methods of preparing nucleoside monomers having corresponding modifications and methods of introducing modified nucleotide groups into siRNA described in the present disclosure can be incorporated by using nucleoside monomers having corresponding modifications as well as methods of introducing modified nucleotide groups into siRNA are well known to those skilled in the art. All modified nucleoside monomers are commercially available or can be prepared using known methods.

Preparation of oligonucleotide conjugates

Any reasonable synthetic route can be used to prepare the oligonucleotide conjugates of the present disclosure.

For example, a method of preparing an oligonucleotide conjugate of the present disclosure may comprise:

(a) Reacting a compound of formula (I) with succinic acid or succinic anhydride to obtain an intermediate of formula (II-M1);

(b) Removing the protecting group on the solid carrier with the protected amino group, and connecting the intermediate of the formula (II-M1) obtained in the step (a) to the solid carrier under the coupling reaction condition and in the presence of a coupling reagent;

(c) Under the condition of the solid phase phosphoramidite solid phase synthesis method of nucleic acid, respectively according to the nucleotide types and sequences of the functional oligonucleotides, sequentially connecting nucleoside monomers according to the 3 'to 5' direction, wherein the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfuration.

As the solid phase carrier, a polymer carrier having a hydroxyl group or an amino group on the surface thereof, for example, commercially available Wang's resin, an Aminomethyl (AM) resin, a benzyl alcohol resin, etc. can be used.

The deprotection, coupling, capping, oxidation, sulfidation, or borohydride reaction may employ the same conditions and reagents as in the solid phase phosphoramidite solid phase synthesis of nucleic acids, the specific reaction conditions and reagents being described in detail below.

In some embodiments, the method further comprises a step of removing the protecting group and cleaving from the solid support, a separation and purification step.

In the context of the present invention, the term "carbonyl- (oxyethyl) _k "means k-O-CH ₂ -CH ₂ -the unit is attached to the carbonyl group, k is an integer from 2 to 7, preferably from 2 to 5, more preferably from 2 to 4, most preferably from 2 to 3; examples include, but are not limited to, carbonyldi (oxyethyl), carbonyltri (oxyethyl), etc.

In the present invention, amino group (ethoxy group) _q2 Represents q 2-CH ₂ -CH ₂ The O-unit is attached to the amino group, q2 is an integer from 1 to 10, in some embodiments from 2 to 7, preferably from 2 to 5, more preferably from 2 to 4, most preferably 2 or 3; examples include, but are not limited to, aminodi (ethoxy), aminotri (ethoxy), and the like.

In some embodiments, the oligonucleotide is a double-stranded oligonucleotide, and the method of making comprises the steps of: contacting the intermediate of formula (II-M1) attached to the solid support with a first nucleoside monomer at the 3' end of the sense strand or antisense strand in the presence of coupling reaction conditions and a coupling reagent to ligate the first nucleotide in the sequence to the intermediate of formula (II-M1) attached to the solid support, and ligating the nucleoside monomers in sequence in a 3' to 5' direction to synthesize the sense strand or antisense strand of the double-stranded oligonucleotide; wherein the intermediate of formula (II-M1) attached to the solid support is deprotected prior to attachment to the first nucleoside monomer; the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or vulcanization reaction; obtaining a sense strand or an antisense strand of the nucleic acid to which the conjugate molecule is attached; sequentially connecting nucleoside monomers in a 3 'to 5' direction to synthesize the other strand of the double-stranded oligonucleotide, wherein the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or vulcanization; removing protecting group, cutting with solid phase carrier, separating and purifying to obtain the sense strand and antisense strand of nucleic acid, and annealing.

In some embodiments, the oligonucleotide is a double-stranded oligonucleotide, and the method of making comprises the steps of: sequentially connecting nucleoside monomers in the 3 'to 5' direction to synthesize a sense strand and an antisense strand, wherein the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or vulcanization reaction to obtain the sense strand connected to the solid phase carrier and the antisense strand connected to the solid phase carrier; contacting a compound represented by the formula (II-M1) with a sense strand attached to a solid support or an antisense strand attached to a solid support in the presence of a coupling reagent and coupling reaction conditions, thereby attaching the compound of the formula (II-M1) to the sense strand or the antisense strand; removing protecting groups, cutting with a solid phase carrier, separating and purifying to obtain a sense strand or an antisense strand of the oligonucleotide, and annealing, wherein the sense strand or the antisense strand of the oligonucleotide is connected with a conjugated molecule.

In some embodiments, a compound of formula (II) is attached to the 3' end of the sense strand in an siRNA, and a method of making an siRNA conjugate of the disclosure comprises:

(1) Removing the hydroxyl protecting group G from the solid support-attached compound of formula (II-M1) (hereinafter also referred to as solid support-attached L conjugate molecule); contacting the L conjugated molecule connected with the solid phase carrier with a nucleoside monomer under the condition of coupling reaction and in the presence of a coupling reagent to obtain the nucleoside monomer connected with the solid phase carrier through the L conjugated molecule;

(2) Synthesizing the sense strand of the siRNA by a phosphoramidite solid phase synthesis method according to the 3'-5' direction starting from the nucleoside monomer attached to the solid phase carrier through the L-conjugated molecule;

(3) Synthesizing antisense strand of siRNA through phosphoramidite solid phase synthesis method;

(4) The sense and antisense strands of the siRNA were isolated and annealed to obtain the siRNA conjugates of the present disclosure.

Wherein, in step (1), the method for removing the protecting group G in the L conjugated molecule attached to the solid support comprises contacting the compound of formula (II-M1) attached to the solid support with a deprotection reagent under deprotection conditions. Deprotection conditions include a temperature of 0 to 50 ℃, in some embodiments 15 to 35 ℃, 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, in some embodiments dichloroacetic acid. The molar ratio of deprotecting reagent to compound of formula (II-M1) is from 2:1 to 100:1, in one embodiment from 3:1 to 50:1, based on the amount of substance of compound of formula (II-M1).

The coupling reaction conditions and coupling reagents may use any suitable conditions and reagents for the coupling reactions described above. In some embodiments, the same conditions and reagents are used as the coupling reaction in the solid phase synthesis method employed.

In some embodiments, the conditions of the coupling reaction include a reaction temperature of 0 to 50 ℃, in some embodiments 15 to 35 ℃. The molar ratio of the compound of formula (II-M1) to nucleoside monomer is from 1:1 to 1:50, in some embodiments from 1:2 to 1:5, calculated as the amount of substance of the compound of formula (II-M1); the molar ratio of the compound of formula (II-M1) to the coupling agent is from 1:1 to 1:50, in some embodiments from 1:3 to 1:10, with a reaction time of from 200 to 3000 seconds, in some embodiments from 500 to 1500 seconds, calculated on the amount of material of the compound of formula (II-M1). The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole, and in some embodiments 5-ethylthio 1H-tetrazole. The coupling reaction may be carried out in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, anhydrous dichloromethane, in some embodiments, anhydrous acetonitrile. The organic solvent is used in an amount of 3 to 50L/mol, in some embodiments 5 to 20L/mol, relative to the amount of the substance of the compound of formula (II-M1).

In step (2), the sense strand S of the siRNA conjugate is synthesized in the 3'-5' direction by the method of solid phase synthesis of phosphoramidite nucleic acid, starting with the nucleoside monomer attached to the solid support via the L-conjugated molecule prepared in the above step. At this time, the L conjugate molecule is attached to the 3' end of the resulting sense strand.

Other conditions for the solid phase synthesis described in steps (2) and (3) include deprotection conditions for nucleoside monomers, types and amounts of deprotection reagents, coupling reaction conditions, types and amounts of coupling reagents, conditions for capping reactions, types and amounts of capping reagents, oxidation reaction conditions, types and amounts of oxidizing reagents, sulfidation reaction conditions, sulfidation reagents and amounts using various reagents, amounts and conditions conventionally used in the art.

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

the nucleoside monomer deprotection conditions include a temperature of from 0 to 50 ℃, in some embodiments from 15 to 35 ℃, for a reaction time of from 30 to 300 seconds, in some embodiments from 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 dichloroacetic acid in some embodiments. The molar ratio of deprotection reagent to 4,4' -dimethoxytrityl protecting group on the solid support is from 2:1 to 100:1, and in some embodiments from 3:1 to 50:1.

Coupling reaction conditions include a temperature of 0 to 50 ℃, in some embodiments 15 to 35 ℃, and a molar ratio of nucleic acid sequence to nucleoside monomer attached to the solid support of 1:1 to 1:50, in some embodiments 1:5 to 1:15; the molar ratio of nucleic acid sequence to coupling reagent attached to the solid support is 1:1 to 1:100, in some embodiments 1:50 to 1:80, and the reaction time and coupling reagent selection is the same as described above.

The capping reaction conditions include a temperature of 0-50 ℃, in some embodiments 15-35 ℃, a reaction time of 5-500 seconds, in some embodiments 10-100 seconds, and the capping reagent is selected as described above. The molar ratio of the total amount of capping reagent to the nucleic acid sequence attached to the solid support is from 1:100 to 100:1, in some embodiments from 1:10 to 10:1. Where equimolar amounts of acetic anhydride to N-methylimidazole are used for the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole, and nucleic acid sequences attached to the solid support is 1:1:10 to 10:10:1, in some embodiments 1:1:2 to 2:2:1.

The oxidation reaction conditions include a temperature of 0-50 ℃, in some embodiments 15-35 ℃, a reaction time of 1-100 seconds, in some embodiments 5-50 seconds, and an oxidizing agent, in some embodiments iodine (in further embodiments provided in the form of iodine water). The molar ratio of oxidizing reagent to nucleic acid sequence attached to the solid support during the coupling step is 1:1 to 100:1, in some embodiments 5:1 to 50:1. In some embodiments, the oxidation reaction is performed in a mixed solvent of tetrahydrofuran: water: pyridine=3:1:1 to 1:1:3. The sulfiding reaction conditions include a temperature of 0-50 ℃, in some embodiments 15-35 ℃, a reaction time of 50-2000 seconds, in some embodiments 100-1000 seconds, and a sulfiding agent of hydrogenation Huang Yuansu in some embodiments. The molar ratio of sulfiding reagent to nucleic acid sequence attached to the solid support during the coupling step is from 10:1 to 1000:1, in some embodiments from 10:1 to 500:1. In some embodiments, the sulfidation reaction is performed in a mixed solvent of acetonitrile: pyridine=1:3-3:1.

In accordance with the methods provided by the present disclosure, after ligating all nucleoside monomers, the methods further comprise isolating the sense strand and the antisense strand of the siRNA prior to annealing. Methods of isolation are well known to those skilled in the art and generally involve cleavage of the synthesized nucleotide sequence from the solid support, removal of protecting groups on the base, phosphate and ligand, purification and desalting.

The nucleotide sequence obtained by synthesis is cut off from the solid phase carrier, and the protecting groups on the base, the phosphate group and the ligand are removed according to the conventional cutting and deprotection method in siRNA synthesis. For example, the obtained nucleotide sequence linked to the solid phase carrier is contacted with concentrated ammonia water; in the deprotection process, the hydroxy protecting group is removed to obtain M ₁ A group, thereby producing a conjugate represented by formula (II). Wherein, the concentrated ammonia water is 25-30 wt% ammonia water, and the dosage of the concentrated ammonia water is 0.2 ml/mu mol-0.8 ml/mu mol compared with the target siRNA sequence.

In the presence of at least one 2'-TBDMS protection on the synthesized nucleotide sequence, the method further comprises contacting the solid support-removed nucleotide sequence with triethylamine trihydrofluoride to remove the 2' -TBDMS protection. At this time, the corresponding nucleoside having a free 2' -hydroxy group in the resulting target siRNA sequence. The amount of pure triethylamine-tricofluoride salt is 0.4 ml/. Mu.mol-1.0 ml/. Mu.mol compared with the target siRNA sequence. This resulted in the siRNA conjugates of the present disclosure.

Methods of purification and desalination are well known to those skilled in the art. For example, purification of nucleic acids can be accomplished by gradient elution with NaBr or NaCl using preparative ion chromatography purification columns; after the product is collected and combined, the desalination can be performed by adopting a reversed phase chromatographic purification column.

The purity and molecular weight of the nucleic acid sequence can be detected at any time during the synthesis process, so that the quality of the synthesis can be better controlled, and the detection method is well known to those skilled in the art. For example, the purity of the nucleic acid can be detected by ion exchange chromatography and the molecular weight can be determined by liquid chromatography.

Methods of annealing are also well known to those skilled in the art. For example, the synthesized sense strand (S strand) and antisense strand (AS strand) may simply be mixed in equimolar ratio in water for injection and heated to 70-95℃and then cooled at room temperature to form a double-stranded structure through hydrogen bonding. This resulted in the siRNA conjugates of the present disclosure.

After obtaining the conjugates of the present disclosure, in some embodiments, the synthesized siRNA conjugates can also be characterized by means of molecular weight detection, etc., using methods such as liquid chromatography, etc., to determine that the synthesized siRNA conjugates are targeted siRNA conjugates, and that the sequences of the synthesized siRNA correspond to the sequences of the siRNA to be synthesized, such as those listed in table 1 above.

In further embodiments, the conjugates of the present disclosure may be prepared by the following method:

synthesizing oligonucleotides according to the phosphoramidite solid phase synthesis method described above, wherein at least one nucleotide in the oligonucleotide sequence is linked to an amino group;

adding a mixture of sodium bicarbonate, acetonitrile and dimethyl sulfoxide at normal temperature, and reacting the synthesized oligonucleotide with a conjugate molecule of the disclosure, wherein in the conjugate molecule of the disclosure, an L2 group has an imido ester group, an acyl group and an alkylene group which are sequentially connected, so that the oligonucleotide and the conjugate molecule of the disclosure are connected through an amide bond; isolation yields conjugates of the present disclosure.

Use of conjugates of the present disclosure

As shown in the present disclosure, the conjugates can deliver an active agent to cells for the treatment or prevention of a disease or condition that may require such delivery. Without wishing to be bound by any theory, we believe that the spatial arrangement of the conjugate molecules is particularly effective in targeting cell surface receptors, thereby bringing the supported active agent into contact with the cells. In some embodiments, such conjugates are oligonucleotide conjugates directed against hepatocytes.

The oligonucleotide conjugate has excellent liver targeting specificity, so that the conjugated functional oligonucleotide and small molecule drugs can be efficiently delivered to the liver at the same time, and the oligonucleotide conjugate has low toxicity, good stability and high gene expression inhibition activity in liver cells, so that a better therapeutic effect is achieved.

The oligonucleotide conjugate of the invention is suitable for use in the preparation of a medicament for preventing and/or treating a pathological condition or disease caused by expression of genes in hepatocytes.

The oligonucleotide conjugates of the invention are useful for preventing and/or treating pathological conditions or diseases caused by the expression of genes in hepatocytes.

The gene may be an endogenous gene expressed in the liver or a pathogen gene propagated in the liver. In some embodiments, the gene is selected from the group consisting of a hepatitis b virus gene, an angiopoietin-like protein 3 gene, or an apolipoprotein C3 gene. Accordingly, the disease is selected from the group consisting of chronic liver disease, hepatitis, liver fibrosis disease, liver hyperplasia disease and dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

The following examples illustrate the invention by way of illustration only and are not limiting.

Examples

Boc: tert-butoxy radical

Fmoc: 9-fluorenylmethoxycarbonyl

TBS, TBDMS: tert-butyldimethylsilyl group

DMTr:4,4' -Dimethoxytrityl radical

Preparation example

Preparation example 1: preparation of liver targeting Compounds of the invention

1.1 Synthesis of PNB-2

Starting material PNB-1 (available from Annaiji Co., ltd., 14.0g,31.0 mmol) and P4-1 (7.0 g,40 mmol) were dissolved in 300 ml of methylene chloride, HATU (2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, available from Melin Co., ltd., 17.7g,46.2 mmol) was added; DIPEA (N, N-diisopropylethylamine, available as Ara Ding Gongsi, 10.2g,77.0 mmol) was then added under ice-bath; the reaction was then stirred at 25℃for 3 hours and quenched with water. The reaction mixture was extracted with dichloromethane multiple times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was distilled under reduced pressure. The residue obtained was purified by column chromatography on 200-300 mesh normal phase silica gel, eluting with petroleum ether: ethyl acetate=20:1-1:1 gradient to give the product PNB-2 as 18.8g in 99.3% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.80–7.73(m,2H),7.56(t,J＝8.9Hz,2H),7.45–7.37(m,2H),7.36–7.30(m,2H),7.15–6.89(m,1H),5.32-5.28(m,2H),4.76-4.73(m,1H),4.47-4.42(m,2H),4.16(d,J＝34.3Hz,3H),3.69-3.64(m,3H),3.39-3.35(m,3H),1.45(s,9H),0.90(s,9H),0.06(s,6H).MS m/z：C ₃₃ H ₄₇ N ₃ O ₆ Si，[M+H] ⁺ Theory: 610.84, found: 611.03

1.2 Synthesis of PNB-3

PNB-2 (18.8 g,30.8 mmol) was dissolved in anhydrous acetonitrile (100 ml), and diethylamine (20 ml) was added to the reaction solution by syringe, and the reaction was stirred at 25℃for 2 hours. Subsequently, the reaction mixture was evaporated to dryness under reduced pressure, and the obtained residue was diluted with methanol and evaporated to dryness under reduced pressure again to give a crude product. Column chromatography (200-300 mesh normal phase silica gel, ethyl acetate: methanol=100:1-10:1 gradient elution) Purification, because the polarity of the target product is relatively large, after eluting the low-polarity compound, eluting with 10:1 ethyl acetate-methanol, concentrating and drying to obtain PNB-3 with a yield of 12.3g (about 99%). MS m/z: c (C) ₁₈ H ₃₇ N ₃ O ₄ Si，[M+H] ⁺ Theory: 388.590, found: 388.830

1.3 Synthesis of PNB-4

PNB-1 (15.8 g,34.9 mmol) and PNB-3 (12.3 g) obtained in 1.2 were dissolved in dichloromethane (310 ml), HATU (18.1 g,47.6 mmol) was added; DIPEA (10.3 g,79.3 mmol) was then added under ice bath, stirred at room temperature for 3 hours, and quenched with water. Dichloromethane was extracted 3 times, the organic phases were combined, concentrated by rotary evaporation, and the target product was collected by purification by column chromatography (200-300 mesh normal phase silica gel, ethyl acetate: methanol=100:1-15:1 gradient elution) and concentrated to give PNB-4 as 26.0g, quantitative yield (about 99%). ¹ H NMR(400MHz,DMSO)δ8.20–8.04(m,3H),7.86(t,J＝6.5Hz,2H),7.69–7.61(m,2H),7.38(t,J＝7.3Hz,2H),7.30(t,J＝7.3Hz,2H),4.34–4.16(m,4H),3.64–3.49(m,8H),3.15–3.04(m,7H),1.21(d,J＝6.0Hz,18H),0.82(s,10H),-0.01(s,6H)。

1.4 Synthesis of PNB-5

PNB-4 (26 g) obtained in the above reaction 1.3 was dissolved in acetonitrile (100 ml), and diethylamine (20 ml) was added to the reaction solution by syringe and stirred at room temperature for 3 hours. Subsequently, the reaction mixture was evaporated to dryness under reduced pressure, and the obtained residue was diluted with methanol and evaporated to dryness under reduced pressure again to give a crude product. Purifying by column chromatography (200-300 mesh normal phase silica gel, ethyl acetate: methanol=100:1-10:1 gradient elution), eluting low polar compound with 10:1 ethyl acetate-methanol, concentrating and drying to obtain PNB-5 (22 g), and quantitatively yield (about 99%). M is M S m/z：C ₂₈ H ₅₃ N ₅ O ₇ Si，[M+H]++, theory: 600.8450, found: 601.14

1.5PNB-6 Synthesis

PNB-5 (17 g,28.3 mmol) and PNB-1 (14.1 g,31.2 mmol) were dissolved in dichloromethane (283 ml) and HATU (16.1 g,42.5 mmol) was added; DIPEA (9.1 g,70.8 mmol) was then added under ice bath, stirred at room temperature for 3 hours, and quenched with water. Extracting with dichloromethane for 3 times, mixing organic phases, concentrating under reduced pressure, purifying by column chromatography (200-300 mesh normal phase silica gel, ethyl acetate: methanol=100:1-8:1 gradient elution) to obtain PNB-6 with 17.0g and 58% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.79(d,J＝7.1Hz,2H),7.60(d,J＝7.3Hz,2H),7.45–7.40(m,2H),7.36–7.32(m,2H),6.24(s,3H),4.57–4.22(m,10H),3.83–3.63(m,9H),3.36–3.16(m,7H),1.47(s,27H),0.94(s,9H),0.12(s,6H)。

1.6 Synthesis of PNB-7 and PNB-8

PNB-6 (17 g,16.3 mmol) was added to 30ml HCl/EtOH (30%), stirred at room temperature for 4 hours, the solvent was distilled off under reduced pressure and pumped dry with an oil pump for 16 hours to give PNB-7, which was used directly in the next reaction without further purification.

PNB-7 was dissolved in 80ml DMF, DIPEA (25.2 g,195 mmol) was added, stirred for 3 minutes, TBSCl (t-butyldimethylchlorosilane, available from Melin Corp., 12.28g,81.5 mmol) was added slowly, the reaction stirred at room temperature for 4 hours, quenched with water, and extracted 3 times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, distilled under reduced pressure and the solvent was drained to give the crude PNB-8, which was used directly in the next reaction without further purification to give crude PNB-8 as 11.9g. The product was identified by LC-MS: MS m/z: c (C) ₃₈ H ₅₅ N ₇ O ₆ Si，[M+H] ⁺ Theory: 734.986, found: 734.39.

1.7 Synthesis of PNB-9

PNB-8 (11.9 g,16.3 mmol) was dissolved in 160 mL of N, N-Dimethylformamide (DMF), gal-5 (customized by Tianjin Jun Yao Co., ltd., 25.5g,57.1 mmol) was added, and HATU (9.3 g,24.5 mmol) was added; DIPEA (6.3 g,48.9 mmol) was then added under ice-water bath, the reaction stirred for 4 hours, quenched with water, and extracted 3 times with dichloromethane; the organic phases were combined, concentrated by distillation under reduced pressure, and purified by column chromatography (200-300 mesh normal phase silica gel, ethyl acetate: methanol=100:1-20:1 gradient elution) to give PNB-9 as 16.1g in 97.5% yield. The product was identified by LC-MS: MS m/z: c (C) ₉₅ H ₁₃₆ N ₁₀ O ₃₆ Si，[M/2+H] ⁺ Theory: 1012.1260, found: 1012.10.

1.8 Synthesis of PNB-10 and PNB-11

PNB-9 (16.1 g,7.96 mmol) was dissolved in anhydrous acetonitrile (available from sigma-aldrich,40 mL) and diethylamine (8 mL, 77.6 mmol) was added to the reaction solution via syringe and stirred at ambient temperature for 2h. The reaction mixture was evaporated to dryness under reduced pressure, the resulting residue was diluted with methanol, and evaporated to dryness again under reduced pressure to give crude PNB-10 (12.1 g), which was directly subjected to the next reaction as a raw material.

PNB-10 (12.1 g) obtained in the above step was dissolved in methylene chloride (70 ml), 5-O-DMTr-5-hydroxyvaleric acid (9.9 g,23.5 mmol) was added, and HATU (3.8 g,10.1 mmol) was added; DIPEA (2.6 g,20.2 mmol) is then added under ice-water bath condition, the reaction is stirred for 4 hours, then water is added to quench the reaction, and dichloromethane is used for extraction; the organic phase is evaporated to dryness under reduced pressure, the residue is purified by column chromatography (normal phase silica gel of 200-300 meshes, ethyl acetate: methanol=100:1-12:1 gradient elution), PNB-11 product was obtained in 6.2g, with a total yield of 41.0% in the two steps. ¹ H NMR(400MHz,DMSO)δ8.38(d,J＝5.5Hz,6H),8.16(d,J＝9.6Hz,8H),7.94(s,2H),7.35(t,J＝9.2Hz,5H),7.23(d,J＝8.8Hz,5H),7.21–7.15(m,7H),6.89(d,J＝8.7Hz,4H),5.23(d,J＝2.8Hz,2H),4.99(d,J＝11.1Hz,2H),4.51(d,J＝7.6Hz,2H),4.31(d,J＝28.7Hz,5H),4.04(t,J＝7.0Hz,10H),3.95–3.81(m,4H),3.74(s,6H),2.91(d,J＝18.4Hz,18H),2.69(s,2H),2.11(s,9H),2.00(s,12H),1.89(s,9H),1.79(s,9H),1.52–1.45(m,10H),0.85(s,9H),0.03(s,6H)。

MS m/z：C ₁₀₆ H ₁₅₂ N ₁₀ O ₃₈ Si，[M-303(DMTr)] ⁺ Theory: 1899.4985, found: 1899.94.

1.9 Synthesis of PNB-12

PNB-11 (6.2 g,2.8 mmol) was dissolved in THF (tetrahydrofuran, 28 ml), 1M TBAF (12 ml,12.6 mmol) was added, and after stirring for 4 hours, the reaction was quenched with water and extracted 3 times with dichloromethane; the organic phases were combined, concentrated by distillation under reduced pressure, and purified by column chromatography (200-300 mesh normal phase silica gel, ethyl acetate: methanol=100:1-8:1 gradient elution) to give PNB-12 as 3.1g in 64% yield. The product was identified by LC-MS: MS m/z: c (C) ₁₀₀ H ₁₃₈ N ₁₀ O ₃₈ ，[M-303] ⁺ Theory: 1785.2360, found: 1785.81.

1.10 Synthesis of PNB-13

PNB-12 (3.1 g,1.5 mmol), succinic anhydride (360 mg,3.6 mmol) and DMAP (4-dimethylaminopyridine, available from Aba Ding Gongsi, 366mg,3.0 mmol) were added to a dichloromethane solution, a DIPEA (968 mg,7.5 mmol) solution in dichloromethane was added dropwise and the reaction stirred for 4 hours; after the complete reaction is monitored by adopting LC-MS, adding water to quench the reaction, and extracting with dichloromethane for 3 times; combining the organic phases andwashing with saturated ammonium chloride solution three times, evaporating under reduced pressure to dryness, to give PNB-13 as 3.3g in quantitative yield (about 99%). The product was identified by LC-MS and used directly for the next step in connection with the solid support. MS m/z: c (C) ₁₀₄ H ₁₄₂ N ₁₀ O ₄₁ ，[M-303] ⁺ Theory: 1885.3090, found: 1885.95.

preparation example 2: preparation of conjugate molecules attached to solid support

PNB-13 (53 mg,0.243 mmol) and HBTU (138 mg,0.365 mmol), and DIPEA (125 mg,0.974 mmol) were added to 5ml acetonitrile, dissolved with the aid of ultrasound, and then added with an Aminomethyl (AM) resin solid-phase carrier (available from Nanko Synthesis, amino content 400. Mu. Mol/g,607 mg), followed by shaking for 16 hours, and after completion of the reaction, suction filtration; the filter cake was rinsed with acetonitrile and drained. The filter cake was then reacted with an acetic anhydride/pyridine (25%) solution for a further half hour, leaving the unconnected amino groups capped with acetyl groups. Filtering again, repeating the washing process, and completely pumping. The target product PNB-14, i.e. the conjugated molecule attached to the solid support, is obtained. Yield 1.03g, loading 135. Mu. Mol/g.

Preparation example 3: preparation of PNB-siRNA Compound (SR 16-X2U 3460-PNB)

In this example, the siRNA of the siRNA conjugate was the sequence numbered SR 16-X23460:

sense strand:

5'-CmsCmsUmUmGfAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3'

(SEQ ID NO:140)

antisense strand:

5'-VPUmsUfsUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmsUmsUm-3'；

(SEQ ID NO:141)

wherein capital C, G, U, A indicates the base composition of the nucleotide; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a 2' -methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a 2' -fluoro modified nucleotide; the lower case letter s indicates that the linkage between two nucleotides adjacent to the letter s is a phosphorothioate linkage; VP means that the nucleotide to the right of the VP is a vinyl phosphate modified nucleotide.

Preparation method

3.1 Synthesis of sense strand (S)

The sense strand was prepared by using PNB-14 prepared in example 2 as a starting material, and nucleoside monomers were linked one by one in the 3'-5' direction according to the sequence order described above. Each nucleoside monomer connected comprises four steps of deprotection, coupling, capping and oxidation. The synthesis conditions were given as follows:

the nucleoside monomer was provided as a 0.1M acetonitrile solution, the deprotection conditions were the same for each step, i.e., the temperature was 25 ℃, the reaction time was 70 seconds, the deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% v/v), and the molar ratio of dichloroacetic acid to 4,4' -dimethoxytrityl protecting group on the solid support was 5:1.

The coupling reaction conditions of each step are the same, the temperature is 25 ℃, the mole ratio of the nucleic acid sequence connected on the solid carrier to the nucleoside monomer is 1:10, the mole ratio of the nucleic acid sequence connected on the solid carrier to the coupling reagent is 1:65, the reaction time is 600 seconds, and the coupling reagent is a 0.5M acetonitrile solution of 5-ethylthio-1H-tetrazole.

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 mol ratio of 1:1, the CapA is a pyridine/acetonitrile mixed solution of 20 volume percent N-methylimidazole, and the volume ratio of pyridine to acetonitrile is 3:5; capB is an acetonitrile solution of 20% acetic anhydride by volume; the molar ratio of the capping reagent to the nucleic acid sequence attached to the solid support is acetic anhydride: N-methylimidazole: nucleic acid sequence attached to the solid support=1:1:1.

The oxidation reaction conditions are the same in each step, the temperature is 25 ℃, the reaction time is 15 seconds, and the oxidizing agent is iodine water with the concentration of 0.05M. The molar ratio of iodine to nucleic acid sequence attached to the solid support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran, water, pyridine=3:1:1.

Wherein, the vinyl phosphate modified 2' -methoxy modified uracil nucleoside monomer (VP-Um) is synthesized according to the following method:

(3 a-1) Synthesis of VP-U-2

VP-U-2 molecules were synthesized according to the following procedure:

2 '-methoxy-modified uridine (2' -OMe-U,51.30g,91.6 mmol), tert-butyldiphenylchlorosilane (TBDPSCl, 50.35g,183.2 mmol), imidazole (12.47 g,183.2 mmol) were dissolved in 450ml of N, N-Dimethylformamide (DMF) and stirred at room temperature for 20h. After removal of DMF by evaporation and dissolution with 600ml of dichloromethane, 300ml of saturated aqueous sodium bicarbonate solution were added and the aqueous phase was extracted 3 times with 300ml of Dichloromethane (DCM) and the organic phases were combined. The combined organic phases were washed with 5% oxalic acid to a pH of the separated aqueous phase of <5 and the solvent in the organic phase was evaporated to dryness to give crude VP-U-1 which was used directly in the subsequent synthesis of VP-U-2.

After the VP-U-1 crude product was dissolved in 100ml of methylene chloride, the mixture was stirred in an ice bath for 10 minutes, 450ml of a 2% p-toluenesulfonic acid solution (solvent: methanol-methylene chloride mixed solvent in a volume ratio of 3:7) previously stored in a refrigerator at 4℃was added thereto, and the mixture was reacted for 10 minutes. The reaction was quenched by adding 200ml of saturated aqueous sodium bicarbonate solution, and the organic and aqueous phases were separated. The organic phase was washed with saturated aqueous sodium bicarbonate to ph=8 and the aqueous phases combined. The combined aqueous phases were extracted 2 times with 200ml portions of dichloromethane and the organic phases were combined. The combined organic phases were washed once more with 200ml of saturated brine and the solvent was evaporated to dryness. Purifying the residue by column chromatography (200-300 mesh normal phase silica gel column, loading petroleum ether into column, gradient eluting with petroleum ether: ethyl acetate: dichloromethane: methanol=1:1:1:0.05-1:1:0.25), collecting the product eluate, evaporating solvent under reduced pressure, and vacuum oil pump Foaming and drying to obtain pure VP-U-2 with total weight of 40.00g. ¹ H NMR(400MHz,DMSO-d6)δ7.96(d,J＝7.8Hz,1H),7.64(dtd,J＝5.1,4.0,2.2Hz,4H),7.41–7.30(m,6H),6.79(d,J＝4.7Hz,1H),5.73(d,J＝7.6Hz,1H),4.94(t,J＝7.0Hz,1H),4.12(td,J＝4.6,3.9Hz,1H),4.05(dd,J＝4.8,4.0Hz,1H),3.96(t,J＝4.7Hz,1H),3.68(ddd,J＝11.8,7.0,4.6Hz,1H),3.57–3.46(m,1H),3.39(s,3H),1.05(s,8H).MS m/z：C ₂₆ H ₃₃ N ₂ O ₆ Si，[M+H] ⁺ Theory: 497.21, found: 497.45.

(3 a-2) Synthesis of VP-U-4:

VP-U-2 (19.84 g,40.0 mmol), dicyclohexylcarbodiimide (DCC, 16.48g,80.0 mmol), pyridine (4.20 g,53.2 mmol), trifluoroacetic acid (6.61 g,53.2 mmol) were mixed and dissolved in 200ml dimethyl sulfoxide (DMSO), and the reaction was stirred at room temperature for 20h. In addition, tetraethyl methylenediphosphate (21.44 g,74.4 mmol) was dissolved in 120ml of THF, cooled in an ice bath, t-BuOK (11.36 g,101.2 mmol) was added at the ice bath temperature, reacted for 10 minutes at the ice bath temperature, then allowed to react at room temperature for 0.5 hours, then added to the above reaction solution, and after about 1 hour, reacted for 1 hour at the ice bath temperature again, and then allowed to react at room temperature for 18 hours. The reaction was quenched with water and the aqueous phase was extracted 3 times with 200ml of dichloromethane. The organic phases were combined, washed once with 200ml of saturated brine and the solvent was evaporated to dryness. Purifying the residue by column chromatography (200-300 mesh normal phase silica gel column, loading petroleum ether into column, gradient eluting with petroleum ether: ethyl acetate=1:1-1:4), collecting the product eluent, evaporating solvent under reduced pressure, and vacuum oil pump foaming and drying to obtain pure VP-U-4 14.00g. ¹ H NMR(400MHz,DMSO-d6)δ7.96(d,J＝7.8Hz,1H),7.64(dtd,J＝5.1,4.0,2.2Hz,4H),7.41–7.30(m,6H),6.82–6.71(m,2H),5.90(ddd,J＝25.9,15.0,1.0Hz,1H),5.73(d,J＝7.6Hz,1H),4.36–4.21(m,3H),4.18(t,J＝4.9Hz,1H),4.05(ddq,J＝9.7,8.5,6.9Hz,2H),3.87(t,J＝4.8Hz,1H),3.39(s,3H),1.32(td,J＝6.9,0.7Hz,6H),1.05(s,8H).MS m/z：C ₃₁ H ₄₂ N ₂ O ₈ PSi，[M+H] ⁺ Theory: 629.24, found: 629.51.

(3 a-3) Synthesis of VP-U-5:

VP-U-4 (14.00 g,22.29 mmol) was dissolved in 100ml tetrahydrofuran, triethylamine trihydrofluoride (17.96 g,111.45 mmol) was added, and the reaction was stirred at room temperature for 20h to completion. The solvent was evaporated directly to dryness, then dissolved with dichloromethane and then evaporated to dryness 2 times, using 50ml of dichloromethane each time, to give the crude product. Purifying the crude product by column chromatography (200-300 mesh normal phase silica gel column, loading petroleum ether into column, gradient eluting with petroleum ether: ethyl acetate: dichloromethane: methanol=1:1:1:0.05-1:1:0.25), collecting the product eluent, evaporating solvent under reduced pressure, and foaming and drying by vacuum oil pump to obtain pure VP-U-5 with total weight of 6.70g. ¹ H NMR(400MHz,DMSO-d6)δ7.96(d,J＝7.8Hz,1H),6.77(dd,J＝15.0,6.2Hz,1H),5.99–5.82(m,2H),5.73(d,J＝7.6Hz,1H),5.27(d,J＝5.1Hz,1H),5.10(dd,J＝5.3,4.7Hz,1H),4.29(ddq,J＝9.8,8.6,7.0Hz,2H),4.17(ddd,J＝6.2,5.2,1.0Hz,1H),4.12–3.98(m,3H),3.39(s,2H),1.32(td,J＝6.9,0.6Hz,6H).MS m/z：C ₁₅ H ₂₄ N ₂ O ₈ P，[M+H] ⁺ Theory: 391.13, found: 391.38.

(3 a-4) Synthesis of VP-U-6:

VP-U-5 (399mg, 1.0 mmol), pyridine trifluoroacetate (0.232 g,1.2 mmol), N-methylimidazole (0.099 g,1.2 mmol) and bis (diisopropylamino) (2-cyanoethoxy) phosphine (0.452 g,1.5 mmol) were added to 10ml of anhydrous dichloromethane under argon atmosphere, and the mixture was stirred at room temperature for 5 hours. Evaporating the solvent to dryness, purifying by column chromatography (200-300 mesh normal phase silica gel, dichloromethane: acetonitrile (containing 0.5wt% triethylamine) =3:1-1:3 gradient elution), collecting product eluent, concentrating to remove the solvent to obtain the target product VP-U-6 of 508mg. ³¹ P NMR(161MHz,DMSO-d6)δ150.34,150.29,17.07,15.50.MS m/z：C ₂₄ H ₄₁ N ₄ O ₉ P ₂ ，[M+H] ⁺ Theory: 591.23, found: 591.55.VP-U-6 is the target product VP-Um, and participates in RNA strand synthesis as a nucleoside monomer.

The cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence with the carrier attached was added to ammonia water at a concentration of 25wt% at an amount of 0.5 ml/. Mu.mol, reacted at 55℃for 16 hours, the liquid was removed, and concentrated to dryness in vacuo. Purifying and desalting: purification of nucleic acids was accomplished by gradient elution with NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the method comprises the following steps: eluent A:20mM sodium phosphate (pH 8.1), solvent water/acetonitrile=9:1 (volume ratio); eluent B:1.5M sodium chloride, 20mM sodium phosphate (pH 8.1), solvent water/acetonitrile=9:1 (volume ratio); elution gradient: eluent a, eluent b=100:0-50:50 gradient elution. Collecting and combining product eluents, desalting by using a reversed phase chromatographic purification column, wherein specific conditions comprise desalting by using a sephadex column, eluting with deionized water by using sephadex G25 as a filler.

And (3) detection: detection was performed using ion exchange chromatography (IEX-HPLC) with a purity of 78%; molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS), theoretical 7662.5, measured 7661.4.

3.2 Synthesis of Antisense Strand (AS)

The antisense strand of the phosphoramidite nucleic acid is prepared by using a commercial universal solid phase carrierHL UnyLinker ^TM 300Oligonucleotide Synthesis Support,Kinovate Life Sciences company, load 300. Mu. Mol/g) as starting material. Deprotection, coupling, capping, oxidation reaction conditions, deprotection and cleavage in the solid phase synthesis method, and separation conditions are the same AS those for synthesizing the sense strand to obtain the antisense strand AS.

And (3) detection: purity was measured by ion exchange chromatography (IEX-HPLC), and as a result, purity was 93.2%; molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS). Antisense strand: 7037.1, found: 7036.2.

3.3 Synthesis of oligonucleotide conjugates of the invention (SR 16-X2U 3460-PNB)

The sense strand and the antisense strand obtained above were mixed in an equimolar ratio, dissolved in water for injection and heated to 50 ℃, cooled at room temperature, and then formed into a double-stranded structure through hydrogen bonding. Thus obtaining the siRNA double-strand SR16-X2U3460-PNB conjugated with PNB. The conjugate has a structure shown in formula (II-1).

Application examples:activity test of oligonucleotide conjugate (SR 16-X2U 3460-PNB) of the invention

Unless otherwise indicated, reagents and media used in the examples below are commercially available, and the procedures for electrophoresis of nucleic acids, real-time PCR, etc. used are performed according to protocols well known to those skilled in the art. For example, the method can be carried out as described in Molecular Cloning (Cold Spring Harbor LBboratory Press (1989)).

Unless otherwise indicated, the reagent ratios provided below are all calculated as volume ratios (v/v).

Experimental example in vivo (in vivo) inhibition efficiency of oligonucleotide conjugate on the expression level of X mRNA from HBV

In this example, the inhibition efficiency of HBV X mRNA expression level in HBV transgenic mice 44BriHBV was examined with respect to the oligonucleotide conjugate (SR 16-X2U 3460-PNB) of the present invention and negative control 1 XPBS (NS).

The HBV transgenic mice 44BriHBV used in this example were purchased from Experimental animal department of science, university of Beijing, approximately 8-12 weeks, male.

First, C57BL/6J-Tg (Alb 1 HBV) 44Bri/J mice were randomly grouped (females) with serum HbsAg content, 4 mice per group, and a negative control group was given PBS solution. All animals were dosed on a weight basis in a single dose (using subcutaneous dosing) and the siRNA conjugate SR16-X2U3460-PNB was formulated in PBS buffer as a solution with final concentrations of 0.2mg/ml and 0.02mg/ml (calculated as siRNA) for subcutaneous dosing at doses of 1mg/kg body weight and 0.1mg/kg body weight, respectively, and at a dosing volume of 5ml/kg. Animals were sacrificed on day 7 post-dose, livers were collected and saved with RNA later (Sigma Aldrich company); homogenizing liver tissue with tissue homogenizer, and extracting with Trizol according to standard procedure of total RNA extraction to obtain total RNA.

The expression level of HBV X mRNA in liver tissue was detected using real-time fluorescent quantitative PCR, specifically: the extracted total RNA was reverse transcribed into cDNA using an ImProm-IITM reverse transcription kit (Promega company) according to the instructions thereof, and then the inhibition efficiency of siRNA on HBV X mRNA expression in liver tissue was examined using a fluorescent quantitative PCR kit (Beijing kang is century Biotech Co.). In the fluorescent quantitative PCR method, the HBV and the beta-actin are detected by using a primer for HBV and a primer for beta-actin, respectively, with the beta-actin (beta-actin) gene as an internal reference gene.

The sequence of the detection primers is shown in Table 2 below.

TABLE 2 sequence of detection primers

In the fluorescent quantitative PCR method, the siRNA inhibitory activity was expressed as the remaining amount of HBV gene expression, calculated as follows:

HBV gene expression residual amount = (copy number of HBV gene in test group/copy number of beta-actin in test group)/(copy number of HBV gene in control group/copy number of beta-actin in control group) ×100%,

mRNA inhibition was then calculated according to the following formula:

mRNA inhibition ratio= (residual amount of 1-HBV gene expression) ×100%,

wherein, the control group is a control group mouse to which PBS is applied in the experiment, and each test group is a dosing group mouse to which different siRNA conjugates are respectively applied. The results are shown in table 3 below.

TABLE 3 inhibition of HBV X mRNA expression by siRNA conjugates in mouse liver

NA indicates that data is not available.

As can be seen from the results of table 3, the oligonucleotide conjugate of the present invention in the application example has a high inhibition rate for inhibition of HBV X mRNA expression; particularly, the inhibition activity is more remarkable at a higher concentration of 1 mg/kg.

Example embodiments have been disclosed herein, and although specific terms are employed, they are used and described in a generic and descriptive sense only and not for purposes of limitation. In some cases, as will be apparent to one of ordinary skill in the art from the filing of the present application, the features, characteristics, and/or elements described in connection with a particular embodiment may be used alone or in combination with the features, characteristics, and/or elements described in connection with other embodiments unless explicitly indicated otherwise. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as set forth in the appended claims.

Sequence listing

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<211> 21

<212> RNA

<213> artifical sequence

<400> 39

uagaagauga ggcauagcag c 21

<210> 40

<211> 21

<212> RNA

<213> artifical sequence

<400> 40

uagaagauga ggcauagcau u 21

<210> 41

<211> 19

<212> RNA

<213> artifical sequence

<400> 41

ugcuaugccu caucuucua 19

<210> 42

<211> 21

<212> RNA

<213> artifical sequence

<400> 42

uagaagauga ggcauagcag c 21

<210> 43

<211> 21

<212> RNA

<213> artifical sequence

<400> 43

uagaagauga ggcauagcau u 21

<210> 44

<211> 21

<212> RNA

<213> artifical sequence

<400> 44

uagaagauga ggcauagcag c 21

<210> 45

<211> 21

<212> RNA

<213> artifical sequence

<400> 45

uagaagauga ggcauagcau u 21

<210> 46

<211> 21

<212> RNA

<213> artifical sequence

<400> 46

uagaagauga ggcauagcag c 21

<210> 47

<211> 21

<212> RNA

<213> artifical sequence

<400> 47

uagaagauga ggcauagcau u 21

<210> 48

<211> 21

<212> RNA

<213> artifical sequence

<400> 48

uagaagauga ggcauagcag c 21

<210> 49

<211> 21

<212> RNA

<213> artifical sequence

<400> 49

uagaagauga ggcauagcau u 21

<210> 50

<211> 21

<212> RNA

<213> artifical sequence

<400> 50

uagaagauga ggcauagcag c 21

<210> 51

<211> 21

<212> RNA

<213> artifical sequence

<400> 51

uagaagauga ggcauagcau u 21

<210> 52

<211> 19

<212> RNA

<213> artifical sequence

<400> 52

ucugugccuu cucaucuga 19

<210> 53

<211> 21

<212> RNA

<213> artifical sequence

<400> 53

ucagaugaga aggcacagac g 21

<210> 54

<211> 19

<212> RNA

<213> artifical sequence

<400> 54

ucugugccuu cucaucuga 19

<210> 55

<211> 21

<212> RNA

<213> artifical sequence

<400> 55

ucagaugaga aggcacagac g 21

<210> 56

<211> 19

<212> RNA

<213> artifical sequence

<400> 56

ucugugccuu cucaucuga 19

<210> 57

<211> 21

<212> RNA

<213> artifical sequence

<400> 57

ucagaugaga aggcacagac g 21

<210> 58

<211> 19

<212> RNA

<213> artifical sequence

<400> 58

ucugugccuu cucaucuga 19

<210> 59

<211> 21

<212> RNA

<213> artifical sequence

<400> 59

ucagaugaga aggcacagac g 21

<210> 60

<211> 19

<212> RNA

<213> artifical sequence

<400> 60

ucugugccuu cucaucuga 19

<210> 61

<211> 21

<212> RNA

<213> artifical sequence

<400> 61

ucagaugaga aggcacagac g 21

<210> 62

<211> 21

<212> RNA

<213> artifical sequence

<400> 62

ucagaugaga aggcacagac g 21

<210> 63

<211> 21

<212> RNA

<213> artifical sequence

<400> 63

ucagaugaga aggcacagac g 21

<210> 64

<211> 21

<212> RNA

<213> artifical sequence

<400> 64

ucagaugaga aggcacagac g 21

<210> 65

<211> 21

<212> RNA

<213> artifical sequence

<400> 65

ucagaugaga aggcacagac g 21

<210> 66

<211> 19

<212> RNA

<213> artifical sequence

<400> 66

cgugugcacu ucgcuucaa 19

<210> 67

<211> 21

<212> RNA

<213> artifical sequence

<400> 67

uugaagcgaa gugcacacgg u 21

<210> 68

<211> 19

<212> RNA

<213> artifical sequence

<400> 68

cgugugcacu ucgcuucaa 19

<210> 69

<211> 21

<212> RNA

<213> artifical sequence

<400> 69

uugaagcgaa gugcacacgg u 21

<210> 70

<211> 19

<212> RNA

<213> artifical sequence

<400> 70

cgugugcacu ucgcuucaa 19

<210> 71

<211> 21

<212> RNA

<213> artifical sequence

<400> 71

uugaagcgaa gugcacacgg u 21

<210> 72

<211> 19

<212> RNA

<213> artifical sequence

<400> 72

cgugugcacu ucgcuucaa 19

<210> 73

<211> 21

<212> RNA

<213> artifical sequence

<400> 73

uugaagcgaa gugcacacgg u 21

<210> 74

<211> 19

<212> RNA

<213> artifical sequence

<400> 74

cgugugcacu ucgcuucaa 19

<210> 75

<211> 21

<212> RNA

<213> artifical sequence

<400> 75

uugaagcgaa gugcacacgg u 21

<210> 76

<211> 21

<212> RNA

<213> artifical sequence

<400> 76

uugaagcgaa gugcacacgg u 21

<210> 77

<211> 21

<212> RNA

<213> artifical sequence

<400> 77

uugaagcgaa gugcacacgg u 21

<210> 78

<211> 21

<212> RNA

<213> artifical sequence

<400> 78

uugaagcgaa gugcacacgg u 21

<210> 79

<211> 21

<212> RNA

<213> artifical sequence

<400> 79

uugaagcgaa gugcacacgg u 21

<210> 80

<211> 19

<212> RNA

<213> artifical sequence

<400> 80

ccaagagcac caagaacua 19

<210> 81

<211> 21

<212> RNA

<213> artifical sequence

<400> 81

uaguucuugg ugcucuuggc u 21

<210> 82

<211> 21

<212> RNA

<213> artifical sequence

<400> 82

agccaagagc accaagaacu a 21

<210> 83

<211> 23

<212> RNA

<213> artifical sequence

<400> 83

uaguucuugg ugcucuuggc uug 23

<210> 84

<211> 19

<212> RNA

<213> artifical sequence

<400> 84

ccaagagcac caagaacua 19

<210> 85

<211> 21

<212> RNA

<213> artifical sequence

<400> 85

uaguucuugg ugcucuuggc u 21

<210> 86

<211> 21

<212> RNA

<213> artifical sequence

<400> 86

agccaagagc accaagaacu a 21

<210> 87

<211> 23

<212> RNA

<213> artifical sequence

<400> 87

uaguucuugg ugcucuuggc uug 23

<210> 88

<211> 21

<212> RNA

<213> artifical sequence

<400> 88

uaguucuugg ugcucuuggc u 21

<210> 89

<211> 23

<212> RNA

<213> artifical sequence

<400> 89

uaguucuugg ugcucuuggc uug 23

<210> 90

<211> 19

<212> RNA

<213> artifical sequence

<400> 90

ccaagagcac caagaacua 19

<210> 91

<211> 21

<212> RNA

<213> artifical sequence

<400> 91

agccaagagc accaagaacu a 21

<210> 92

<211> 19

<212> RNA

<213> artifical sequence

<400> 92

ccaagagcac caagaacua 19

<210> 93

<211> 21

<212> RNA

<213> artifical sequence

<400> 93

uaguucuugg ugcucuuggc u 21

<210> 94

<211> 21

<212> RNA

<213> artifical sequence

<400> 94

agccaagagc accaagaacu a 21

<210> 95

<211> 23

<212> RNA

<213> artifical sequence

<400> 95

uaguucuugg ugcucuuggc uug 23

<210> 96

<211> 21

<212> RNA

<213> artifical sequence

<400> 96

uaguucuugg ugcucuuggc u 21

<210> 97

<211> 23

<212> RNA

<213> artifical sequence

<400> 97

uaguucuugg ugcucuuggc uug 23

<210> 98

<211> 19

<212> RNA

<213> artifical sequence

<400> 98

ccaagagcac caagaacua 19

<210> 99

<211> 21

<212> RNA

<213> artifical sequence

<400> 99

agccaagagc accaagaacu a 21

<210> 100

<211> 21

<212> RNA

<213> artifical sequence

<400> 100

uaguucuugg ugcucuuggc u 21

<210> 101

<211> 23

<212> RNA

<213> artifical sequence

<400> 101

uaguucuugg ugcucuuggc uug 23

<210> 102

<211> 21

<212> RNA

<213> artifical sequence

<400> 102

uaguucuugg ugcucuuggc u 21

<210> 103

<211> 23

<212> RNA

<213> artifical sequence

<400> 103

uaguucuugg ugcucuuggc uug 23

<210> 104

<211> 21

<212> RNA

<213> artifical sequence

<400> 104

uaguucuugg ugcucuuggc u 21

<210> 105

<211> 23

<212> RNA

<213> artifical sequence

<400> 105

uaguucuugg ugcucuuggc uug 23

<210> 106

<211> 21

<212> RNA

<213> artifical sequence

<400> 106

uaguucuugg ugcucuuggc u 21

<210> 107

<211> 23

<212> RNA

<213> artifical sequence

<400> 107

uaguucuugg ugcucuuggc uug 23

<210> 108

<211> 19

<212> RNA

<213> artifical sequence

<400> 108

caauaaagcu ggacaagaa 19

<210> 109

<211> 21

<212> RNA

<213> artifical sequence

<400> 109

uucuugucca gcuuuauugg g 21

<210> 110

<211> 21

<212> RNA

<213> artifical sequence

<400> 110

cccaauaaag cuggacaaga a 21

<210> 111

<211> 23

<212> RNA

<213> artifical sequence

<400> 111

uucuugucca gcuuuauugg gag 23

<210> 112

<211> 19

<212> RNA

<213> artifical sequence

<400> 112

caauaaagcu ggacaagaa 19

<210> 113

<211> 21

<212> RNA

<213> artifical sequence

<400> 113

uucuugucca gcuuuauugg g 21

<210> 114

<211> 21

<212> RNA

<213> artifical sequence

<400> 114

cccaauaaag cuggacaaga a 21

<210> 115

<211> 23

<212> RNA

<213> artifical sequence

<400> 115

uucuugucca gcuuuauugg gag 23

<210> 116

<211> 19

<212> RNA

<213> artifical sequence

<400> 116

caauaaagcu ggacaagaa 19

<210> 117

<211> 21

<212> RNA

<213> artifical sequence

<400> 117

uucuugucca gcuuuauugg g 21

<210> 118

<211> 21

<212> RNA

<213> artifical sequence

<400> 118

cccaauaaag cuggacaaga a 21

<210> 119

<211> 23

<212> RNA

<213> artifical sequence

<400> 119

uucuugucca gcuuuauugg gag 23

<210> 120

<211> 19

<212> RNA

<213> artifical sequence

<400> 120

caauaaagcu ggacaagaa 19

<210> 121

<211> 21

<212> RNA

<213> artifical sequence

<400> 121

uucuugucca gcuuuauugg g 21

<210> 122

<211> 21

<212> RNA

<213> artifical sequence

<400> 122

cccaauaaag cuggacaaga a 21

<210> 123

<211> 23

<212> RNA

<213> artifical sequence

<400> 123

uucuugucca gcuuuauugg gag 23

<210> 124

<211> 19

<212> RNA

<213> artifical sequence

<400> 124

caauaaagcu ggacaagaa 19

<210> 125

<211> 21

<212> RNA

<213> artifical sequence

<400> 125

uucuugucca gcuuuauugg g 21

<210> 126

<211> 21

<212> RNA

<213> artifical sequence

<400> 126

cccaauaaag cuggacaaga a 21

<210> 127

<211> 23

<212> RNA

<213> artifical sequence

<400> 127

uucuugucca gcuuuauugg gag 23

<210> 128

<211> 21

<212> RNA

<213> artifical sequence

<400> 128

uucuugucca gcuuuauugg g 21

<210> 129

<211> 23

<212> RNA

<213> artifical sequence

<400> 129

uucuugucca gcuuuauugg gag 23

<210> 130

<211> 21

<212> RNA

<213> artifical sequence

<400> 130

uucuugucca gcuuuauugg g 21

<210> 131

<211> 23

<212> RNA

<213> artifical sequence

<400> 131

uucuugucca gcuuuauugg gag 23

<210> 132

<211> 21

<212> RNA

<213> artifical sequence

<400> 132

uucuugucca gcuuuauugg g 21

<210> 133

<211> 23

<212> RNA

<213> artifical sequence

<400> 133

uucuugucca gcuuuauugg gag 23

<210> 134

<211> 21

<212> RNA

<213> artifical sequence

<400> 134

uucuugucca gcuuuauugg g 21

<210> 135

<211> 23

<212> RNA

<213> artifical sequence

<400> 135

uucuugucca gcuuuauugg gag 23

<210> 136

<211> 20

<212> DNA

<213> artifical sequence

<400> 136

ccgtctgtgc cttctcatct 20

<210> 137

<211> 20

<212> DNA

<213> artifical sequence

<400> 137

taatctcctc ccccaactcc 20

<210> 138

<211> 24

<212> DNA

<213> artifical sequence

<400> 138

agcttctttg cagctccttc gttg 24

<210> 139

<211> 24

<212> DNA

<213> artifical sequence

<400> 139

ttctgaccca ttcccaccat caca 24

<210> 140

<211> 19

<212> RNA

<213> artifical sequence

<400> 140

ccuugaggca uacuucaaa 19

<210> 141

<211> 21

<212> RNA

<213> artifical sequence

<400> 141

uuugaaguau gccucaaggu u 21

Claims

1. A compound having a structure represented by formula (I-1):

Wherein DMTr represents 4,4' -dimethoxytrityl.

2. An oligonucleotide conjugate having a structure represented by formula (II-1):

where Nu represents siRNA.

3. The oligonucleotide conjugate of claim 2, wherein the siRNA contains a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises nucleotide sequence 1 and the antisense strand comprises nucleotide sequence 2, the nucleotide sequence 1 and the nucleotide sequence 2 each being 19 nucleotides in length, the nucleotide sequence 1 being equal to the first stretch of nucleotide sequence in length and not more than 1 nucleotide difference, the nucleotide sequence 2 being equal to the nucleotide sequence B in length and not more than 1 nucleotide difference;

the first nucleotide sequence is a nucleotide sequence in target mRNA, and the target mRNA is mRNA corresponding to a gene which is abnormally expressed in liver cells; the nucleotide sequence B is a nucleotide sequence which is equal to the first segment of nucleotide in length and is completely complementary in reverse direction;

the nucleotide difference between the nucleotide sequence 2 and the nucleotide sequence B is in the direction from the 5 'end to the 3' end, the difference at the first nucleotide Z 'position on the nucleotide sequence 2, the last nucleotide Z on the nucleotide sequence 1 being the nucleotide complementary to Z'; p in the formula (II-1) is attached to the end of the sense strand or the antisense strand.

4. The oligonucleotide conjugate according to claim 3, wherein P in formula (II-1) is attached to the 3' end of the sense strand.

5. The oligonucleotide conjugate of claim 3, wherein P in formula (II-1) is linked to the 2' position, 3' position, or 5' position of a nucleotide in the oligonucleotide conjugate by formation of a phosphodiester bond.

6. The oligonucleotide conjugate of claim 3, wherein the sense strand further comprises nucleotide sequence 3, the antisense strand further comprises nucleotide sequence 4, the nucleotide sequence 3 and the nucleotide sequence 4 are equal in length and each are 1-4 nucleotides, the nucleotide sequence 3 is linked at the 5 'end of the nucleotide sequence 1, and the nucleotide sequence 4 is linked at the 3' end of the nucleotide sequence 2, the nucleotide sequence 4 is complementary to a second nucleotide sequence, the second nucleotide sequence being a nucleotide sequence adjacent to the first nucleotide sequence in the target mRNA and identical in length to the nucleotide sequence 4, and the nucleotide sequence 3 and the nucleotide sequence 4 are fully reverse-complementary.

7. The oligonucleotide conjugate of claim 3, wherein the siRNA further comprises a nucleotide sequence 5, the nucleotide sequence 5 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; the nucleotide sequence 5 is 2 nucleotides in length and in the direction from the 5 'end to the 3' end, the nucleotide sequence 5 is a continuous 2 deoxythymine nucleotides, a continuous 2 uracil nucleotides, or is complementary to a third nucleotide sequence, which is adjacent to the first nucleotide sequence in the target mRNA and is equal in length to the nucleotide sequence 5.

8. The oligonucleotide conjugate of claim 6, wherein the siRNA further comprises a nucleotide sequence 5, the nucleotide sequence 5 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; the nucleotide sequence 5 is 2 nucleotides in length and in the direction from the 5 'end to the 3' end, the nucleotide sequence 5 is a continuous 2 deoxythymine nucleotides, a continuous 2 uracil nucleotides, or is complementary to a third nucleotide sequence, which refers to a nucleotide sequence in the target mRNA adjacent to the second nucleotide sequence and having the same length as the nucleotide sequence 5.

9. The oligonucleotide conjugate according to claim 3, wherein at least one nucleotide in the sense strand or the antisense strand is a modified nucleotide and/or at least one phosphate group is a phosphate group having a modification group.

10. The oligonucleotide conjugate of claim 9, wherein each nucleotide in the sense strand and the antisense strand is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide, the fluoro-modified nucleotide being a nucleotide formed by substitution of the hydroxyl group at the 2' -position of the ribosyl of the nucleotide with fluorine;

Wherein in the sense strand, the nucleotides at positions 7, 8, 9 or 5, 7, 8, 9 of the nucleotide sequence 1 are fluoro-modified nucleotides in the direction from the 5 'end to the 3' end, and the nucleotides at the remaining positions in the sense strand are non-fluoro-modified nucleotides; in the antisense strand, the nucleotides at positions 2, 6, 14 and 16 or positions 2, 6, 8, 9, 14 and 16 of the nucleotide sequence 2 are fluoro-modified nucleotides, and the nucleotides at the rest positions in the antisense strand are non-fluoro-modified nucleotides;

wherein each non-fluoro modified nucleotide is a methoxy modified nucleotide, which refers to a nucleotide formed by substituting a 2' -hydroxy group of a ribosyl in the nucleotide with a methoxy group.

11. The oligonucleotide conjugate according to claim 9, wherein the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom of a phosphodiester bond in the phosphate group with a sulfur atom.

12. The oligonucleotide conjugate according to claim 11, wherein the phosphate group having a modifying group is a phosphorothioate group having a structure as shown in formula (201):

13. The oligonucleotide conjugate according to claim 11, wherein the phosphorothioate group is present at least one of:

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

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

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

the 3' -terminal end of the sense strand is between nucleotide 2 and nucleotide 3;

the 5' terminal end of the antisense strand is between nucleotide 1 and nucleotide 2;

the 5' terminal end of the antisense strand is between nucleotide 2 and nucleotide 3;

the 3' -terminal end of the antisense strand is between nucleotide 1 and nucleotide 2; and

the 3' -terminal end of the antisense strand is between nucleotide 2 and nucleotide 3.

14. The oligonucleotide conjugate of claim 9, wherein the 5' terminal nucleotide of the antisense strand is a 5' -phosphonucleotide or a 5' -phosphoanalog modified nucleotide.

15. The oligonucleotide conjugate according to claim 14, wherein the 5 '-phosphonucleotide or 5' -phosphoanalogue modified nucleotide is a nucleotide represented by one of the following formulas (202) - (206):

Wherein R represents a group selected from the group consisting of H, OH, F and methoxy, and Base represents a Base selected from A, U, C, G or T.

16. The oligonucleotide conjugate according to claim 15, wherein the 5 '-phosphonucleotide or 5' -phosphoanalogue modified nucleotide is a nucleotide represented by formula (202), formula (203) or formula (205).

17. The oligonucleotide conjugate of claim 3, wherein the siRNA is selected from any one of siHBa1, siHBa2, siHBa1M1, siHBa1M2, siHBa2M1, siHBa2M2, siHBa1M1S, siHBa1M2S, siHBa M1S, siHBa M2S, siHBa1M1P1, siHBa1M2P1, siHBa2M1P1, siHBa2M2P1, siHBa1M1SP1, siHBa1M2SP1, siHBa2M1SP1, siHBa2M2SP1, or SR 16-X23460:

siHBa1

sense strand: 5'-CCUUGAGGCAUACUUCAAA-3' (SEQ ID NO: 1),

antisense strand: 5'-UUUGAAGUAUGCCUCAAGGUU-3' (SEQ ID NO: 2);

siHBa2

sense strand: 5'-GACCUUGAGGCAUACUUCAAA-3' (SEQ ID NO: 3),

antisense strand: 5'-UUUGAAGUAUGCCUCAAGGUCGG-3' (SEQ ID NO: 4);

siHBa1M1

sense strand: 5 '-CmUmUmGmGfCfAmUmAmCmUmUmCmAmAmAmAmAmAmam-3'

(SEQ ID NO:5)，

Antisense strand: 5 '-UmUfUmGmAfmAfGmUmAmUmMcMcMfCmAfAmGmUmUmUm-3'

(SEQ ID NO:6)；

siHBa1M2

Sense strand: 5 '-CmUmUmGfAmGfCfAmUmAmUmUmUmCmAmAmAmAmAmam-3'

(SEQ ID NO:7)，

Antisense strand: 5 '-UmUfUmGmAfmAfGmUfUmGmCmUfCmUfCmAfAmGmUmUmUmUm-3'

(SEQ ID NO:8)；

siHBa2M1

Sense strand: 5 '-GmAmCmUmUmTMAMmGfCfAmUmAmUmUmUmCmAmAmAmAmAmAmam-3'

(SEQ ID NO:9)，

Antisense strand:

5'-UmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmCmGmGm-3'

(SEQ ID NO:10)；

siHBa2M2

sense strand: 5 '-GmAmCmUmUmGfAmGfCfAmUmAmUmUmCmAmAmAmAmAmAmAmAmAmam-3'

(SEQ ID NO:11)，

Antisense strand:

5'-UmUfUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmUmCmGmGm-3'

(SEQ ID NO:12)；

siHBa1M1S

sense strand: 5 '-CmsCmsUmUmTMAMmGfCfAmUmAmUmUmCmAmAmAmAmAmAmam-3'

(SEQ ID NO:13)，

Antisense strand: 5 '-UmsUfsUmGmAmAfGmUmAmXmCmUfCmAfAmGmSUmsum-3' (SEQ ID NO: 14);

siHBa1M2S

sense strand: 5 '-CmsCmsUmUmGfAmGfCfAmUmAmUmUmCmAmAmAmAmAmAmAmAmAmam-3'

(SEQ ID NO:15)，

Antisense strand: 5 '-UmsUfsUmGmAfGmUfUmCmUfCmUfCmAfAmGmSUmsum-3' (SEQ ID NO: 16);

siHBa2M1S

sense strand: 5 '-GmsAMSmCmUmMUmmAmGfCfAmUmAmUmUmCmAmAmAmAmAmAmAmAmAm-3' (SEQ ID NO: 17),

antisense strand:

5'-UmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmCmsGmsGm-3'(SEQ ID NO:18)；

siHBa2M2S

sense strand: 5 '-GmsAMSCmUmUmGfGfCfAmUmAmUmUmCmAmAmAmAmAmAmAmAmAmAm-3' (SEQ ID NO: 19),

antisense strand:

5'-UmsUfsUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmUmCmsGmsGm-3'(SEQ ID NO:20)；

siHBa1M1P1

sense strand: 5 '-CmUmUmGmGfCfAmUmAmCmUmUmCmAmAmAmAmAmAmam-3'

(SEQ ID NO:5)，

Antisense strand: 5 '-P1-UmUfUmGmAfGmUmAmGmMCmUfCmCmAfAmGmUmUm-3' (SEQ ID NO: 21);

siHBa1M2P1

sense strand: 5 '-CmUmUmGfAmGfCfAmUmAmUmUmUmCmAmAmAmAmAmam-3'

(SEQ ID NO:7)，

Antisense strand: 5 '-P1-UmUfUmGmAfGmUfAfUmGmCmUfCmCmAfAmGmUmUm-3' (SEQ ID NO: 22);

siHBa2M1P1

sense strand: 5 '-GmAmCmUmUmTMAMmGfCfAmUmAmUmUmMcMAmAmAmAmAmam-3' (SEQ ID NO: 9),

Antisense strand:

5'-P1-UmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmCmGmGm(SEQ ID NO:23)；

siHBa2M2P1

sense strand: 5 '-GmAmCmUmUmGfGfCfAmUmAmUmUmCmUmAmAmAmAmAmAmAmAmam-3' (SEQ ID NO: 11),

antisense strand:

5'-P1-UmUfUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmUmCmGmGm-3'(SEQ ID NO:24)；

siHBa1M1SP1

sense strand: 5 '-CmsCmsUmUmTMAMmGfCfAmUmAmUmUmCmAmAmAmAmAmAmam-3'

(SEQ ID NO:13)，

Antisense strand:

5'-P1-UmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmsUmsUm-3'

(SEQ ID NO:25)；

siHBa1M2SP1

sense strand: 5 '-CmsCmsUmUmGfAmGfCfAmUmAmUmUmCmAmAmAmAmAmAmAmAmAmam-3'

(SEQ ID NO:15)，

Antisense strand:

5'-P1-UmsUfsUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmsUmsUm-3'

(SEQ ID NO:26)；

siHBa2M1SP1

sense strand: 5 '-GmsAMSCmMMUmUmTMAMmFGfCfAmUmAmUmUmMcMAmAmAmAmAmAmAmam-3'

(SEQ ID NO:17)，

Antisense strand:

5'-P1-UmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmUmCmsGms

Gm-3'

(SEQ ID NO:27)；

siHBa2M2SP1

sense strand: 5 '-GmsAMSCmMMUmUmGfGfCfAmUmAmUmMUmCmAmAmAmAmAmAmAmAmam-3'

(SEQ ID NO:19)，

Antisense strand:

5'-P1-UmsUfsUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmUmCmsGmsGm-3'

(SEQ ID NO:28)；

SR16-X23460

sense strand: 5 '-CmsCmsUmUmGfAmGfCfAmUmAmUmUmCmAmAmAmAmAmAmAmAmAmam-3'

(SEQ ID NO:140)，

Antisense strand:

5'-VPUmsUfsUmGmAmAfGmUfAfUmGmCmCmUfCmAfAmGmGmsUmsUm-3'；

(SEQ ID NO:141)；

wherein capital C, G, U, A indicates the base composition of the nucleotide; the lower case letter m indicates that the adjacent nucleotide to the left of the letter m is a 2' -methoxy modified nucleotide; the lower case letter f indicates that the adjacent nucleotide to the left of the letter f is a 2' -fluoro modified nucleotide; the lower case letter s indicates that the linkage between two nucleotides adjacent to the letter s is a phosphorothioate linkage; p1 represents that one nucleotide adjacent to the right side of the P1 is a nucleotide modified with 5 '-phosphonucleotide or 5' -phosphoanalog; VP means that the adjacent nucleotide to the right of the VP is a vinyl phosphate modified nucleotide.

18. Use of the oligonucleotide conjugate of claim 17 in the manufacture of a medicament for treating hepatitis caused by expression of hepatitis b virus genes.

19. A method of inhibiting hepatitis b virus gene expression in a hepatocyte in vitro, wherein the method comprises contacting an effective amount of the oligonucleotide conjugate of any one of claims 2-17 with the hepatocyte.

20. A kit comprising the conjugate of any one of claims 2-17.