CN112876534A - Liver targeting compounds and conjugates - Google Patents

Liver targeting compounds and conjugates Download PDF

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CN112876534A
CN112876534A CN201911198284.9A CN201911198284A CN112876534A CN 112876534 A CN112876534 A CN 112876534A CN 201911198284 A CN201911198284 A CN 201911198284A CN 112876534 A CN112876534 A CN 112876534A
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alkyl
group
nucleotide
nucleotide sequence
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CN112876534B (en
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张鸿雁
杨志伟
黄金宇
王秀莲
黄敏印
何涛
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Suzhou Ruibo Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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, and oligonucleotide conjugates for modulating gene expression in hepatocytes, and the conjugatesThe use of a compound in the manufacture of a medicament for the prevention and/or treatment of a related disorder. The conjugates of the present disclosure are capable of specifically targeting hepatocytes, thereby effectively solving problems associated with in vivo delivery of oligonucleotide drugs, are low in toxicity, and have excellent delivery efficiency while maintaining high stability of delivered oligonucleotides.

Description

Liver targeting compounds and conjugates
Technical Field
The present invention relates to the field of delivery of active agents using targeting ligands. In particular, the invention relates to a novel liver targeting compound and a conjugate containing the compound, in particular to an oligonucleotide conjugate, and a preparation method and application thereof.
Background
Oligonucleotide compounds have medically important therapeutic applications. Oligonucleotides can be used to regulate specific genes. Such oligonucleotide compounds include, but are not limited to, oligonucleotide compounds such as small interfering RNA (siRNA), antisense oligonucleotide (ASO), small activating RNA (sarna), and microRNA (microRNA). In particular therapeutic applications, oligonucleotides and their analogs that are expressed only in specific tissues or locations may be selected to target the relevant disease or condition at a specific 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 is still a need for improvements in their pharmacological properties, such as targeted delivery to the lesion to increase the selectivity of the therapeutic agent, to increase its biological activity and efficacy. Targeted conjugation delivery technology has recently become the most widely studied class of delivery systems, focusing primarily on liver-targeted delivery.
Disclosure of Invention
In a first aspect, the present invention provides a novel liver targeting compound (hereinafter sometimes also referred to as "conjugate molecule") having a structure represented by formula (I):
Figure BDA0002295222110000011
wherein the content of the first and second substances,
m represents an integer of 1 to 6;
each R2Independently selected from H, C1-C10Alkyl radical, C1-C10Haloalkyl or C1-C10An alkoxy group;
L1represents a carbonyl group, a carbonyl group (C)1-C10Alkylene) or carbonyl- (oxyethyl)kWherein k represents an integer of 1 to 10;
g represents a first hydroxyl protecting group;
L2represents 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
L2Comprising a solid support attached by said covalent bond;
e represents a ligand M having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells1Or is or
Represents said ligand M1The reactive hydroxyl groups in (1), if any, are all substituted with a second hydroxyl protecting group;
each L3Is a straight chain 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、C2-C10Alkenylene radical, C2-C10Alkynylene, C6-C10Arylene radical, C3-C18Heterocyclylene and C5-C10A heteroarylene group; and the number of the first and second electrodes,
each L3Optionally having any one or more substituents selected from the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, aryl, heteroaryl, and heteroaryl,-OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Alkyl halides, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl), -N (C)1-C10Alkyl) (C1-C10Alkylphenyl), -NH (C)1-C10Alkylphenyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON (C)1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2，-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl radical C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl group C (O) (phenyl), -C (O) C1-C10Alkyl, -C (O) C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -SO2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl).
In a second aspect, the present invention provides a novel oligonucleotide conjugate having the structure represented by formula (II):
Figure BDA0002295222110000021
wherein the content of the first and second substances,
E1is OH, SH or BH2Nu represents a functional oligonucleotide;
m represents an integer of 1 to 6;
each R2Independently selected from H, C1-C10Alkyl radical, C1-C10Haloalkyl or C1-C10An alkoxy group;
L1represents a carbonyl group, a carbonyl group (C)1-C10Alkylene) or carbonyl- (oxyethyl)kWherein k represents an integer of 1 to 10;
E2represents amino, hydroxy, -amino (C)1-C10Alkylene) amino, -amino (C)1-C10Alkylene) hydroxy, -hydroxy (C)1-C10Alkylene) amino or hydroxy (C)1-C10Alkylene) hydroxyl, the alkylene optionally bearing one or more substituents selected from the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Alkyl halides, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON (C)1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2，-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl radical C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl group C (O) (phenyl), -C (O) C1-C10Alkyl radical、-C(O)C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -SO2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl);
M1represents a ligand having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells;
each L3Is a straight chain alkyl 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、C2-C10Alkenylene radical, C2-C10Alkynylene, C6-C10Arylene radical, C3-C18Heterocyclylene and C5-C10A heteroarylene group; and is
Each L3May optionally have any one or more substituents selected from the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Alkyl halides, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl), -N (C)1-C10Alkyl) (C1-C10Alkylphenyl), -NH (C)1-C10Alkylphenyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON (C)1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2，-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl radical C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl group C (O) (phenyl), -C (O) C1-C10Alkyl, -C (O) C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -SO2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl).
In a third aspect, the 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 present invention provides a method of modulating the expression of a gene in a hepatocyte, the modulation comprising inhibiting or enhancing the expression of the gene, the method comprising contacting an oligonucleotide conjugate of the invention with the hepatocyte.
The oligonucleotide conjugate of the invention shows higher gene expression inhibition activity in hepatocytes, and is expected to effectively improve the in vivo delivery efficiency of siRNA, and has low toxicity, 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.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Incorporation by reference all publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
Definition of
In the above and the following, particularly in describing the method for preparing the delivery compound of the present disclosure (hereinafter, sometimes also referred to as "conjugate molecule of the present disclosure" or simply "conjugate molecule") or the method for preparing an oligonucleotide conjugate, unless otherwise specified, the nucleoside monomer (nucleoside monomer) means, depending on the RNA sequence to be prepared, the "unmodified or modified RNA phosphoramidite" used for so-called solid phase phosphoramidite synthesis, respectively, which is a method well known in the art for synthesizing RNA. RNA phosphoramidites are also referred to herein as nucleoside phosphoramidites. Unless otherwise indicated, the nucleotide monomers used in the present disclosure are commercially available.
As used herein, a dash ("-") that is not between two letters or two symbols is used to indicate a position of a point of attachment for a substituent. For example: -C1-C10alkyl-NH2Through C1-C10Alkyl groups are attached.
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. It will be understood by those skilled in the art that, for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically non-feasible, and/or inherently unstable.
As used herein, "alkyl" refers to straight and branched chains having the specified number of carbon atoms, typically from 1 to 20 carbon atoms, for example from 1 to 10 carbon atoms, such as from 1 to 8 or from 1 to 6 carbon atoms. E.g. C1-C6Alkyl groups include straight and branched chain alkyl groups of 1 to 6 carbon atoms. When referring to an alkyl residue having a particular 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 and refers to the same residue as alkyl but with two points of attachment.
As used herein, "alkenyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond derived by the loss of one hydrogen atom from each of the 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: a vinyl group; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl; butenyl, e.g., but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-2-yl, but-1, 3-dien-1-yl, but-1, 3-dien-2-yl, and the like. In certain embodiments, alkenyl groups have 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to the same residue as alkenyl, but with 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: an ethynyl group; propynyl groups, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl groups such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl and the like. In certain embodiments, alkynyl groups have 2 to 20 carbon atoms, and in other embodiments 2 to 10, 2 to 8, or 2 to 6 carbons.
As used herein, "alkoxy" refers to an alkyl group of the indicated 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 monocyclic or polycyclic hydrocarbon ring system formed by the removal of a hydrogen atom from a ring carbon atom. The aromatic monocyclic or polycyclic hydrocarbon ring systems contain only hydrogen and carbon of 6 to 18 carbon atoms, wherein at least one ring in 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, naphthyl, and the like. Arylene is a subset of aryl and refers to the same residue as aryl, but with two points of attachment.
As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring, typically having 3 to 7 cyclic carbon atoms. The rings 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 (norbonane).
As used herein, "halogen substituent" or "halo" refers to fluoro, chloro, bromo, and iodo, and the term "halogen" includes fluoro, chloro, bromo, and iodo.
As used herein, "haloalkyl" refers to an alkyl group having the specified number of carbon atoms substituted with one or more, up to the maximum allowable number of halogen atoms. Examples of haloalkyl groups 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 stated in the specification, a heterocyclyl group is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, and may include fused or bridged ring systems. The heteroatoms in the heterocyclic group may be optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. Heterocyclyl groups are partially or fully saturated. The heterocyclyl group may be attached to the remainder of the molecule through any ring atom. Examples of such heterocyclic groups include, but are not limited to: dioxanyl, thienyl [1,3] dithioyl (thienyl [1,3] dithianyl), decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithioyl (trithionyl), tetrahydropyranyl, thiomorpholinyl (thiomorpholinyl), 1-oxothiomorpholinyl (1 oxothiomorpholinyl), and 1, 1-dioxothiomorpholinyl (1, 1-dioxothiomorpholinyl).
"heteroaryl" refers to a group derived from a 3-to 18-membered aromatic ring radical containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, a heteroaryl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., comprises a cyclic delocalized (4n +2) pi-electron system, according to huckel theory. Heteroaryl includes fused or bridged ring systems. The heteroatoms in the heteroaryl group are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heteroaryl group is attached through any atom in the ring. Examples of heteroaryl groups include, but are not limited to: azacyclotrienoyl, acridinyl, benzimidazolyl, benzindolyl, 1, 3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo [ d ] thiazolyl, benzothiadiazolyl, benzo [ b ] [1,4] dioxepinyl (benzo [ b ] [1,4] dioxepinyl), benzo [ b ] [1,4] oxazinyl (benzo [ b ] [1,4] oxazinyl), 1,4-benzodioxanyl (1,4-benzodioxanyl), benzonaphthofuranyl, benzoxazolyl, benzodioxolyl (benzodioxolyl), benzodioxinyl (benzodioxanyl), benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothiophenyl, benzothieno [3,2-d ] pyrimidinyl, benzotriazolyl, benzo [4,6] imidazo [1,2-a ] pyridinyl, Carbazolyl, cinnolinyl, cyclopenta [ d ] pyrimidinyl, 6, 7-dihydro-5H-cyclopenta [4,5] thieno [2,3-d ] pyrimidinyl, 5,6-dihydrobenzo [ H ] quinazolinyl (5,6-dihydrobenzo [ H ] quinazolinyl), 5,6-dihydrobenzo [ H ] cinnolinyl (5,6-dihydrobenzo [ H ] cinnolinyl), 6, 7-dihydro-5H-benzo [6,7] cyclohepta [1,2-c ] pyridazinyl, dibenzofuranyl, dibenzothienyl, furanyl, furanonyl, furo [3,2-c ] pyridinyl, 5,6,7,8,9, 10-hexahydrocycloocta [ d ] pyrimidinyl, 5,6,7,8,9, 10-hexahydrocycloocta [ d ] pyridazinyl, 5,6,7,8,9, 10-hexahydrocycloocta [ d ] pyridazinyl, 7,8,9, 10-hexahydrocycloocta [ d ] pyridyl, isothiazolyl, imidazolyl, indazolyl (indazolyl), indolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5, 8-methanol-5, 6,7,8-tetrahydroquinazolinyl (5,8-methano-5,6,7,8-tetrahydroquinazolinyl), naphthyridinyl (naphthyridinyl), 1,6-naphthyridinonyl (1,6-naphthyridinonyl), oxadiazolyl, 2-oxazepinyl (2-oxoazepinyl), oxazolyl, oxacyclopropane (oxacinnanyl), 5,6,6a,7,8,9,10,10 a-octahydrobenzo [ H ] quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, and oxazolyl, Phthalazinyl (phthalazinyl), pteridinyl (pteridinyl), purinyl, pyrrolyl, pyrazolyl, pyrazolo [3,4-d ] pyrimidinyl, pyridyl, pyrido [3,2-d ] pyrimidinyl, pyrido [3,4-d ] pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7, 8-tetrahydrobenzo [4,5] thieno [2,3-d ] pyrimidinyl, 6,7,8, 9-tetrahydro-5H-cyclohepta [4,5] thieno [2,3-d ] pyrimidinyl, 5,6,7, 8-tetrahydropyrido [4,5-c ] pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridinyl, Triazinyl, thieno [2,3-d ] pyrimidinyl, thieno [3,2-d ] pyrimidinyl, thieno [2,3-c ] pyridinyl (thieno [2,3-c ] pridinyl) and thienyl (thiophenyl/thiophenyl).
Various hydroxyl protecting groups may be used in the present disclosure. In general, protecting groups render a chemical functional group insensitive to the particular reaction conditions, and may be appended to 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 Greenea and Wuts, Protective Groups in Organic Synthesis, Chapter 2,2d ed, John Wiley & Sons, New York, 1991, which are incorporated herein by reference in their entirety. In some embodiments, the protecting group is stable under basic conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups that may be used herein include Dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthine-9-yl (Pixyl), and 9- (p-methoxyphenyl) xanthine-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 tert-butyldimethylsilyl (TBS or TBDMS). Non-exclusive examples of hydroxy protecting groups that may be used herein include hydrocarbyl acyl groups.
The term "subject", as used herein, refers to any animal, e.g., a mammal or a marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any species of poultry.
As used herein, "treat," "alleviate," or "improve" may be used interchangeably herein. These terms refer to methods of achieving beneficial or desired results, including but not limited to therapeutic benefits. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.
As used herein, "prevent" and "prevention" are used interchangeably. These terms refer to methods of achieving beneficial or desired results, including but not limited to prophylactic benefits. To obtain a "prophylactic benefit," the conjugate or composition can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more pathological symptoms of a disease, even though a diagnosis of the disease may not have been made.
Liver targeting compounds
In one aspect, the present invention discloses a conjugate molecule for delivery of an active agent or 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 part 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 hepatocyte surface receptors and thus specifically target liver tissue. In some embodiments, the conjugate molecules of the present disclosure specifically target cell surface receptors specific to hepatocytes. In some embodiments, the conjugate molecules of the present disclosure specifically target asialoglycoprotein receptors (ASGPR) on the surface of hepatocytes.
As used herein, "active agent" and "active drug" are used interchangeably and both refer to a molecule capable of being delivered by a conjugate molecule of the present disclosure. In some embodiments, the active agent is an agent capable of delivering to a hepatocyte. Such agents 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 the structure shown in formula (I):
Figure BDA0002295222110000051
wherein the content of the first and second substances,
m represents an integer of 1 to 6;
each R2Independently selected from H, C1-C10Alkyl radical, C1-C10Haloalkyl or C1-C10An alkoxy group;
L1represents a carbonyl group, a carbonyl group (C)1-C10Alkylene) or carbonyl- (oxyethyl)kWherein k represents an integer of 1 to 10;
g represents a first hydroxyl protecting group;
L2represents 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
L2Comprising a solid support attached by said covalent bond;
e represents a ligand M having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells1Or is or
Represents said ligand M1The reactive hydroxyl groups in (1), if any, are all substituted with a second hydroxyl protecting group;
each L3Is a straight chain 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、C2-C10Alkenylene radical, C2-C10Alkynylene, C6-C10Arylene radical, C3-C18Heterocyclylene and C5-C10A heteroarylene group; and the number of the first and second electrodes,
each L3Optionally having any one or more substituents selected from the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Alkyl halides, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl), -N (C)1-C10Alkyl) (C1-C10Alkylphenyl), -NH (C)1-C10Alkylphenyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON (C)1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2，-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl radical C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl group C (O) (phenyl), -C (O) C1-C10Alkyl, -C (O) C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -SO2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl).
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 "conjugate molecule") is at least 2; in some embodiments, m.gtoreq.3, which allows for the formation of oligonucleotide conjugates (II) formed from the conjugate molecules,M1the number of ligands may be at least 3, such that M1The ligand binds more readily to the hepatic surface asialoglycoprotein receptor, thereby facilitating the entry of the oligonucleotide conjugate into the cell by endocytosis. Experiments show that when M is used1When the number of ligands is more than 3, M1The increased ease with which ligands bind to the liver surface asialoglycoprotein receptor is not significant, and thus, in some embodiments m is an integer from 2 to 4, taken together from the aspects of ease of synthesis, structure/process cost, and delivery efficiency.
In some embodiments, each R is2Independently of one another, H, methyl or ethyl; in some embodiments, each R is2Are all H;
L1the function of (a) is to provide a suitable spatial location and chemical environment for the attachment of the liver targeting compound to the oligonucleotide. To this end, in some embodiments, L1Represents a carbonyl group, a carbonyl group (C)1-C10Alkylene) or carbonyl- (oxyethyl)kWherein k represents an integer of 1 to 10. In some embodiments, L1Is carbonyl (C)2-C6Alkylene) or carbonyl (oxyethyl)kWherein k is an integer of 2 to 7, preferably an integer of 2 to 4. In some embodiments, L1Is carbonyl butylene.
G represents a first hydroxyl protecting group. In some embodiments, the first hydroxyl protecting group functions to liberate a reactive hydroxyl group under suitable conditions for forming a covalent linkage with 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 '-bismethoxytrityl (DMTr), 4' -trimethoxybenzyl and tert-butyldimethylsilyl (TBS or TBDMS); in some embodiments, G is 4,4' -bis-methoxytrityl (DMTr).
In some embodiments, L2Comprising a group capable of attachment to a solid support by a covalent bond. In some embodiments, L2Comprising a bridge capable of being bridged by a hydrocarbyl diacid, thereby being supported on a solid phaseThe hydroxyl group or the amino group on the body forms an amide bond or an ester bond to be connected. In some embodiments, L2Represents-amino (C)1-C10Alkyl alcohol) or-amino (ethoxy)q2Ethanol, wherein q2 represents an integer from 1 to 10, preferably an integer from 2 to 7. In other embodiments, L2A group capable of forming an ester bond or an amide bond with a hydroxyl group or an amino group on a solid support; in some embodiments, L2Comprising a carboxyl group or a salt of a cationic carboxylic acid, which cation 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, L2Comprising an oxyacylalkylenecarboxy (or carboxylate) or an aminoacylalkylenecarboxy (or carboxylate). In some embodiments, L2Comprising a solid support linked by forming an ester or amide bond with a hydroxyl or amino group. In some embodiments, L2Containing amino or hydroxy groups, or L2Comprises a structure represented by formula (C1), (C2), (C3), (C4), (C1'), (C3') or (C4 '):
Figure BDA0002295222110000061
Figure BDA0002295222110000071
in the formula, each q1Independently an integer of 1 to 5, each X independently is O or NH, M+Is a cation; in some embodiments, M+Selected from any one of hydrogen ions, ammonium ions, alkali metal ions or alkaline earth metal ions, SPS represents a solid phase carrier,
Figure BDA0002295222110000072
indicates the site at which the group is covalently attached.
E represents a ligand M having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells1Or is or
Represents said ligand M1In (1)All reactive hydroxyl groups, if any, are protected with a second hydroxyl protecting group. In some embodiments of the present disclosure, the hydroxy group protected by the second hydroxy protecting group has the form YCOO-, wherein each Y is independently selected from the group consisting of: c1-C10Alkyl and C6-C10Aryl radical, said C1-C10Alkyl or C6-C10Aryl optionally has one or more substituents selected from the group consisting of halogen substituents and C1-C6Alkyl groups. In some embodiments, each Y is independently selected from methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and C1-C6One of alkyl phenyl;
the ligand M having affinity for asialoglycoprotein receptor on the surface of mammalian liver cell1May be any ligand having affinity for asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes, the class of such ligands being well known to those skilled in the art. In some embodiments, at least one ligand M1Is a saccharide. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand M1Is monosaccharide, disaccharide, trisaccharide or polysaccharide. In some embodiments, each ligand M1Is monosaccharide, disaccharide, trisaccharide or polysaccharide. In some embodiments, at least one ligand M1Is a modified sugar. In some embodiments, each ligand M1Is a modified sugar. In some embodiments, each ligand M1Independently selected from polysaccharides, modified polysaccharides, monosaccharides or monosaccharide derivatives. In some embodiments, each or at least one ligand M1May be independently selected from the group consisting of: glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose, maltose and its derivatives, arabinose and its derivatives, fructoseAnd derivatives thereof, and sialic acid.
In some embodiments, the ligand M1May 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-mannofuranose, beta 0-D-mannopyranose, beta 1-D-mannopyranose, beta 2-D-glucopyranose, beta 3-D-glucopyranose, alpha-D-glucopyranose, beta-D-glucofuranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, alpha-D-galactopyranose, beta-D-galactofuranose, alpha-D-galactofuranose, beta-D-galactopyranose, alpha-D, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionyl galactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [ (R) -1-carboxyethyl]-2-deoxy-beta-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-glycolyl-alpha-neuraminic acid, 5-thio-beta-D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranoside methyl ester, 4-thio-beta-D-galactopyranose, beta-glucopyranose, beta, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio-alpha-D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allose nitrile, ribose, D-4-thioribose, L-ribose, L-4-thioribose. In some embodiments, at least one M1Is N-acetylgalactosamine (GalNAc); in some embodiments, each M is1Are all N-acetylgalactosamine. In some embodiments, ligand M1See, for example, the disclosure of 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 being selected from the group consisting of a polysaccharide, a modified polysaccharide, mannose, galactose, a mannose derivative, a galactose derivative, D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranoseSaccharides, D-glucose, L-glucose, D-galactose, L-galactose, alpha-D-mannofuranose, beta 0-D-mannopyranose, beta 1-D-mannopyranose, beta 2-D-glucopyranose, beta 3-D-glucopyranose, beta 4-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructofuranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, alpha-D-galactosamine, N-acetylgalactosamine, beta-D-galactopyranose, beta-4-D-fructofuranose, beta-D-glucofurano, 2-amino-3-O- [ (R) -1-carboxyethyl]-2-deoxy-beta-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-glycolyl-alpha-neuraminic acid, 5-thio-beta-D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranoside methyl ester, 4-thio-beta-D-galactopyranose, beta-glucopyranose, beta, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio-alpha-D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allose nitrile, ribose, D-4-thioribose, L-ribose 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 for their delivery, and enhances serum stability by modifying the nucleotides of the sirnas. This 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 the same, and enhances serum stability by modifying the nucleotides of the sirnas. The document also discloses siRNA conjugates.
N-acetylgalactosamine (GalNAc), a ligand that binds to the hepatic surface asialoglycoprotein receptor. Asialoglycoprotein receptor (ASGPR) is an endocytotic receptor specifically expressed by hepatocytes. In recent years, the high affinity ligand of ASGPR, N-acetylgalactosamine (GalNAc), has been used as a targeting molecule for nucleic acidsThe liver targeting delivery of the drug achieves better effect. For example, alnilamel corporation (alanam pharmaceuticals, Inc.) first reported that sirnas based on GalNAc conjugation technology exert interfering activity in mice (Nair et al, j.am.chem.so., 2014,136, 1695-. The article reports that three clusters of GalNAc conjugated sirnas exhibit good delivery activity in vitro and in vivo experiments. Single dose ED by subcutaneous administration in vivo experiments in mice501mg/kg, and the single injection dosage is less than 1 ml. In long-term administration experiments, stable interfering activity for up to 9 months can be obtained by subcutaneous injection once a week.
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-butyrylgalactosamine, N-isobutyrylgalactosamine, 4-thio-beta-D-galactopyranose.
In some preferred embodiments, each E is independently selected from one of the groups of formula a46-a 54:
Figure BDA0002295222110000081
in some embodiments, E is formula a49 or a 50. In some embodiments, each Y is methyl.
L3The function of (a) is to provide a link to the N atom on the pyrrolidine ring in formula (I) of the present disclosure for providing liver targeting functions for the oligonucleotide conjugates of the present disclosure. In some embodiments, L3Independently selected from the group consisting of groups of formula A1-A26 and any combination thereof:
Figure BDA0002295222110000091
Figure BDA0002295222110000101
wherein each j1 is independently an integer from 1-20;
each j2 is independently an integer from 1-20;
each R' is independently C1-C10-an alkyl group;
each Ra is independently selected from the group consisting of groups of formula a27-a 45:
Figure BDA0002295222110000102
Figure BDA0002295222110000111
each Rb is independently C1-C10-an alkyl group;
Figure BDA0002295222110000113
indicates the site at which the group is covalently attached.
In some embodiments, L3A combination of one or more linkages selected from a1, a4, a5, a6, A8, a10, a11, and a 13. In some embodiments, L3A linked combination of at least 2 selected from a1, a4, A8, a10, and a 11. In some embodiments, L3At least 2 connecting combinations selected from A1, A8 and A10.
In some embodiments, L3May be 3-25, 3-20, 4-15, or 5-12 atoms in length. In some embodiments, L3Is 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. Said L3The length of (b) is the number of chain-forming atoms in the longest atom chain formed from the atom attached to the N atom of the pyrrolidine ring in the compound of formula (I) to the atom attached to E.
According to some embodiments of the present disclosure, each j1 is independently an integer from 2 to 10, and 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 C1-C4In 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 a 28. In some embodiments, each Rb is independently C1-C5In some embodiments, each Rb is independently one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', Ra, Rb in formulas a1-a26 are each selected to achieve stable attachment of the ligand to N on the pyrrolidine ring and to make the spatial location between the ligands more suitable for binding of the ligands to hepatic surface asialoglycoprotein receptors.
In one embodiment, the liver targeting compound of the present invention has the structure represented by formula (I-1), (I-2), (I-3), (I-4), (I-5) or (I-6):
Figure BDA0002295222110000112
Figure BDA0002295222110000121
Figure BDA0002295222110000131
wherein SPS represents a solid support;
the solid phase carrier may be one known in the art and usable for solid phase synthesis of nucleic acids, for example, commercially available general solid phase carrier: (
Figure BDA0002295222110000133
HL UnyLinkerTM300oligonucleotid Synthesis Support, Kinovate Life Sciences, having the structure shown in formula B80):
Figure BDA0002295222110000132
wherein Resin represents Resin.
In some embodiments, SPS represents a resin. In some embodiments, the SPS may be a hydroxyl or amino resin, or the SPS further comprises a linking group, when L1The covalent bond can be formed directly, or through the linking group, with a hydroxyl or amino group 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)2The group has a hydroxyl group, and in this case, the compound of the formula (I) can be obtained by the following production method: the method comprises contacting a compound of formula (X-11) with a deprotecting agent in an organic solvent to remove a hydroxy-protecting group X from the compound of formula (X-11)11And (4) separating.
Figure BDA0002295222110000141
Wherein L is1、L3、G、E、R2M is as defined above;
L11is a linking group which obtains the above-mentioned L by linking a hydroxyl group2A group; x11Is a hydroxyl protecting group.
The organic solvent can be halogenated alkane, ether or nitrile solvent; in some embodiments, the organic solvent is Tetrahydrofuran (THF). The organic solvent may be used in an amount of 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 used11The deprotecting agent is selected. For example, in some embodiments, a hydroxyl protecting group X11Tetramethylsilyl (TMS) or tert-butyldimethylsilyl (TBDMS), and in this case, the deprotecting agent may be, for example, tetrabutylammonium fluoride (TBAF). The deprotection agent may be used in a molar ratio of 2:1 to 10:1, for example 4:1 to 6:1, with respect to the compound of formula (X-11). The reaction may be carried out at a suitable temperature, such as 0-25 deg.C, for 2-8 hours. In some embodiments, the reaction is performed at room temperature for 4 h. In some embodiments, the reaction is carried out in an ice bath for 8 h. In some embodiments, the compound of formula (I) produced by the reaction may be isolated using, for example, column chromatography, under conditions such as a 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, a compound shown in PNB-11 below.
The compound 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 the compound of formula (X-10) with the compound of formula (X-22) in an organic solvent in the presence of a condensation catalyst and an organic base to perform a condensation reaction, and separating.
Figure BDA0002295222110000142
Wherein L is1、L3、G、E、m、R2、L11、X11As defined above;
the organic solvent can be halogenated alkane, ether or nitrile solvent; in some embodiments, the organic solvent is dichloromethane. The organic solvent may be used in an amount of 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, it may be, for example, 2- (7-benzotriazole oxide) -N, N, 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, with respect to the compound of formula (X-10). The organic base may be, for example, an amine-type 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 of 1.5:1 to 8:1, for example 2:1 to 5:1, relative to the compound of formula (X-10). The reaction may be carried out at a suitable temperature, such as 0-25 deg.C, for 2-8 hours. In some embodiments, the reaction is carried out in an ice bath for 4 h. In some embodiments, the end of the reaction can be monitored by, for example, LC-MS. In some embodiments, the compound of formula (X-11) produced by the reaction may be separated using, for example, column chromatography, under conditions such as a gradient elution using ethyl acetate: methanol-100: 1-12: 1.
The compound of formula (X-22) can be prepared by any method by those skilled in the art, or can be obtained commercially. For example, when L is1In the case of carbonylbutylene, the compound of formula (X-22) can be obtained by protecting the hydroxyl group with commercially available hydroxypentanoic acid by a method known to those skilled in the art.
The compound 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 remove an amino protecting group G in the compound of formula (X-9) via a deprotection reactionNThe crude product is isolated or used directly in the subsequent reaction.
Figure BDA0002295222110000151
Wherein L is3、E、m、R2、L11、X11As defined above;
GNis an amino protecting group as described above; in some embodiments, GNMay be, for example, an Fmoc protecting group.
The organic solvent can be halogenated alkane, ether or nitrile solvent; in some embodiments, the organic solvent is acetonitrile. The organic solvent may be used in an amount of 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 of 5:1 to 30:1L/mol, for example 6:1 to 20:1, relative to the compound of formula (X-9). The deprotection reaction may be carried out at 0-40 ℃ for 1-4h, for example at room temperature for 2h, and/or the end of the reaction 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 a 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, under conditions such as a gradient elution using ethyl acetate: methanol-100: 1-10: 1.
The compound 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 the steps of 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 perform a condensation reaction, and separating.
Figure BDA0002295222110000161
Wherein L is3、E、m、R2、L11、GN、X11As defined above.
In some embodiments, the compound of formula (X-21) is a carboxylic acid, i.e., L3Having a carbonyl group attached to the hydroxyl group shown in formula (X-21). At this time, the compound of formula (X-21) may use, for example, compounds disclosed in j.am. chem.soc.2014,136, 169581-16961, or, the compound of formula (X-21) may be prepared by various methods by those skilled in the art, for example, certain compounds of formula (X-21) may be prepared by reference to the methods disclosed in US 8,106,022B2, example 1, the entire contents of which are incorporated herein by reference in their entirety.
The organic solvent may be one or more of acetonitrile, an epoxy-based solvent, which is dioxane and/or tetrahydrofuran in some embodiments, an ether-based solvent, which is diethyl ether and/or methyl tert-butyl ether in some embodiments, an ether-based solvent, which is one or more of dichloromethane, chloroform and 1, 2-dichloroethane in some embodiments, N-Dimethylformamide (DMF), dimethylsulfoxide, N-Dimethylformamide (DMF) in some embodiments. The organic solvent may be used in an amount of 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, and in this case, the condensing agent is an amide-forming reaction condensing agent. In some embodiments, the amide-forming reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (DEPBT), O-benzotriazol-tetramethyluronium hexafluorophosphate or 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, and in further embodiments 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine Hydrochloride (HATU). The molar ratio of the amide-forming condensation agent to the compound of formula (X-8) is from 1:1 to 5:1, and 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, and in some embodiments, from 3:1 to 10: 1. The reaction may be carried out, for example, under ice bath conditions for 2 to 6 hours. The compound of formula (X-9) may be isolated from the reaction mixture using any suitable separation method. In some embodiments, the compound of formula (X-9) may be isolated by extraction with water and a haloalkane-based solvent, washing with a salt-based solvent after combining the organic phases, drying with sulfate, and subsequent chromatographic methods, e.g., using the following chromatographic conditions: (1) normal phase purification of silica gel: 200-mesh 300-mesh silica gel filler is subjected to gradient elution by using ethyl acetate and methanol in a ratio of 100:1-20: 1; or (2) reversed-phase purification: c18, C8 reversed phase packing, eluting with a gradient of methanol to acetonitrile 0.1:1 to 1: 0.1. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (X-9) which may be used directly in a subsequent reaction.
In some embodiments, a compound of formula (X-8) is reacted with a sufficient amount of one compound of formula (X-21) in a single reaction to form the desired compound of formula (X-9), in which case each E-L is3The portions are identical to each other. In some embodiments, the compound of formula (X-8) may be reacted with a different compound of formula (X-21), i.e., L, by batching the compound of formula (X-8) as desired3And/or the compound of formula (X-21) having different E is reacted so that the compound of formula (X-9) produced contains two or more kinds of E and/or L3. For example, for 1eq of a compound of formula (X-8), it may be contacted first with 2eq of a first compound of formula (X-21) to which the first E-L is attached at both amino groups in the compound of formula (X-8)3And subsequently, continuing its contact with (m-2) eq of a second compound of formula (X-21) (m being defined and having the values given above), thereby linking a second E-L to the (m-2) amino groups in the compound of formula (7)3And (4) partial.
The compound 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-OX11And (4) separating.
Figure BDA0002295222110000171
Wherein m and L11、R2、GNAs defined above.
The organic solvent may be one or more of acetonitrile, epoxy-based solvents, which are dioxane and/or tetrahydrofuran in some embodiments, ether-based solvents, haloalkane-based solvents, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine, or mixtures thereofThe ethereal solvent is in some embodiments diethyl ether and/or methyl tert-butyl ether, the haloalkane solvent is in one embodiment one or more of dichloromethane, chloroform and 1, 2-dichloroethane, and in some embodiments the organic solvent is N, N-Dimethylformamide (DMF). The organic solvent may be used in an amount of 2 to 15L/mol, for example, 3 to 8L/mol, relative to the compound of formula (X-7). In some embodiments, the hydroxy protecting agent provides a hydroxy protecting group X11In some embodiments, it may be, for example, tert-butyldimethylsilyl chloride (TBSCl), in which case X11Is 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, and 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 is 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, and 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 separation method. In some embodiments, the compound of formula (X-8) may be isolated by extraction with water and a haloalkane-based solvent, combining the organic phases, washing with a salt-based solvent, drying with a sulfate salt, and then chromatographically. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (X-8) which may be used directly in a subsequent reaction.
The compound 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)'NAnd a hydroxy-protecting group X11And (4) separating.
Figure BDA0002295222110000172
Wherein m and L11、R2、GN、X11As defined above.
G’NIs a reaction of with GNA second amino protecting group which is removed by reaction with an acid more readily than when G is used, e.g.NWhen it is a Fmoc protecting group, G'NIt may be, for example, tert-butyloxycarbonyl (Boc). The solvent may be one or more of an alcohol solvent, an ether solvent, and 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 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 significant excess and is related to the value of m. In the case where m is 2 to 5, the molar ratio of the acid to the compound represented by formula (X-6) may be, for example, 50:1 to 400:1, and in some embodiments, 100:1 to 250: 1. The reaction may be carried out, for example, at room temperature for 3 to 6 hours, as long as sufficient reaction is possible, and/or the end of the reaction is monitored using TLC. The compound of formula (X-7) may be isolated from the reaction mixture using any suitable separation method. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (X-7) which may be used directly in a subsequent reaction.
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).
Figure BDA0002295222110000181
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 a compound represented by formula (X-1) and formula (X-20) in an organic solvent in the presence of a condensing agent to cause a condensation reaction:
X1-L20-OX11
formula (X-20)
Wherein m and L11、GN、G’N、X11As defined above.
L20To correspond to L11A moiety of a group; x1Is amino or hydroxy, condensed with a carboxyl group in a compound of formula (X-1), together with L20The moieties together obtaining a linking group L11. The organic solvent and the condensing agent may be selected according to amide-forming or ester-forming methods well known in the art. For example, in some embodiments, the organic solvent may be dichloromethane, the organic solvent 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 the organic base to the compound of formula (X-1) of 1:1 to 10:1, e.g., 1.5:1 to 5: 1. The condensation reaction can be carried out, for example, at room temperature for 3h, and/or the end of the reaction monitored by TLC. In some embodiments, the compound of formula (X-6) may be separated by column chromatography, for example, using 200-.
The compound of formula (X-1) can be easily prepared by a person skilled in the art according to well-known preparation methods, or can be purchased commercially. For example, when each R is2Are all hydrogen, GNIs Fmoc protecting group, G'NFor the Boc protecting group, the compound of formula (X-1) is commercially available from Annagel corporation.
In some embodiments, m is an integer ≧ 2. In this case, it is necessary to specifically remove the amino-protecting group G in the previous intermediate (for example, in the case of producing an X-6 compound (m ═ 2) (similarly, also referred to as a compound of formula (X-6-2)), the aforementioned X-6-1 compound is used as the "previous intermediate"; in the case of producing an X-6 compound (m ═ 3) (hereinafter, also referred to as a compound of formula (X-6-3)), the X-6-2 compound is used as the "previous intermediate", and so on) in addition to the above reactionNAnd the obtained compound is subjected to the condensation reaction with the compound of the formula (X-1). The solvent, the reaction reagent, the conditions and the separation method for the condensation reaction may be the same as or different from those described above, and may be selected according to the structure of the desired productAppropriate reagents, reaction conditions and methods.
In some embodiments, m is 3, and in this case, the compound of formula (X-6) (the compound of formula (X-6-3)) can be prepared by:
(i) preparing a compound of formula (X-6-1) as described previously;
(ii) in an organic solvent, the compound of the formula (X-6-1) is contacted with a base to remove the amino protecting group GNSeparating; wherein the organic solvent may be, for example, acetonitrile, and 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 column chromatography with a gradient elution, for example, ethyl acetate: methanol-100: 1-10:1(V: V), to finally obtain the compound of formula (X-6-1 a);
Figure BDA0002295222110000182
(iii) contacting the compound of formula (X-1) with the compound of formula (X-6-1a) in an organic solvent in the presence of a condensing agent to perform condensation reaction, and separating to obtain the compound of formula (X-6-2); wherein the solvents, condensing agents and reaction conditions are selected the same as in step (i) and the column chromatography is performed using, for example, a gradient elution column of ethyl acetate: methanol-100: 1-15: 1:
Figure BDA0002295222110000191
(iv) contacting the compound of formula (X-6-2) with a base in an organic solvent to remove the amino protecting group GNIsolating to obtain the compound of formula (X-6-2 a); (iii) the solvents, reagents and reaction conditions are the same as in step (ii) and the column chromatography is performed using, for example, a gradient eluent of ethyl acetate: methanol-100: 1-10: 1;
Figure BDA0002295222110000192
(v) contacting the compound of formula (X-1) with the compound of formula (X-6-2a) in an organic solvent in the presence of a condensing agent to perform condensation reaction, and separating to obtain the compound of formula (X-6-3); wherein the solvents, condensing agents and reaction conditions are selected the same as in step (i) and the column chromatography is performed using, for example, a gradient elution column of ethyl acetate: methanol-100: 1-8: 1:
Figure BDA0002295222110000193
oligonucleotide conjugates
In one aspect, the present disclosure provides an oligonucleotide conjugate having a structure represented by formula (II):
Figure BDA0002295222110000194
wherein the content of the first and second substances,
E1is OH, SH or BH2；
Nu represents a functional oligonucleotide;
m、L1、L3、R2the definitions and alternative ranges of (a) are the same as above;
M1represents a ligand having affinity for asialoglycoprotein receptor on the surface of mammalian liver cells, said ligand being as defined and optionally within the scope of E above;
E2represents amino, hydroxy, -amino (C)1-C10Alkylene) amino, -amino (C)1-C10Alkylene) hydroxy, -hydroxy (C)1-C10Alkylene) amino or hydroxy (C)1-C10Alkylene) hydroxyl, the alkylene optionally bearing one or more substituents selected from the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Alkyl halides, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON (C)1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2，-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl radical C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl group C (O) (phenyl), -C (O) C1-C10Alkyl, -C (O) C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -SO2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl); in some embodiments, E2Represents-amino (C)1-C10Alkylene) hydroxy; in some embodiments, E2Is hydroxyethylamino (-NHCH)2CH2OH)。
By forming an oligonucleotide conjugate of formula (II) the functional oligonucleotide is covalently linked to one or more M having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells1The ligand is easy to enrich to the surface of the liver cell and further enter the liver cell, so that the specific targeted delivery of the functional oligonucleotide is realized.
In the present disclosure, preference is given to oligonucleotide conjugates of the formula (II), in which
E1Is OH, m is an integer of 2 to 4;
each R2Independently H, methyl or ethyl; or each R2Are all H;
L1is carbonyl (C)2-C6Alkylene) or carbonyl (oxyethyl)kWherein k is an integer of 2 to 7;
E2represents amino or-amino (C)2-C6Alkylene) hydroxy; and/or
Nu、M1、L3The definitions and alternative ranges of (a) are the same as above.
In one embodiment, the oligonucleotide conjugate of the present invention has a structure represented by formula (II-1), (II-2) or (II-3):
Figure BDA0002295222110000201
Figure BDA0002295222110000211
wherein Nu represents a functional oligonucleotide.
In the context of the present disclosure, "conjugated," means that two or more chemical moieties, each having a particular function, are linked to each other in a covalent linkage, unless otherwise indicated; accordingly, "conjugate" refers to a compound formed by covalent linkage between the various chemical moieties. Further, "oligonucleotide conjugate" means a compound formed by covalently attaching one or more chemical moieties having a specific function to an oligonucleotide. Hereinafter, the oligonucleotide conjugate of the present disclosure is also sometimes simply referred to as "conjugate". More specifically, in the context of the present disclosure, a "conjugate molecule" should be understood as a specific compound that can be conjugated to an oligonucleotide by a reaction, ultimately forming an oligonucleotide conjugate of the present disclosure. In some embodiments, the oligonucleotide is an siRNA, and when the conjugate of the present disclosure is an siRNA conjugate. More specifically, for the purposes of the present disclosure, "conjugate molecule" may refer to a compound represented by formula (I), and correspondingly, "oligonucleotide conjugate" may refer to a compound represented by formula (II).
In some embodiments, the oligonucleotide in the oligonucleotide conjugates of the present disclosure is a functional oligonucleotide. Functional oligonucleotide refers to an oligonucleotide that: the oligonucleotide can up-regulate or down-regulate the expression of a target gene or cause alternative splicing of mRNA by generating stable and specific hybridization with a target sequence and utilizing the principles of RNA activation (RNAa), RNA interference (RNAi), an antisense nucleic acid technology, an exon skipping (exon skipping) technology and the like. 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 understood 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 the expression of proteins transcribed from the 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 a target sequence to affect the normal function of the target sequence molecule, such as causing mRNA fragmentation or translational repression or exon skipping triggering mRNA alternative splicing, and the like. 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 complementary to more than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bases of 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 oligonucleotide comprises a deoxyribonucleotide or a ribonucleotide, as well as a nucleotide having a modification. In some embodiments, the functional oligonucleotide may be a single-stranded DNA, RNA, or DNA-RNA chimera (chimera), or a double-stranded DNA, RNA, or DNA-RNA hybrid (hybrids).
Thus, in some embodiments, a functional oligonucleotide suitable for inclusion in an oligonucleotide conjugate of the present disclosure may be one of small interfering RNA (sirna), microRNA (microRNA), anti-microRNA (antimir), microRNA antagonist (antimir), microRNA mimics (microRNA mimics), decoy oligonucleotide (decoy), immune stimulator (immune stimulator), G-quadrupole (G-quadruplex), variable splice variant (splice alteration), single stranded RNA (ssrna), antisense Nucleic Acid (antisense), Nucleic Acid Aptamer (Nucleic Acid Aptamer), small activating RNA (small activating RNA, saRNA), stem-loop RNA (stem-loop RNA), or DNA. WO2015/006740a2 discloses a conjugate in which different ligands are conjugated to an oligonucleotide, wherein the ligand is linked to the oligonucleotide by a linker (linker), said oligonucleotide being selected from one of small interfering RNA (sirna), microRNA (microRNA), anti-microRNA (antimir), microRNA antagonist (antagomir), microRNA mimics (microRNA mimics), decoy oligonucleotide (decoy), immune stimulant (immune stimulator), G-quadrupole (G-quadrupulplex), variable splice body (splice alteration), single stranded RNA (ssrna), antisense nucleic acid (antisense), aptamer (aptamer), stem-loop RNA (stem-loop RNA) or DNA. These conjugates exhibit good stability on in vivo delivery of the oligonucleotide. In further embodiments, a functional oligonucleotide suitable for inclusion in an oligonucleotide conjugate of the present disclosure may be an oligonucleotide 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 aberrant expression of a particular gene in a particular cell, such as a hepatocyte, by increasing the efficiency of liver-targeted delivery of an active agent, such as a functional oligonucleotide, thereby enhancing the interaction between the functional oligonucleotide and the targeted sequence in the cell. In some embodiments, the specific gene may be an endogenous gene expressed in the liver, or a pathogen gene that proliferates in the liver. The gene which is abnormally expressed in the hepatocyte may be, for example, ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, HCV, or the like gene. In some embodiments, the gene that is aberrantly expressed in hepatocytes is an HBV gene, an ANGPTL3 gene, or an APOC3 gene. In the context of the present disclosure, an HBV gene refers to a gene whose sequence is as shown in Genbank accession number NC _ 003977.1; the ANGPTL3 gene refers to a gene with mRNA sequence shown in Genbank registration number 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 mRNA corresponding to a gene that is overexpressed or mRNA corresponding to a gene that is underexpressed. Since most diseases result from overexpression of mRNA, in the present disclosure, target mRNA refers to, inter alia, mRNA corresponding to the overexpressed gene. In some embodiments of the present disclosure, the target mRNA may be mRNA corresponding to genes of ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1a1, FVII, STAT3, p53, HBV, HCV, etc., corresponding to the above-described aberrantly expressed genes. In some embodiments, the target mRNA may be mRNA transcribed from a corresponding HBV gene, or mRNA corresponding to ANGPTL3 gene, or mRNA corresponding to 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 attachment of the P to the nucleotide by formation of a phosphodiester bond (also sometimes referred to simply as a phosphoester bond). 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), in which case P in formula (II) may be attached to the end of the single-stranded oligonucleotide, which refers to the first 4 nucleotides from one end of the single-stranded oligonucleotide. In some embodiments, P in formula (II) is attached to the end of the single stranded oligonucleotide.
P in formula (II) may be attached to any possible position on a 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 linked to the 2', 3' or 5' position of a nucleotide in the oligonucleotide sequence by forming 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, P and the corresponding phosphate group can be considered to be both P and phosphate group in the double-stranded oligonucleotide), or P in formula (II) is attached to a nucleotide by replacement of a 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 a nucleotide by replacement of a hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide in the sense strand in the double-stranded oligonucleotide sequence.
Without wishing to be bound, in the following embodiments and examples, the case where the functional oligonucleotide in the oligonucleotide conjugate of the present disclosure is a small interfering rna (sirna) is described in detail. At this time, the oligonucleotide conjugate of the present disclosure is an siRNA conjugate. In the context herein, for convenience of description, the siRNA conjugates in these embodiments are also referred to as siRNA conjugates of the present disclosure. This does not mean that the oligonucleotide in the oligonucleotide conjugates of the present disclosure may be simply an siRNA, rather the oligonucleotide may be a substitute drug as disclosed herein or as would be known to one of skill in the art. Based on the detailed description of siRNA conjugates, it is contemplated that other functional oligonucleotides will work similarly when conjugated to the conjugation molecules provided by the present disclosure.
As is well known to those skilled in the art, siRNA contains, as a basic structural unit, a nucleotide group containing a phosphate group, a ribose group and a base. Generally active, i.e., functional, siRNAs are about 12 to 40 nucleotides in length, and in some embodiments about 15 to 30 nucleotides in length, each nucleotide in the siRNA may independently be a modified or unmodified nucleotide, and at least one nucleotide in the siRNA is a modified nucleotide for added stability.
The inventors of the present disclosure found that the siRNA described in the following embodiments has higher activity and/or stability, and thus may be an object of the invention of the siRNA in the present disclosure.
In some embodiments, each nucleotide in the siRNA conjugates of the present disclosure (hereinafter, also referred to as siRNA of the present disclosure) is independently a modified or unmodified nucleotide, and the siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence 1, the antisense strand comprises a nucleotide sequence 2, the nucleotide sequences 1 and 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 double-stranded region, at least a portion of the nucleotide sequence 2 is complementary to a first nucleotide sequence, which is a stretch of nucleotide sequence in a target mRNA.
In some embodiments, the siRNA of the present disclosure is an siRNA capable of inhibiting at least 50% of hepatitis b virus gene expression, at least 50% of angiopoietin-like protein 3 gene expression, or at least 50% of 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% of HBV gene expression at a concentration of 3 mg/kg.
In some embodiments, the nucleotide sequence 1 is the same length as the first nucleotide sequence and does not differ by more than 3 nucleotides; the nucleotide sequence 2 and the nucleotide sequence B are equal in length and have no more than 3 nucleotide differences; the nucleotide sequence B is a nucleotide sequence which is completely reverse complementary to the first nucleotide sequence. Without wishing to be bound, these specific nucleotide differences do not significantly reduce the target gene inhibition ability of the siRNA conjugates, and 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 complete reverse complementary, or complete reverse complementary.
In some embodiments, the nucleotide sequence 1 differs from the first stretch of nucleotide sequence 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 the 5' end to 3' end direction. In some embodiments, the last nucleotide Z on the nucleotide sequence 1 is the nucleotide complementary to Z ' in the 5' to 3' direction.
In some embodiments, the sense strand further comprises nucleotide sequence 3, the antisense strand further comprises nucleotide sequence 4, the length of each of the nucleotide sequences 3 and 4 is equal and is 1-4 nucleotides, the nucleotide sequence 3 is linked to the 5 'end of the nucleotide sequence 1, and the nucleotide sequence 4 is linked to the 3' end of the nucleotide sequence 2, the nucleotide sequence 4 is complementary to a second nucleotide sequence, and the second nucleotide sequence is a nucleotide sequence adjacent to the first nucleotide sequence and having the same length as 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, said nucleotide sequence 5 being 1 to 3 nucleotides in length, attached to the 3 'end of said antisense strand, thereby constituting a 3' overhang of said antisense strand; in some embodiments, the nucleotide sequence 5 is 1 or 2 nucleotides in length. As such, in some embodiments, the ratio of the lengths of the sense and antisense strands of the sirnas of the present disclosure may be 19/20, 19/21, 20/21, 20/22, 21/22, 21/23, 22/23, 22/24, 23/24, or 23/25.
In one embodiment, 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 2 consecutive deoxythymine nucleotides, 2 consecutive uracil nucleotides, or is complementary to a third nucleotide sequence that is adjacent to the first nucleotide sequence or the second nucleotide sequence in the target mRNA and that is equal in length to the nucleotide sequence 5. In one embodiment, the siRNA of the present disclosure has a ratio of the length of the sense strand to the length of the antisense strand of 19/21 or 21/23, when the siRNA of the present disclosure has better hepatocyte mRNA silencing activity.
In some embodiments, the nucleotides in the sirnas of the present disclosure are each independently modified or unmodified nucleotides. In some embodiments, the sirnas of the present disclosure do not contain modified nucleotide groups; in some embodiments, the sirnas of the present disclosure contain modified nucleotide groups.
Currently, there are a variety of ways in which sirnas can be modified, including backbone modifications (also known as internucleotide linkage modifications, such as phosphate group modifications), ribose group modifications, base modifications, and the like (see, e.g., Watts and applications, drug discovery Today, 2008.13 (19-20): p.842-55, incorporated herein by reference in its entirety).
In the context of the present disclosure, the term "modified nucleotide" is used to refer to a nucleotide or nucleotide analog in which the ribosyl group of the nucleotide is modified, such as by substituting the hydroxyl group at the 2' position with another group, or a nucleotide in which the base on the nucleotide is a modified base.
In some embodiments of the present 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 modifying group, in other words, at least a portion of the phosphate groups and/or ribosyl groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand are phosphate groups having a modifying group and/or ribosyl groups (or modified phosphate groups and/or modified ribosyl groups) having a modifying group. 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 and antisense strands 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).
The non-fluorinated modified nucleotide refers to a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group. In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group.
Nucleotides in which the hydroxyl group at the 2 '-position of the ribosyl group is substituted with a non-fluorine group are known to those skilled in the art, and these nucleotides may be one selected from the group consisting 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, and 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 according to formula (211).
Figure BDA0002295222110000241
Figure BDA0002295222110000251
A nucleotide analog refers to a group that can replace a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine. In some embodiments, the nucleotide analog can be, for example, a heteronucleotide, a Bridged Nucleic Acid (BNA) nucleotide, or an acyclic nucleotide.
BNA nucleotides refer to constrained or inaccessible nucleotides. BNAs may contain five-membered, six-membered, or seven-membered ring bridged structures with "fixed" C3' -endo-sugar pull-down. The bridge is typically incorporated at the 2'-, 4' -position of the ribose ring to provide a 2',4' -BNA nucleotide, such as LNA, ENA, cET BNA, etc., wherein LNA is as shown in formula (212), ENA is as shown in formula (213) and cET BNA is as shown in formula (214).
Figure BDA0002295222110000252
Acyclic nucleotides are nucleotides in which the sugar ring of the nucleotide is opened, such as Unlocked Nucleic Acid (UNA) nucleotides or Glycerol Nucleic Acid (GNA) nucleotides, wherein UNA is represented by formula (215) and GNA is represented by formula (216).
Figure BDA0002295222110000253
Wherein R is selected from H, OH or alkoxy (O-alkyl).
An isonucleotide refers to a compound formed by changing the position of a base on a ribose ring in a nucleotide, for example, a compound formed by moving the base from the 1' -position to the 2' -position or the 3' -position of the ribose ring, as shown in formula (217) or (218).
Figure BDA0002295222110000254
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 a heteronucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluorinated modified nucleotide is a methoxy modified nucleotide, which refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group.
In the above and the following, the terms "fluoro-modified nucleotide", "2 '-fluoro-modified nucleotide", "nucleotide in which 2' -hydroxyl group of ribose group is substituted with fluorine" and "2 '-fluoro-ribosyl group" are the same, and refer to a compound having a structure represented by formula (207) in which 2' -hydroxyl group of nucleotide is substituted with fluorine; the terms "methoxy-modified nucleotide", "2 '-methoxy-modified nucleotide", "nucleotide in which 2' -hydroxyl group of ribose group is substituted with methoxy group" and "2 '-methoxy ribosyl group" have the same meaning, and refer to that 2' -hydroxyl group of ribose group of nucleotide is substituted with methoxy group to form a structure as shown in formula (208).
In some embodiments, the siRNA of the present disclosure is an siRNA with the following modifications: the sense strand and the antisense strand both comprise fluoro-modified nucleotides and non-fluoro-modified nucleotides, the fluoro-modified nucleotides are located in the aforementioned nucleotide sequence 1 and nucleotide sequence 2, the fluoro-modified nucleotides in the nucleotide sequence 1 are not more than 5, and the nucleotides at positions 7,8 and 9 of the nucleotide sequence 1 are 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 the 2 nd, 6 th, 14 th and 16 th positions 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 the following modifications: according to the direction from the 5 'end to the 3' end, the nucleotides at the 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 the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence 2 are fluorine-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 the following modifications: or according to the direction from 5 'end to 3' end, the 5 th, 7 th, 8 th and 9 th nucleotides of the nucleotide sequence 1 in the sense strand of the siRNA are fluorine-modified nucleotides, and the rest nucleotides in the sense strand are methoxy-modified nucleotides; in the antisense strand, the nucleotides at the 2 nd, 6 th, 8 th, 9 th, 14 th and 16 th positions of the nucleotide sequence 2 are fluorine-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 the following modifications: according to the direction from the 5 'end to the 3' end, the nucleotides at the 7 th, 8 th and 9 th positions of the nucleotide sequence 1 in the sense strand of the siRNA are fluorine-modified nucleotides, the nucleotides at the rest positions in the sense strand are methoxy-modified nucleotides, and according to the direction from the 5 'end to the 3' end, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence 2 in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the rest positions in the antisense strand are methoxy-modified nucleotides.
In some embodiments of the sirnas of the present disclosure, the nucleotide comprises a phosphate group modification. In the context of the present disclosure, the phosphate group modification is in one embodiment a phosphorothioate (phosphothioate) modification as shown in formula (201) below, i.e., replacing the non-bridging oxygen atom in the phosphodiester linkage with a sulfur atom, thereby replacing the phosphodiester linkage with a phosphorothioate diester linkage. In some embodiments, the modification stabilizes the structure of the siRNA, maintaining high specificity and high affinity for base pairing.
Figure BDA0002295222110000261
According to some embodiments of the disclosure, the siRNA wherein 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 or antisense strand; between the second and third nucleotides at either end of the sense or antisense strand; or any combination of the above. In some embodiments, phosphorothioate-based linkages are present at all of the above positions except at the 5' end of the sense strand. In some embodiments, phosphorothioate-based linkages are present at all of the above positions except at the 3' end of the sense strand. In some embodiments, the phosphorothioate-based linkage is present in at least one of the following positions:
a linkage between the 1 st and 2 nd nucleotides at the 5' terminal end of the sense strand;
a linkage between the 2 nd and 3 rd nucleotides at the 5' terminal end of the sense strand;
a linkage between the 1 st and 2 nd nucleotides at the 3' terminal end of the sense strand;
a linkage between the 2 nd and 3 rd nucleotides at the 3' terminal end of the sense strand;
a linkage between the 1 st and 2 nd nucleotides at the 5' terminal end of the antisense strand;
a linkage between the 2 nd and 3 rd nucleotides at the 5' terminal end of the antisense strand;
a linkage between the 1 st and 2 nd nucleotides at the 3' terminal end of the antisense strand; and
a linkage between the 2 nd and 3 rd nucleotides at the 3' terminal end of the antisense strand.
According to some embodiments of the disclosure, the 5' terminal nucleotide of the antisense strand sequence of the siRNA molecule is a 5' -phosphate nucleotide or a 5' -phosphate analog modified nucleotide.
In some embodiments, the nucleotide 5' -phosphate can have a structure represented by formula (202):
Figure BDA0002295222110000262
meanwhile, The types of The 5' -phosphate analogue-modified nucleotides which are commonly used are well known to those skilled in The art, for example, 4 nucleotides as shown in The following formulas (203) to (206) disclosed in Anastasia Khvorova and Jonathan K.Watts, The chemical evaluation of oligonucleotide therapeutics of clinical utility, Nature Biotechnology,2017,35(3): 238-48:
Figure BDA0002295222110000263
Figure BDA0002295222110000271
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 nucleotide modified with a5 '-phosphate or a 5' -phosphate analog is a nucleotide containing a vinyl phosphate (E-VP) represented by formula (203), a nucleotide containing a5 '-phosphate modification represented by formula (202), or a nucleotide containing a 5' -phosphorothioate modification 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 exhibited target mRNA silencing activity that is not significantly reduced and excellent gene expression inhibition effect; thus, it was shown that the siRNA conjugates of the present disclosure have higher in vivo delivery efficiency. According to some embodiments of the disclosure, the oligonucleotide conjugates of the disclosure are siRNA conjugates comprising sirnas, such as the sirnas shown in tables 1A-1F:
table 1 siRNA sequences in some embodiments
TABLE 1A
Figure BDA0002295222110000272
Figure BDA0002295222110000281
TABLE 1B
Figure BDA0002295222110000282
Figure BDA0002295222110000291
TABLE 1C
Figure BDA0002295222110000292
TABLE 1D
Figure BDA0002295222110000293
Figure BDA0002295222110000301
TABLE 1E
Figure BDA0002295222110000302
Figure BDA0002295222110000311
TABLE 1F
Figure BDA0002295222110000321
S: a sense strand; AS: antisense strand
Wherein, the capital letters C, G, U, A represent the base composition of nucleotides; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a 2' -methoxy modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side 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 left and right of the letter s is a phosphorothioate-based linkage; p1 indicates that the nucleotide adjacent to the right side of P1 is a 5' -phosphate nucleotide or a 5' -phosphate analog modified nucleotide, in some embodiments a vinyl phosphate modified nucleotide (indicated by VP in the examples below), a 5' -phosphate modified nucleotide (indicated by P in the examples below), or a phosphorothioate modified nucleotide (indicated by Ps in the examples below).
It is clear to those skilled in the art that modified nucleotide groups can be introduced into the sirnas described in the present disclosure by using nucleoside monomers with corresponding modifications, and methods of preparing nucleoside monomers with corresponding modifications and methods of introducing modified nucleotide groups into sirnas are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or can be prepared by known methods.
Preparation of oligonucleotide conjugates
Oligonucleotide conjugates of the present disclosure can be prepared using any reasonable synthetic route.
For example, a method of preparing an oligonucleotide conjugate of the present disclosure may comprise:
(a) reacting the compound of formula (I) with succinic acid or succinic anhydride to give an intermediate of formula (II-M1);
Figure BDA0002295222110000331
(b) removing the protecting group from the solid support bearing the protected amino group and attaching the intermediate of formula (II-M1) obtained in step (a) to the solid support in the presence of a coupling reagent under coupling reaction conditions;
(c) under the condition of the nucleic acid solid phase phosphoramidite solid phase synthesis method, nucleoside monomers are connected in sequence according to the nucleotide type and sequence of the functional oligonucleotide and the 3 'to 5' direction, and the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfuration.
As the solid phase carrier to be used, a high molecular polymer carrier having a hydroxyl group or an amino group on the surface thereof, for example, commercially available Wang resin, Aminomethyl (AM) resin, benzyl alcohol resin, etc. can be used.
The deprotection, coupling, capping, oxidation, sulfurization or hydroboration reaction may use the same conditions and reagents as those used in the solid phase phosphoramidite solid phase synthesis method of nucleic acid, and specific reaction conditions and reagents will be described in detail later.
In some embodiments, the method further comprises the steps of removing the protecting group and cleaving with the solid support, isolating and purifying.
In the context of the present invention, the term "carbonyl- (oxyethyl)k"denotes k-O-CH2-CH2-the units are linked to a 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, carbonylbis (oxyethyl), carbonyltris (oxyethyl), and the like.
In the present invention, amino (ethoxy)q2Represents q 2-CHs2-CH2-O-units are linked to amino groups, 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 the first nucleoside monomer at the 3' end of the sense strand or the antisense strand under coupling reaction conditions and in the presence of a coupling reagent to attach the first nucleotide in the attached sequence to the intermediate of formula (II-M1) attached to the solid support, and sequentially attaching the nucleoside monomers in the 3' to 5' direction to synthesize the sense strand or the antisense strand of the double-stranded oligonucleotide; wherein the intermediate of formula (II-M1) attached to the solid support as described above 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 sulfuration; obtaining a sense or antisense strand of the nucleic acid to which the conjugate molecule is attached; connecting nucleoside monomers in sequence according to the 3 'to 5' direction to synthesize the other chain of the double-chain oligonucleotide, wherein the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfuration; removing protecting group, cutting with solid phase carrier, separating and purifying to obtain 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: connecting nucleoside monomers in sequence according to the direction from 3 'to 5' 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 sulfuration to obtain the sense strand connected to a solid phase carrier and the antisense strand connected to the solid phase carrier; contacting the compound represented by 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 under coupling reaction conditions to thereby link the compound represented by formula (II-M1) to the sense strand or the antisense strand; removing the protecting group, cutting with a solid phase carrier, respectively 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 conjugate molecule.
In some embodiments, the compound represented by formula (II) is linked to the 3' end of the sense strand in the siRNA, and the method for preparing the siRNA conjugate of the present 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 coupling reaction condition 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 a sense strand of the siRNA by a phosphoramidite solid phase synthesis method in a 3'-5' direction starting with the nucleoside monomer linked to the solid phase support by the L-conjugated molecule;
(3) synthesizing an antisense strand of the siRNA by a phosphoramidite solid phase synthesis method;
(4) the sense and antisense strands of the siRNA are isolated and annealed to obtain the siRNA conjugates of the present disclosure.
Wherein, in step (1), the method for removing the protecting group G from the solid support-linked L-conjugated molecule comprises contacting the solid support-linked compound of formula (II-M1) with a deprotection reagent under deprotection conditions. Deprotection conditions include temperatures of 0 to 50 deg.C, in some embodiments 15 to 35 deg.C, reaction times of 30 to 300 seconds, in some embodiments 50 to 150 seconds, and the deprotection reagent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments dichloroacetic acid. The molar ratio of deprotecting reagent to compound of formula (II-M1) is 2:1 to 100:1, and in one embodiment 3:1 to 50:1, calculated on the amount of material of compound of formula (II-M1).
The coupling reaction conditions and coupling reagents may use any conditions and reagents suitable for the above-described coupling reaction. In some embodiments, the same conditions and reagents are used as for the coupling reaction in the solid phase synthesis method employed.
In some embodiments, the conditions of the coupling reaction include a reaction temperature of from 0 to 50 ℃, in some embodiments from 15 to 35 ℃. The molar ratio of the compound of formula (II-M1) to nucleoside monomer, calculated as the amount of material of the compound of formula (II-M1), is from 1:1 to 1:50, in some embodiments from 1:2 to 1: 5; the molar ratio of the compound of formula (II-M1) to the coupling reagent, calculated as the amount of material of the compound of formula (II-M1), is from 1:1 to 1:50, in some embodiments from 1:3 to 1:10, and the reaction time is from 200 to 3000 seconds, in some embodiments from 500 to 1500 seconds. The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, and 5-benzylthio 1H-tetrazole, and in some embodiments is 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, and in some embodiments, anhydrous acetonitrile. The organic solvent is used in an amount of 3 to 50L/mol, and in some embodiments 5 to 20L/mol, relative to the amount of species 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 by the L-conjugate molecule prepared in the above step. At this point, the L-conjugate molecule is attached to the 3' end of the resulting sense strand.
Other conditions of the solid phase synthesis in the steps (2) and (3) include deprotection conditions of nucleoside monomers, types and amounts of deprotection reagents, coupling reaction conditions, types and amounts of coupling reagents, capping reaction conditions, types and amounts of capping reagents, oxidation reaction conditions, types and amounts of oxidation reagents, vulcanization reaction conditions, and vulcanization reagents and amounts, and various reagents, amounts and conditions conventionally used in the art are adopted.
For example, in some embodiments, the solid phase synthesis in steps (2) and (3) may use the following conditions:
the nucleoside monomer deprotection conditions include a temperature of 0 to 50 deg.C, in some embodiments 15 to 35 deg.C, a reaction time of 30 to 300 seconds, in some embodiments 50 to 150 seconds, and the deprotection reagent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments dichloroacetic acid. The molar ratio of deprotecting reagent to 4,4' -dimethoxytrityl protecting group on solid support is from 2:1 to 100:1, and in some embodiments from 3:1 to 50: 1.
The coupling reaction conditions include a temperature of 0-50 deg.C, in some embodiments 15-35 deg.C, and a molar ratio of nucleic acid sequence attached to the solid support to nucleoside monomer of 1:1 to 1:50, in some embodiments 1:5 to 1: 15; the molar ratio of nucleic acid sequence attached to the solid support to coupling reagent is from 1:1 to 1:100, and in some embodiments from 1:50 to 1:80, and the reaction time and choice of coupling reagent are the same as described above.
Capping reaction conditions include a temperature of 0-50 deg.C, in some embodiments 15-35 deg.C, and a reaction time of 5-500 seconds, in some embodiments 10-100 seconds, with the same selection of capping reagents as previously described. The molar ratio of the total amount of capping reagent to the nucleic acid sequence attached to the solid support is 1:100-100:1, and in some embodiments 1:10-10: 1. In the case where equimolar amounts of acetic anhydride and N-methylimidazole are used as the capping reagent, the molar ratio of acetic anhydride, N-methylimidazole and nucleic acid sequence attached to the solid support is 1:1:10 to 10:10:1, and in some embodiments 1:1:2 to 2:2: 1.
The oxidation reaction conditions include a temperature of from 0 to 50 deg.C, in some embodiments from 15 to 35 deg.C, a reaction time of from 1 to 100 seconds, in some embodiments from 5 to 50 seconds, and the oxidizing agent, in some embodiments, iodine (provided in the form of iodine water in further embodiments). The molar ratio of oxidizing reagent to nucleic acid sequence attached to the solid support in the coupling step is from 1:1 to 100:1, and in some embodiments from 5:1 to 50: 1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran, water, pyridine ═ 3:1:1-1:1: 3. The sulfurization reaction conditions include a temperature of from 0 to 50 deg.C, in some embodiments from 15 to 35 deg.C, a reaction time of from 50 to 2000 seconds, in some embodiments 100 and 1000 seconds, and the sulfurizing agent, in some embodiments hydrogenated flavonones. The molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling step is from 10:1 to 1000:1, and in some embodiments from 10:1 to 500: 1. In some embodiments, the sulfurization reaction is carried out in a mixed solvent of acetonitrile and pyridine 1:3-3: 1.
According to the methods provided by the present disclosure, after all nucleoside monomers are linked, and prior to annealing, the method further comprises isolating the sense and antisense strands of the siRNA. Isolation procedures are well known to those skilled in the art and generally involve cleaving the synthesized nucleotide sequence from the solid support, removing protecting groups on the base, phosphate and ligand, purification and desalting.
The nucleotide sequence obtained by synthesis is cut from the solid phase carrier, and the removal of the protecting groups on the base, the phosphate group and the ligand can be carried out according to the conventional cutting and deprotection method in the siRNA synthesis. For example, the obtained nucleotide sequence with the solid support attached thereto is contacted with concentrated ammonia water; during deprotection, the hydroxyl protecting group is removedObtaining M1And (II) to produce a conjugate of 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.
When there is at least one 2'-TBDMS protection on the synthesized nucleotide sequence, the method further comprises contacting the nucleotide sequence with the solid support removed with triethylamine trihydrofluoride to remove the 2' -TBDMS protection. In this case, the corresponding nucleoside having a free 2' -hydroxyl group in the target siRNA sequence was obtained. The dosage of the triethylamine trihydrofluoride salt pure product is 0.4 ml/mu mol-1.0 ml/mu mol compared with the target siRNA sequence. This gives 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 a preparative ion chromatography purification column; the products can be desalted by adopting a reverse phase chromatographic purification column after being collected and combined.
The purity and molecular weight of the nucleic acid sequence can be readily determined during synthesis to better control the quality of the synthesis, methods of detection being well known to those skilled in the art. For example, nucleic acid purity can be detected by ion exchange chromatography and molecular weight determined by LC-MS.
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) can be simply mixed in equimolar ratio in water for injection and heated to 70-95 ℃ followed by cooling at room temperature to allow formation of a double-stranded structure by hydrogen bonding. This gives the siRNA 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, for example, using methods such as mass spectrometry, etc., to determine that the synthesized siRNA conjugates are the siRNA conjugates designed for the target, and the sequences of the synthesized sirnas correspond to the sequences of the sirnas to be synthesized, for example, the sequences listed in table 1 above.
In further embodiments, the conjugates of the present disclosure can be prepared by the following method:
synthesizing an oligonucleotide according to the above-described phosphoramidite solid phase synthesis method, wherein at least one nucleotide in the oligonucleotide sequence has an amino group attached thereto;
at normal temperature, adding a mixture of sodium bicarbonate, acetonitrile and dimethyl sulfoxide, and reacting the synthesized oligonucleotide with the conjugated molecule of the disclosure, wherein in the conjugated molecule of the disclosure, an L2 group has an imine ester group, an acyl group and an alkylene group which are sequentially connected, so that the oligonucleotide and the conjugated molecule of the disclosure are connected through an amide bond; the conjugates of the present disclosure are isolated.
Use of conjugates of the disclosure
As shown in the present disclosure, the conjugates can deliver an active agent to a cell 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 loaded active agent into contact with the cell. 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 the small molecular drug can be efficiently delivered to the liver simultaneously, and the oligonucleotide conjugate has the advantages of low toxicity, good stability and high gene expression inhibition activity in liver cells as much as possible, so as to achieve better treatment effect.
The oligonucleotide conjugates of the invention are suitable for the preparation of a medicament for the prevention and/or treatment of pathological conditions or diseases caused by the expression of genes in hepatocytes.
The oligonucleotide conjugates of the invention are suitable for use in the prevention and/or treatment of 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 gene of a pathogen that proliferates 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 chronic liver disease, hepatitis, liver fibrosis disease, liver proliferative disease and dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.
The following examples are merely illustrative of the invention and are not intended to be limiting.
Examples
Boc: tert-butoxy radical
Fmoc: 9-fluorenylmethoxycarbonyl group
TBS, TBDMS: tert-butyldimethylsilyl group
DMTr: 4,4' -Bimethoxy trityl
Preparation examples
Preparation example 1: preparation of liver-targeting Compounds of the invention
1.1 Synthesis of PNB-2
Figure BDA0002295222110000361
Dissolving the starting material PNB-1 (14.0 g, 31.0mmol, available from Annaige corporation) and P4-1(7.0g,40mmol) in 300ml of dichloromethane, adding HATU (2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate, 17.7g,46.2mmol, available from Michelin corporation); DIPEA (N, N-diisopropylethylamine, from Aladdin, 10.2g,77.0mmol) was then added while cooling on ice; the reaction was then stirred at 25 ℃ for 3 hours and quenched by addition of water. The reaction mixture was extracted with dichloromethane several times, and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was distilled under reduced pressure. The obtained residue was purified by column chromatography, and eluted on 200-300 mesh normal phase silica gel with a gradient of petroleum ether and ethyl acetate at 20:1-1:1 to obtain 18.8g of PNB-2, the yield of which was 99.3%.1H NMR(400MHz,CDCl3)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：C33H47N3O6Si，[M+H]+Theory: 610.84, actually measuring: 611.03
1.2 Synthesis of PNB-3
Figure BDA0002295222110000362
PNB-2(18.8g, 30.8mmol) was dissolved in anhydrous acetonitrile (100ml), and diethylamine (20 ml) was added to the reaction solution by syringe and the reaction was stirred at 25 ℃ for 2 h. Subsequently, the reaction mixture was evaporated to dryness under reduced pressure, and the resulting residue was diluted with methanol and evaporated to dryness again under reduced pressure to give a crude product. And (3) performing column chromatography (200-300 mesh normal phase silica gel, gradient elution with ethyl acetate and methanol being 100:1-10: 1), eluting a low-polarity compound, eluting with 10:1 ethyl acetate-methanol, concentrating, and drying to obtain 12.3g of PNB-3, wherein the quantitative yield is about 99%. MS m/z: c18H37N3O4Si，[M+H]+Theory: 388.590, actually measuring: 388.830
1.3 Synthesis of PNB-4
Figure BDA0002295222110000371
PNB-1(15.8g,34.9mmol) and 1.2 PNB-3(12.3g) were dissolved in dichloromethane (310ml) and HATU (18.1g,47.6mmol) was added; DIPEA (10.3g,79.3mmol) was then added to the reaction mixture while cooling on ice, and the mixture was stirred at room temperature for 3 hours and quenched by addition of water. Extracting with dichloromethane for 3 times, combining organic phases, performing rotary evaporation and concentration on the organic phases, purifying and collecting a target product through column chromatography (200-300 meshes of normal phase silica gel, and performing gradient elution with ethyl acetate: methanol being 100:1-15: 1), and concentrating to obtain 26.0g of PNB-4 with quantitative yield (about 99%).1H 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
Figure BDA0002295222110000372
PNB-4(26g) obtained in the above reaction 1.3 was dissolved in acetonitrile (100ml), and then 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 resulting residue was diluted with methanol and evaporated to dryness again under reduced pressure to give a crude product. Purifying by column chromatography (200-300 mesh normal phase silica gel, gradient elution with ethyl acetate: methanol 100:1-10: 1), eluting low polarity compounds, eluting with 10:1 ethyl acetate-methanol, concentrating and draining to obtain 22g PNB-5, and quantitative yield (about 99%). MS m/z: c28H53N5O7Si，[M+H]Theory: 600.8450, actually measuring: 601.14
1.5PNB-6 Synthesis
Figure BDA0002295222110000373
PNB-5(17g,28.3mmol) and PNB-1(14.1g,31.2mmol) were dissolved in dichloromethane (283ml) and HATU (16.1g,42.5mmol) was added; DIPEA (9.1g,70.8mmol) was then added to the reaction solution while cooling on ice, and the mixture was stirred at room temperature for 3 hours and quenched by addition of water. Extracting with dichloromethane for 3 times, combining organic phases, distilling under reduced pressure and concentrating to dryness, and purifying by column chromatography (200-300 mesh normal phase silica gel, gradient elution with ethyl acetate: methanol: 100:1-8: 1) to obtain 17.0g of PNB-6, with a yield of 58%.1H NMR(400MHz,CDCl3)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
Figure BDA0002295222110000381
PNB-6(17g,16.3mmol) was added to 30ml HCl/EtOH (30%) and stirred at room temperature for 4 hours, the solvent was removed by distillation under reduced pressure, and PNB-7 was obtained after 16 hours of oil pump drying and used in the next reaction without further purification.
PNB-7 was dissolved in 80ml DMF, DIPEA (25.2g,195mmol) was added, stirring was carried out for 3 minutes, TBSCl (tert-butyldimethylchlorosilane, Michelin, 12.28g,81.5mmol) was slowly added, the reaction was stirred at room temperature for 4 hours, water was added to quench the reaction, and extraction was carried out 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 crude PNB-8, which was used directly in the next reaction without further purification to give 11.9g of crude PNB-8. The product was identified by LC-MS: MS m/z: c38H55N7O6Si，[M+H]+Theory: 734.986, actually measuring: 734.39.
1.7 Synthesis of PNB-9
Figure BDA0002295222110000382
PNB-8(11.9g,16.3mmol) was dissolved in 160 ml of N, N-Dimethylformamide (DMF), Gal-5 (custom made from Tianjin Yao Co., 25.5g,57.1mmol) was added, and HATU (9.3g,24.5mmol) was added; then adding DIPEA (6.3g,48.9mmol) under the condition of ice-water bath, reacting and stirring for 4 hours, adding water to quench the reaction, and extracting for 3 times by using dichloromethane; the organic phases were combined, concentrated by distillation under reduced pressure, and purified by column chromatography (200-300 mesh normal phase silica gel, gradient elution with ethyl acetate: methanol-100: 1-20: 1) to give 16.1g of PNB-9 in 97.5% yield. The product was identified by LC-MS: MS m/z: c95H136N10O36Si，[M/2+H]+Theory: 1012.1260, actually measuring: 1012.10.
1.8 Synthesis of PNB-10 and PNB-11
Figure BDA0002295222110000391
PNB-9(16.1g, 7.96mmol) was dissolved in anhydrous acetonitrile (from sigma-aldrich, 40 mL), and diethylamine (8 mL, 77.6mmol) was added to the reaction solution via syringe and stirred at room temperature for 2 h. The reaction mixture was evaporated to dryness under reduced pressure, and the resulting residue was diluted with methanol and evaporated to dryness again under reduced pressure to give crude PNB-10(12.1g) which was used as a starting material for the next reaction.
PNB-10(12.1g) obtained in the above step was dissolved in methylene chloride (70 ml), and 5-O-DMTr-5-hydroxypentanoic acid (9.9g,23.5mmol) and HATU (3.8g,10.1mmol) were added thereto; then adding DIPEA (2.6g,20.2mmol) under the condition of ice-water bath, adding water to quench the reaction after stirring for 4 hours, and extracting with dichloromethane; the organic phase was evaporated to dryness under reduced pressure and the residue was purified by column chromatography (200-300 mesh normal phase silica gel, gradient elution with ethyl acetate: methanol 100:1-12: 1) to give 6.2g of PNB-11 product, with a total yield of 41.0% over the two steps.1H 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：C106H152N10O38Si，[M-303(DMTr)]+Theory: 1899.4985, actually measuring: 1899.94.
1.9 Synthesis of PNB-12
Figure BDA0002295222110000401
PNB-11(6.2g,2.8mmol) was dissolved in THF (tetrahydrofuran, 28ml), 1M TBAF (12ml,12.6mmol) was added, and after stirring for 4 hours, the reaction was quenched with water and extracted 3 times with dichloromethane; combining the organic phases, distilling and concentrating under reduced pressure, and performing column chromatography (200-300 mesh normal phase silica gel, ethyl acetate: methanol: 100: 1-8)1 gradient elution) gave 3.1g of PNB-12 in 64% yield. The product was identified by LC-MS: MS m/z: c100H138N10O38，[M-303]+Theory: 1785.2360, actually measuring: 1785.81.
1.10 Synthesis of PNB-13
Figure BDA0002295222110000402
PNB-12(3.1g,1.5mmol), succinic anhydride (360mg,3.6mmol) and DMAP (4-dimethylaminopyridine, 366mg,3.0mmol from Alatin) were added to a dichloromethane solution, a solution of DIPEA (968mg,7.5mmol) in dichloromethane was added dropwise, and the reaction was stirred for 4 hours; monitoring complete reaction by LC-MS, adding water to quench the reaction, and extracting with dichloromethane for 3 times; the organic phases were combined and washed three times with saturated ammonium chloride solution and evaporated to dryness under reduced pressure to give 3.3g of PNB-13 in quantitative yield (about 99%). The product is identified by LC-MS and directly used for next step of connecting with a solid phase carrier. MS m/z: c104H142N10O41，[M-303]+Theory: 1885.3090, actually measuring: 1885.95.
preparation example 2: preparation of conjugate molecules attached to solid supports
Figure BDA0002295222110000411
PNB-13(533mg,0.243mmol), HBTU (138mg,0.365mmol) and DIPEA (125mg,0.974mmol) are added into 5ml acetonitrile, after ultrasonic wave-assisted dissolution, Aminomethyl (AM) resin solid phase carrier (purchased from Nankai synthesis company, with the amino content of 400 mu mol/g, 607mg) is added, shaking reaction is carried out for 16 hours, and after the reaction is finished, suction filtration is carried out; the filter cake was rinsed with acetonitrile and drained. The filter cake was then reacted for a further half hour with acetic anhydride/pyridine (25%) solution, allowing the unattached amino group to be capped with acetyl. Filtering again, repeating the above washing process, and fully draining. The target product PNB-14, i.e. the conjugated molecule attached to the solid support, is obtained. Yield 1.03g, load 135. mu. mol/g.
Preparation example 3: preparation of PNB-siRNA Compound (SR16-X2U3460-PNB)
In this example, the siRNA of the siRNA conjugate is 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, the capital letters C, G, U, A represent the base composition of nucleotides; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a 2' -methoxy modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side 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 left and right of the letter s is a phosphorothioate-based linkage; VP indicates that one nucleotide on the right side of the VP is a vinyl phosphate modified nucleotide.
Preparation method
3.1 Synthesis of sense chain (S)
Sense Strand Using PNB-14 prepared in example 2 as a starting material, nucleoside monomers were linked one by one in the 3'-5' direction in the above sequence order, respectively. Each nucleoside monomer is connected by four steps of deprotection, coupling, capping and oxidation. The synthesis conditions are given as follows:
the nucleoside monomer was supplied as a 0.1M acetonitrile solution, the deprotection conditions were the same for each step, i.e., temperature was 25 deg.C, reaction time was 70 seconds, the deprotection reagent was dichloroacetic acid in dichloromethane (3% v/v), and the molar ratio of dichloroacetic acid to 4,4' -dimethoxytrityl protecting group on the solid support was 5: 1.
The coupling reaction conditions in each step are the same, and the coupling reaction conditions comprise that the temperature is 25 ℃, the molar ratio of the nucleic acid sequence connected on the solid phase carrier to the nucleoside monomer is 1:10, the molar ratio of the nucleic acid sequence connected on the solid phase carrier to the coupling reagent is 1:65, the reaction time is 600 seconds, and the coupling reagent is 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 molar ratio of 1:1, the CapA is a pyridine/acetonitrile mixed solution of 20 volume percent N-methylimidazole, and the volume ratio of the pyridine to the acetonitrile is 3: 5; CapB is a 20% by volume acetic anhydride solution in acetonitrile; the molar ratio of the capping reagent to the nucleic acid sequence attached to the solid phase carrier is acetic anhydride, N-methylimidazole and the nucleic acid sequence attached to the solid phase carrier is 1:1: 1.
The oxidation reaction conditions in each step are the same, including the temperature of 25 ℃, the reaction time of 15 seconds, and the oxidizing agent of 0.05M iodine water. The molar ratio of iodine to nucleic acid sequence attached to the solid support in the coupling step is 30: 1. The reaction was carried out in a mixed solvent of tetrahydrofuran, water and pyridine in a ratio of 3:1: 1.
Wherein, the 2' -methoxyl modified uridine monomer (VP-Um) modified by vinyl phosphate is synthesized according to the following method:
Figure BDA0002295222110000421
(3a-1) Synthesis of VP-U-2
The VP-U-2 molecule was synthesized as follows:
Figure BDA0002295222110000422
2 '-methoxy-modified uridine (2' -OMe-U, 51.30g, 91.6mmol), tert-butyldiphenylchlorosilane (TBDPSCl, 50.35g, 183.2mmol), and imidazole (12.47g, 183.2mmol) were mixed and dissolved in 450ml of N, N-Dimethylformamide (DMF), and the reaction was stirred at room temperature for 20 hours. DMF was evaporated, taken up in 600ml of dichloromethane, washed with 300ml of saturated aqueous sodium bicarbonate solution, the aqueous phase was extracted 3 times with 300ml of Dichloromethane (DCM), and the organic phases were combined. The combined organic phases are washed with 5% oxalic acid until the pH of the separated aqueous phase is <5, and the crude VP-U-1 is obtained after evaporation of the solvent from the organic phase to dryness and is used directly in the subsequent synthesis of VP-U-2.
After dissolving the crude VP-U-1 product in 100ml of dichloromethane, stirring the solution for 10 minutes in ice bath, adding 450ml of 2% p-toluenesulfonic acid solution (the solvent is a methanol-dichloromethane mixed solvent with the volume ratio of 3: 7) which is refrigerated in a refrigerator at 4 ℃ in advance, and reacting the solution for 10 minutes. The reaction was quenched by addition of 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 were combined. The combined aqueous phases were extracted 2 times with 200ml 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-mesh 300-mesh normal phase silica gel column, loading petroleum ether into the column, gradient eluting with petroleum ether, ethyl acetate, dichloromethane and methanol at a ratio of 1:1:1:0.05-1:1:1: 0.25), collecting the product eluate, evaporating the solvent under reduced pressure, and foaming and drying by a vacuum oil pump to obtain 40.00g of pure VP-U-2.1H 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：C26H33N2O6Si，[M+H]+Theory: 497.21, actually measuring: 497.45.
(3a-2) Synthesis of VP-U-4:
Figure BDA0002295222110000431
VP-U-2(19.84g, 40.0mmol), dicyclohexylcarbodiimide (DCC, 16.48g, 80.0mmol), pyridine (4.20g, 53.2mmol), and trifluoroacetic acid (6.61g, 53.2mmol) were mixed and dissolved in 200ml of dimethyl sulfoxide (DMSO), and the reaction was stirred at room temperature for 20 hours. Separately, tetraethyl methylenediphosphonate (21.44g, 74.4mmol) was dissolved in 120ml of THF, cooled in an ice bath, t-BuOK (11.36g, 101.2mmol) was added thereto at the ice bath temperature, and the mixture was reacted at the ice bath temperature for 10min, then warmed to room temperature and reacted for 0.5h, and then added to the reaction mixtureAfter about 1h, the reaction was carried out for another 1h at ice bath temperature, and then the reaction was allowed to warm to room temperature for 18 h. The reaction was quenched with water and the aqueous phase was extracted 3 times with 200ml of dichloromethane each time. The organic phases are combined, washed once with 200ml of saturated saline and the solvent is evaporated to dryness. Purifying the residue by column chromatography (200-mesh 300-mesh normal phase silica gel column, loading petroleum ether into column, gradient eluting with petroleum ether and ethyl acetate at ratio of 1:1-1: 4), collecting product eluate, evaporating solvent under reduced pressure, and foaming and drying by vacuum oil pump to obtain pure product VP-U-4 of 14.00 g.1H 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：C31H42N2O8PSi，[M+H]+Theory: 629.24, actually measuring: 629.51.
(3a-3) Synthesis of VP-U-5:
Figure BDA0002295222110000432
VP-U-4(14.00g, 22.29mmol) was dissolved in 100ml tetrahydrofuran, triethylamine trihydrofluoride (17.96g, 111.45mmol) 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. And (3) purifying the crude product by column chromatography (200-mesh 300-mesh normal phase silica gel column, loading petroleum ether into the column, performing gradient elution by using petroleum ether, ethyl acetate, dichloromethane and methanol in a ratio of 1:1:1:0.05-1:1:1: 0.25), collecting product eluent, evaporating the solvent to dryness under reduced pressure, and performing foaming drying by using a vacuum oil pump to obtain 6.70g of a pure product VP-U-5.1H 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：C15H24N2O8P，[M+H]+Theory: 391.13, actually measuring: 391.38.
(3a-4) Synthesis of VP-U-6:
Figure BDA0002295222110000433
VP-U-5(391mg, 1.0mmol), pyridinium trifluoroacetate (0.232g, 1.2mmol), N-methylimidazole (0.099g, 1.2mmol), bis (diisopropylamino) (2-cyanoethoxy) phosphine (0.452g, 1.5mmol) and the reaction mixture was added to 10ml of anhydrous dichloromethane under protection of argon, and the mixture was stirred at room temperature for 5 hours. The solvent was evaporated to dryness, purified by column chromatography (200-300 mesh normal phase silica gel, dichloromethane: acetonitrile (containing 0.5 wt% triethylamine) ═ 3:1-1:3 gradient elution), the product eluate was collected and concentrated to remove the solvent, yielding a total of 508mg of the desired product, VP-U-6.31P NMR(161MHz,DMSO-d6)δ150.34,150.29,17.07,15.50.MS m/z：C24H41N4O9P2，[M+H]+Theory: 591.23, actually measuring: 591.55. VP-U-6 is the target product VP-Um, and participates in RNA strand synthesis as a nucleoside monomer.
Cleavage and deprotection conditions were as follows: the synthesized nucleotide sequence with the attached carrier was added to 25 wt% ammonia water in an amount of 0.5ml/μmol, reacted at 55 ℃ for 16 hours, the liquid was removed, and concentrated to dryness in vacuo. Purification and desalting: purification of nucleic acids was accomplished by gradient elution of NaCl using a preparative ion chromatography purification column (Source 15Q). Specifically, the method comprises the following steps: eluent A: 20mM sodium phosphate (pH 8.1) in water/acetonitrile 9:1 (volume ratio); eluent B: 1.5M sodium chloride, 20mM sodium phosphate (pH 8.1) and solvent water/acetonitrile 9:1 (volume ratio); elution gradient: eluting with eluent A and eluent B in gradient of 100:0-50: 50. Collecting product eluates, mixing, desalting with reverse phase chromatography purification column, specifically desalting with Sephadex column as filler (Sephadex G25), and eluting with deionized water.
And (3) detection: purity was 78% as determined by ion exchange chromatography (IEX-HPLC); molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS) with theoretical value 7662.5, found value 7661.4.
3.2 Synthesis of Antisense Strand (AS)
Method for solid phase synthesis of phosphoramidite nucleic acid using commercially available universal solid phase support for antisense strand: (
Figure BDA0002295222110000442
HL UnyLinkerTM300Oligonucleotide Synthesis Support, Kinovate Life Sciences, 300. mu. mol/g loading) as starting material. Deprotection, coupling, capping, oxidation reaction conditions, deprotection and cutting in the solid phase synthesis method, and separation conditions are the same AS those of the synthesis of the sense strand, so that the antisense strand AS is obtained.
And (3) detection: the purity was measured by ion exchange chromatography (IEX-HPLC), and as a result, the purity was 93.2%; molecular weights were analyzed by liquid chromatography-mass spectrometry (LC-MS). Antisense strand: 7037.1, found: 7036.2.
3.3 Synthesis of oligonucleotide conjugates of the invention (SR16-X2U3460-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 ℃ and cooled at room temperature, and then they were allowed to form a double-stranded structure by hydrogen bonding. Thus obtaining the siRNA double strand SR16-X2U3460-PNB conjugated with PNB. The conjugate has a structure shown in formula (II-1).
The application example is as follows:activity test of the oligonucleotide conjugate of the invention (SR16-X2U3460-PNB)
Unless otherwise specified, reagents and media used in the following examples are commercially available, and procedures such as nucleic acid electrophoresis and real-time PCR are performed according to protocols well known to those skilled in the art. For example, the method can be carried out according to the method described in Molecular Cloning (Cold Spring Harbor LBlaboratory Press (1989)).
Unless otherwise stated, the reagent ratios provided below are calculated as volume ratios (v/v).
Experimental example inhibition efficiency of oligonucleotide conjugate against expression amount of X mRNA from HBV in vivo (in vivo)
In this example, the inhibitory efficiency of the oligonucleotide conjugate of the present invention (SR16-X2U3460-PNB) and negative control 1 XPBS (NS) on the expression level of HBV X mRNA in HBV transgenic mouse 44BriHBV was examined.
HBV transgenic mouse 44BriHBV used in this example was purchased from the laboratory animal department of medicine, Beijing university, approximately 8-12 weeks, male.
First, C57BL/6J-Tg (Alb1HBV)44Bri/J mice were randomly grouped (both female) by serum HbsAg content, 4 mice each group, and a negative control group was given PBS solution. All animals were dosed by weight, single dose (subcutaneous dose) and the siRNA conjugate SR16-X2U3460-PNB was formulated in PBS buffer to give solutions with final concentrations of 0.2mg/ml and 0.02mg/ml (calculated as siRNA) for subcutaneous administration at doses of 1mg/kg body weight and 0.1mg/kg body weight, respectively, and at a dose volume of 5 ml/kg. Animals were sacrificed on day 7 after administration, livers were collected and stored with RNA later (Sigma Aldrich company); homogenizing the liver tissue by a tissue homogenizer, and extracting by Trizol according to the standard operation steps of total RNA extraction to obtain the total RNA.
Detecting the expression level of HBV X mRNA in the liver tissue by adopting real-time fluorescent quantitative PCR, specifically: the extracted total RNA was reverse-transcribed into cDNA using ImProm-IITM reverse transcription kit (Promega corporation) according to its instructions, and then the inhibitory efficiency of siRNA against HBV X mRNA expression in liver tissue was examined using fluorescent quantitative PCR kit (Beijing kang, century Biotechnology Co., Ltd.). In the fluorescent quantitative PCR method, a β -actin (β -actin) gene is used as an internal reference gene, and HBV and β -actin are detected using a primer for HBV and a primer for β -actin, respectively.
See table 2 below for sequences of detection primers.
TABLE 2 detection of primer sequences
Figure BDA0002295222110000441
Figure BDA0002295222110000451
In the fluorescent quantitative PCR method, siRNA inhibitory activity is expressed by the amount of remaining HBV gene expression and is calculated according to the following equation:
the remaining amount of HBV gene expression (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%,
the mRNA inhibition rate was then calculated according to the following formula:
the mRNA inhibition rate is (1-HBV gene expression residual amount) × 100%,
the control group was mice administered with PBS in this experiment, and each test group was mice administered with different siRNA conjugates. The results are shown in table 3 below.
TABLE 3 inhibition of HBV X mRNA expression in mouse liver by siRNA conjugates
Figure BDA0002295222110000452
NA indicates that data is not available.
As can be seen from the results of Table 3, the oligonucleotide conjugates of the present invention in the application examples had a high inhibitory rate against the expression of HBV X mRNA; especially, the inhibitory 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 are to be interpreted in a generic and descriptive sense only and not for purposes of limitation. In some cases, features, and/or elements described in connection with a particular embodiment may be used alone or in combination with features, and/or elements described in connection with other embodiments, unless explicitly indicated otherwise, as will be apparent to those skilled in the art from the present disclosure. It will therefore 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
<110> Sa Ribo Biotechnology Ltd
<120> liver targeting compounds and conjugates
<160> 141
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> RNA
<213> artifical sequence
<400> 1
ccuugaggca uacuucaaa 19
<210> 2
<211> 21
<212> RNA
<213> artifical sequence
<400> 2
uuugaaguau gccucaaggu u 21
<210> 3
<211> 21
<212> RNA
<213> artifical sequence
<400> 3
gaccuugagg cauacuucaa a 21
<210> 4
<211> 23
<212> RNA
<213> artifical sequence
<400> 4
uuugaaguau gccucaaggu cgg 23
<210> 5
<211> 19
<212> RNA
<213> artifical sequence
<400> 5
ccuugaggca uacuucaaa 19
<210> 6
<211> 21
<212> RNA
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<400> 6
uuugaaguau gccucaaggu u 21
<210> 7
<211> 19
<212> RNA
<213> artifical sequence
<400> 7
ccuugaggca uacuucaaa 19
<210> 8
<211> 21
<212> RNA
<213> artifical sequence
<400> 8
uuugaaguau gccucaaggu u 21
<210> 9
<211> 21
<212> RNA
<213> artifical sequence
<400> 9
gaccuugagg cauacuucaa a 21
<210> 10
<211> 23
<212> RNA
<213> artifical sequence
<400> 10
uuugaaguau gccucaaggu cgg 23
<210> 11
<211> 21
<212> RNA
<213> artifical sequence
<400> 11
gaccuugagg cauacuucaa a 21
<210> 12
<211> 23
<212> RNA
<213> artifical sequence
<400> 12
uuugaaguau gccucaaggu cgg 23
<210> 13
<211> 19
<212> RNA
<213> artifical sequence
<400> 13
ccuugaggca uacuucaaa 19
<210> 14
<211> 21
<212> RNA
<213> artifical sequence
<400> 14
uuugaaguau gccucaaggu u 21
<210> 15
<211> 19
<212> RNA
<213> artifical sequence
<400> 15
ccuugaggca uacuucaaa 19
<210> 16
<211> 21
<212> RNA
<213> artifical sequence
<400> 16
uuugaaguau gccucaaggu u 21
<210> 17
<211> 21
<212> RNA
<213> artifical sequence
<400> 17
gaccuugagg cauacuucaa a 21
<210> 18
<211> 23
<212> RNA
<213> artifical sequence
<400> 18
uuugaaguau gccucaaggu cgg 23
<210> 19
<211> 21
<212> RNA
<213> artifical sequence
<400> 19
gaccuugagg cauacuucaa a 21
<210> 20
<211> 23
<212> RNA
<213> artifical sequence
<400> 20
uuugaaguau gccucaaggu cgg 23
<210> 21
<211> 21
<212> RNA
<213> artifical sequence
<400> 21
uuugaaguau gccucaaggu u 21
<210> 22
<211> 21
<212> RNA
<213> artifical sequence
<400> 22
uuugaaguau gccucaaggu u 21
<210> 23
<211> 23
<212> RNA
<213> artifical sequence
<400> 23
uuugaaguau gccucaaggu cgg 23
<210> 24
<211> 23
<212> RNA
<213> artifical sequence
<400> 24
uuugaaguau gccucaaggu cgg 23
<210> 25
<211> 21
<212> RNA
<213> artifical sequence
<400> 25
uuugaaguau gccucaaggu u 21
<210> 26
<211> 21
<212> RNA
<213> artifical sequence
<400> 26
uuugaaguau gccucaaggu u 21
<210> 27
<211> 23
<212> RNA
<213> artifical sequence
<400> 27
uuugaaguau gccucaaggu cgg 23
<210> 28
<211> 23
<212> RNA
<213> artifical sequence
<400> 28
uuugaaguau gccucaaggu cgg 23
<210> 29
<211> 19
<212> RNA
<213> artifical sequence
<400> 29
ugcuaugccu caucuucua 19
<210> 30
<211> 21
<212> RNA
<213> artifical sequence
<400> 30
uagaagauga ggcauagcag c 21
<210> 31
<211> 21
<212> RNA
<213> artifical sequence
<400> 31
uagaagauga ggcauagcau u 21
<210> 32
<211> 19
<212> RNA
<213> artifical sequence
<400> 32
ugcuaugccu caucuucua 19
<210> 33
<211> 21
<212> RNA
<213> artifical sequence
<400> 33
uagaagauga ggcauagcag c 21
<210> 34
<211> 21
<212> RNA
<213> artifical sequence
<400> 34
uagaagauga ggcauagcau u 21
<210> 35
<211> 19
<212> RNA
<213> artifical sequence
<400> 35
ugcuaugccu caucuucua 19
<210> 36
<211> 21
<212> RNA
<213> artifical sequence
<400> 36
uagaagauga ggcauagcag c 21
<210> 37
<211> 21
<212> RNA
<213> artifical sequence
<400> 37
uagaagauga ggcauagcau u 21
<210> 38
<211> 19
<212> RNA
<213> artifical sequence
<400> 38
ugcuaugccu caucuucua 19
<210> 39
<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 (22)

1. A compound having the structure shown in formula (I):
Figure FDA0002295222100000011
wherein the content of the first and second substances,
m represents an integer of 1 to 6;
each R2Independently selected from H, C1-C10Alkyl radical, C1-C10Haloalkyl or C1-C10An alkoxy group;
L1represents a carbonyl group, a carbonyl group (C)1-C10Alkylene) or carbonyl- (oxyethyl)kWherein k represents an integer of 1 to 10;
g represents a first hydroxyl protecting group;
L2represents 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
L2Comprising a solid support attached by said covalent bond;
e represents a ligand M having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells1Or is or
Represents said ligand M1All of the active hydroxyl groups in (1), if any, are protected with a second hydroxyl protecting group;
each L3Is a straight chain 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、C2-C10Alkenylene radical, C2-C10Alkynylene, C6-C10Arylene radical, C3-C18SuterocyclesRadical and C5-C10A heteroarylene group; and the number of the first and second electrodes,
each L3Optionally having any one or more substituents selected from the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Alkyl halides, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl), -N (C)1-C10Alkyl) (C1-C10Alkylphenyl), -NH (C)1-C10Alkylphenyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON (C)1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2，-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl radical C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl group C (O) (phenyl), -C (O) C1-C10Alkyl, -C (O) C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -SO2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl group)。
2. The compound of claim 1, wherein each L3Independently selected from the group consisting of groups of formula A1-A26 and any combination thereof:
Figure FDA0002295222100000012
Figure FDA0002295222100000021
wherein each j1 is independently an integer from 1-20;
each j2 is independently an integer from 1-20;
each R' is independently C1-C10-an alkyl group;
each Ra is independently selected from the group consisting of groups of formula a27-a 45:
Figure FDA0002295222100000022
Figure FDA0002295222100000031
each Rb is independently C1-C10-an alkyl group;
Figure FDA0002295222100000032
represents the site of covalent attachment of a group;
alternatively, L3Is a connection combination of one or more of A1, A4, A5, A6, A8, A10, A11 and A13;
alternatively, L3Is a linked combination of at least 2 of A1, A4, A8, A10 and A11;
alternatively, L3Is a combination of at least 2 of A1, A8 and A10;
alternatively, L3Is 3-25 atoms in length, said L3The length of (a) is the number of chain-forming atoms in the longest atom chain from the atom attached to the N atom of the pyrrolidine ring to the atom attached to E in the compound of formula (I);
alternatively, L3Is 4-15 atoms in length.
3. The compound of claim 2, wherein each j1 is independently an integer from 2-10, each j2 is independently an integer from 2-10, and each R' is independently C1-C4Each Ra is independently one of a27, a28, a29, a30, and a31, and each Rb is independently C1-C5Alkyl groups of (a);
alternatively, each j1 is independently an integer from 3 to 5, each j2 is independently an integer from 3 to 5, each R' is independently one of methyl, ethyl, and isopropyl, each Ra is independently a27 or a28, and each Rb is independently one of methyl, ethyl, isopropyl, and butyl.
4. The compound of claim 1, wherein m is an integer from 2 to 4;
alternatively, each R2Independently H, methyl or ethyl; alternatively, each R2Are all H;
optionally, the first hydroxyl protecting group G is selected from any one of trityl, 4-methoxytrityl, 4 '-bismethoxytrityl, 4', 4 "-trimethoxytrityl and tert-butyldimethylsilyl;
optionally, the hydroxy group protected by the second hydroxy protecting group has the form YCOO-, wherein each Y is independently selected from the group consisting of: c1-C10Alkyl and C6-C10Aryl radical, said C1-C10Alkyl or C6-C10Aryl optionally has one or more substituents selected from the group consisting of halogen substituents and C1-C6Alkyl groups; alternatively, each Y is independently selected from the group consisting of: methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and C1-C6An alkyl phenyl group;
alternatively, L1Is carbonyl (C)2-C6Alkylene) or carbonyl (oxyethyl)kWherein k is an integer of 2 to 7; and/or
Alternatively, L2Containing amino or hydroxy groups, or L2Comprises a structure represented by formula (C1), (C2), (C3), (C4), (C1'), (C3') or (C4 '):
Figure FDA0002295222100000041
in the formula, each q1Independently an integer of 1 to 5, each X independently is O or NH, M+Is a cation, optionally any of a hydrogen ion, an ammonium ion, an alkali metal ion, or an alkaline earth metal ion; SPS represents the solid support for the solid phase,
Figure FDA0002295222100000042
indicates the site at which the group is covalently attached.
5. The compound of claim 1, wherein each M is1Independently a sugar; optionally, each M1Independently a monosaccharide, disaccharide, trisaccharide or polysaccharide;
optionally, at least one M1Is modified;
optionally, each M1Independently 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-mannofuranose, beta-D-mannofuranose, alpha-D-mannopyranose, beta-D-mannopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-glucopyranose, beta-D-furametoseGlucopyranose, alpha-D-fructofuranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [ (R) -1-carboxyethyl group]-2-deoxy-beta-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-glycolyl-alpha-neuraminic acid, 5-thio-beta-D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranoside methyl ester, 4-thio-beta-D-galactopyranose, beta-glucopyranose, beta, One of 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio-alpha-D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allose nitrile, ribose, D-4-thioribose, L-ribose and L-4-thioribose;
optionally, at least one M1Is N-acetylgalactosamine GalNAc;
optionally, each M1Are all N-acetylgalactosamine.
6. The compound of claim 4, wherein each E is independently selected from one of the groups of formula A46-A54:
Figure FDA0002295222100000051
alternatively, E is formula a49 or a 50; alternatively, each Y is methyl.
7. The compound according to claim 1, wherein the compound has a structure represented by formula (I-1), (I-2), (I-3), (I-4), (I-5) or (I-6):
Figure FDA0002295222100000052
Figure FDA0002295222100000061
Figure FDA0002295222100000071
wherein SPS represents a solid support; in some embodiments, the solid support is a resin.
8. An oligonucleotide conjugate having the structure of formula (II):
Figure FDA0002295222100000072
wherein the content of the first and second substances,
E1is OH, SH or BH2Nu represents a functional oligonucleotide;
m represents an integer of 1 to 6;
each R2Independently selected from H, C1-C10Alkyl radical, C1-C10Haloalkyl or C1-C10An alkoxy group;
L1represents a carbonyl group, a carbonyl group (C)1-C10Alkylene) or carbonyl- (oxyethyl)kWherein k represents an integer of 1 to 10;
E2represents amino, hydroxy, -amino (C)1-C10Alkylene) amino, -amino (C)1-C10Alkylene) hydroxy, -hydroxy (C)1-C10Alkylene) amino or hydroxy (C)1-C10Alkylene) hydroxyl, the alkylene optionally having one or more substituents selected from the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Alkyl halides, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON (C)1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2，-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl radical C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl group C (O) (phenyl), -C (O) C1-C10Alkyl, -C (O) C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -SO2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl);
M1represents a ligand having affinity for asialoglycoprotein receptors on the surface of mammalian liver cells;
each L3Is a straight chain alkyl 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、C2-C10Alkenylene radical, C2-C10Alkynylene, C6-C10Arylene radical, C3-C18Heterocyclylene and C5-C10A heteroarylene group; and is
Each L3May optionally have any one or more substituents selected from the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Alkyl halides, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10alkyl-SH, -SC1-C10Haloalkyl, halogen substituents, -OH, -SH, -NH2、-C1-C10alkyl-NH2、-N(C1-C10Alkyl) (C1-C10Alkyl), -NH (C)1-C10Alkyl), -N (C)1-C10Alkyl) (C1-C10Alkylphenyl), -NH (C)1-C10Alkylphenyl), cyano, nitro, -CO2H、-C(O)O(C1-C10Alkyl), -CON (C)1-C10Alkyl) (C1-C10Alkyl), -CONH (C)1-C10Alkyl), -CONH2，-NHC(O)(C1-C10Alkyl), -NHC (O) (phenyl), -N (C)1-C10Alkyl radical C (O) (C)1-C10Alkyl), -N (C)1-C10Alkyl group C (O) (phenyl), -C (O) C1-C10Alkyl, -C (O) C1-C10Alkylphenyl, -C (O) C1-C10Haloalkyl, -OC (O) C1-C10Alkyl, -SO2(C1-C10Alkyl), -SO2(phenyl), -SO2(C1-C10Haloalkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl).
9. The conjugate of claim 8, wherein each L is3Independently selected from the group consisting of groups of formula A1-A26 and any combination thereof:
Figure FDA0002295222100000081
Figure FDA0002295222100000091
wherein each j1 is independently an integer from 1-20;
each j2 is independently an integer from 1-20;
each R' is independently C1-C10-an alkyl group;
each Ra is independently selected from the group consisting of groups of formula a27-a 45:
Figure FDA0002295222100000092
Figure FDA0002295222100000101
each Rb is independently C1-C10-an alkyl group;
Figure FDA0002295222100000102
represents the site of covalent attachment of a group;
alternatively, L3Is a connection combination of one or more of A1, A4, A5, A6, A8, A10, A11 and A13;
alternatively, L3Is a linked combination of at least 2 of A1, A4, A8, A10 and A11;
alternatively, L3Is a combination of at least 2 of A1, A8 and A10;
alternatively, L3Is 3-25 atoms in length, said L3The length of (b) is the number of chain-forming atoms in the longest atom chain from the atom attached to the N atom on the pyrrolidine ring to the atom attached to E;
alternatively, L3Is 4-15 atoms in length.
10. The conjugate of claim 9, wherein each j1 is independently an integer from 2 to 10, each j2 is independently an integer from 2 to 10, and each R' is independently C1-C4Each Ra is independently one of a27, a28, a29, a30, and a31, and each Rb is independently C1-C5Alkyl groups of (a);
alternatively, each j1 is independently an integer from 3 to 5, each j2 is independently an integer from 3 to 5, each R' is independently one of methyl, ethyl, and isopropyl, each Ra is independently a27 or a28, and each Rb is independently one of methyl, ethyl, isopropyl, and butyl.
11. The conjugate of claim 8, wherein E1Is OH, m is an integer of 2 to 4;
alternatively, each R2Independently H, methyl or ethyl;
alternatively, each R2Are all H;
alternatively, L1Is carbonyl (C)2-C6Alkylene) or carbonyl (oxyethyl)kWherein k is an integer of 2 to 7; and/or
Alternatively, E2Represents amino or-amino (C)2-C6Alkylene) hydroxyl.
12. The conjugate of claim 8, wherein each M is1Independently a sugar; optionally, each M1Independently a monosaccharide, disaccharide, trisaccharide or polysaccharide;
optionally, at least one M1Is modified;
optionally, each M1Independently 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-mannofuranose, beta 0-D-mannopyranose, beta 1-D-mannopyranose, beta 2-D-glucopyranose, beta 3-D-glucopyranose, alpha-D-glucopyranose, beta-D-glucopyranose, alpha-D-fructopyranose, alpha-D-galactopyranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactopyranose, beta-D-galactofuranose, beta-D-galactopyranose, alpha-D-galactofuranose, beta-D-galactopyranose, beta-D-, beta-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionyl galactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O- [ (R) -1-carboxyethyl]-2-deoxy-beta-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-glycolyl-alpha-neuraminic acid, 5-thio-beta-D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl-alpha-D-glucopyranoside methyl ester, 4-thio-beta-D-galactopyranose, beta-glucopyranose, beta, One of 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio-alpha-D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allose nitrile, ribose, D-4-thioribose, L-ribose and L-4-thioribose;
optionally, at least one M1Is N-acetylgalactosamine GalNAc;
optionally, each M1Are all N-acetylgalactosamine.
13. The conjugate of claim 8, wherein the conjugate has a structure represented by formula (II-1), (II-2), or (II-3):
Figure FDA0002295222100000111
Figure FDA0002295222100000121
wherein Nu represents a functional oligonucleotide.
14. The oligonucleotide conjugate of claim 8, wherein the functional oligonucleotide is selected from one of a small interfering RNA, a microrna, an anti-microrna, a microrna antagonist, a microrna mimetic, a decoy oligonucleotide, an immunostimulatory substance, a G-quadrupole, a variable spliceosome, a single-stranded RNA, an antisense nucleic acid, an aptamer, a stem-loop RNA, an mRNA fragment, an activating RNA, or a DNA; optionally, the functional oligonucleotide is a single-stranded oligonucleotide or a double-stranded oligonucleotide; optionally, the functional oligonucleotide is a single-stranded oligonucleotide, P in formula (II) is attached to the end of the single-stranded oligonucleotide, which refers to the first 4 nucleotides from one end of the single-stranded oligonucleotide; alternatively, P in formula (II) is attached to the end of the single stranded oligonucleotide; optionally, P in formula (II) is attached to the 3' end of the single stranded oligonucleotide;
optionally, the functional oligonucleotide is a double-stranded oligonucleotide comprising a sense strand and an antisense strand, P in formula (II) is ligated to the end of the double-stranded oligonucleotide, which refers to the first 4 nucleotides from one end of the sense strand or the antisense strand; preferably, P in formula (II) is attached to the end of the sense strand or the antisense strand; preferably, P in formula (II) is attached to the 3' end of the sense strand; preferably, P in formula (II) is linked to the 2', 3' or 5' position of the nucleotide in the oligonucleotide conjugate by forming a phosphodiester bond.
15. The conjugate of claim 14, wherein the double-stranded oligonucleotide is an siRNA;
optionally, each nucleotide in the siRNA is independently a modified or unmodified nucleotide, the siRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence 1, the antisense strand comprises a nucleotide sequence 2, the nucleotide sequence 1 and the nucleotide sequence 2 are both 19 nucleotides in length and are at least partially reverse-complementary to form a double-stranded region, the nucleotide sequence 2 is at least partially complementary to a first nucleotide sequence, the first nucleotide sequence is a nucleotide sequence in a target mRNA, and the target mRNA refers to mRNA corresponding to a gene abnormally expressed in a hepatocyte;
optionally, the target mRNA is selected from one of the mrnas corresponding to the following genes: ApoB, ApoC, ANGPTL3, PCSK9, SCD1, FVII, p53, HBV, HCV; optionally, the target mRNA is selected from the group consisting of hepatitis b virus mRNA, angiopoietin-like protein 3 gene expressed mRNA, or apolipoprotein C3 gene expressed mRNA.
Optionally, the nucleotide sequence 1 is equal in length to the first nucleotide sequence and does not differ by more than 3 nucleotides; the nucleotide sequence 2 and the nucleotide sequence B are equal in length and have no more than 3 nucleotide differences; the nucleotide sequence B is a nucleotide sequence which is equal to the length of the first section of nucleotide and is completely reverse complementary to the sequence;
alternatively, said nucleotide sequence 1 differs from said first stretch of nucleotide sequence by no more than 1 nucleotide, and/or said nucleotide sequence 2 differs from said nucleotide sequence B by no more than 1 nucleotide;
alternatively, 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 the 5' end to 3' end direction;
alternatively, the last nucleotide Z on said nucleotide sequence 1 is the nucleotide complementary to Z ' in the 5' to 3' direction.
Alternatively, said nucleotide sequence 1 and said nucleotide sequence 2 are substantially reverse complementary, substantially complete reverse complementary or complete reverse complementary.
Optionally, the sense strand further comprises a nucleotide sequence 3, the antisense strand further comprises a nucleotide sequence 4, the nucleotide sequence 3 and the nucleotide sequence 4 are equal in length and are each 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 is a nucleotide sequence adjacent to the first nucleotide sequence in the target mRNA and has the same length as the nucleotide sequence 4, and the nucleotide sequence 3 and the nucleotide sequence 4 are substantially completely reverse complementary or completely reverse complementary;
optionally, the siRNA further comprises a nucleotide sequence 5, wherein the nucleotide sequence 5 is 1 to 3 nucleotides in length, and is linked to the 3 'end of the antisense strand, thereby forming a 3' overhang of the antisense strand;
optionally, the length of the nucleotide sequence 5 is 2 nucleotides, and the nucleotide sequence 5 is 2 consecutive deoxythymine nucleotides, 2 consecutive uracil nucleotides, or is complementary to a third nucleotide sequence which is adjacent to the first nucleotide sequence or the second nucleotide sequence and has the same length as the nucleotide sequence 5 in the direction from the 5 'end to the 3' end.
16. The conjugate of claim 14, 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 with a modifying group;
optionally, 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 the non-fluoro-modified nucleotide refers to a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorine group;
optionally, the sense strand and the antisense strand both comprise fluoro-modified nucleotides and non-fluoro-modified nucleotides, the fluoro-modified nucleotides are located in the nucleotide sequence 1 and the nucleotide sequence 2, the fluoro-modified nucleotides in the nucleotide sequence 1 are not more than 5, and the nucleotides at the 7 th, 8 th and 9 th positions of the nucleotide sequence 1 are fluoro-modified nucleotides according to the direction from the 5 'end to the 3' end; no more than 7 fluorinated modified nucleotides in the nucleotide sequence 2, and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence 2 are fluorinated modified nucleotides according to the direction from the 5 'end to the 3' end;
optionally, in the direction from 5 'end to 3' end, in the sense strand, the 7 th, 8 th, 9 th or 5 th, 7 th, 8 th, 9 th nucleotide of the nucleotide sequence 1 is a fluorinated modified nucleotide, and the rest of the nucleotides in the sense strand are non-fluorinated modified nucleotides; in the antisense strand, the 2 nd, 6 th, 14 th, 16 th or 2 nd, 6 th, 8 th, 9 th, 14 th, 16 th nucleotide of the nucleotide sequence 2 is a fluorinated modified nucleotide, and the rest of the nucleotides in the sense strand are non-fluorinated modified nucleotides.
Optionally, the nucleotide formed by substituting the hydroxyl at the 2 '-position of the ribosyl of the nucleotide by a non-fluorine group is selected from one of 2' -alkoxy modified nucleotide, 2 '-substituted alkoxy modified nucleotide, 2' -alkyl modified nucleotide, 2 '-substituted alkyl modified nucleotide, 2' -amino modified nucleotide, 2 '-substituted amino modified nucleotide, 2' -deoxynucleotide; the nucleotide analogue is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA;
alternatively, each of the non-fluorinated modified nucleotides is a methoxy modified nucleotide, which means a nucleotide in which the 2' -hydroxyl group of the ribose group of the nucleotide is substituted with a methoxy group.
17. The conjugate according to claim 16, wherein the phosphate group having a modifying group is a phosphorothioate group in which at least one oxygen atom in a phosphodiester bond in the phosphate group is substituted with a sulfur atom;
optionally, the phosphate group with the modification group is a thiophosphate group with a structure shown as formula (201):
Figure FDA0002295222100000141
optionally, phosphorothioate linkages are present in at least one of:
between the 1 st and 2 nd nucleotides at the 5' terminal end of the sense strand;
between the 2 nd and 3 rd nucleotides at the 5' terminal end of the sense strand;
between the 1 st and 2 nd nucleotides at the 3' terminal end of the sense strand;
between the 2 nd and 3 rd nucleotides at the 3' terminal end of the sense strand;
between the 1 st and 2 nd nucleotides at the 5' terminal end of the antisense strand;
between the 2 nd and 3 rd nucleotides at the 5' terminal end of the antisense strand;
between the 1 st and 2 nd nucleotides at the 3' terminal end of the antisense strand; and
the 3' terminal end of the antisense strand is between the 2 nd and 3 rd nucleotides.
18. The conjugate of any one of claims 12-15, wherein the 5' terminal nucleotide of the antisense strand is a 5' -phosphate nucleotide or a 5' -phosphate analogue modified nucleotide;
alternatively, the nucleotide 5 '-phosphate or nucleotide 5' -phosphate analogue modified is a nucleotide represented by one of the following formulae (202) to (206):
Figure FDA0002295222100000142
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;
preferably, the nucleotide 5 '-phosphate or nucleotide 5' -phosphate analogue modified is a nucleotide represented by formula (202), formula (203) or formula (205);
optionally, the nucleotide sequence is selected from one of tables 1A, 1B, 1C, 1D, 1E or 1F.
19. Use of a conjugate according to any one of claims 8 to 18 in the manufacture of a medicament for the treatment and/or prevention of a pathological condition or disease caused by the expression of a specific gene in a hepatocyte; preferably, the specific gene is selected from a hepatitis b virus gene, an angiopoietin-like protein 3 gene or an apolipoprotein C3 gene.
20. The use according to claim 19, wherein the disease is selected from chronic liver disease, hepatitis, liver fibrosis disease, liver proliferative disease and dyslipidemia;
optionally, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.
21. A method of inhibiting the expression of a specific gene in a hepatocyte, wherein the method comprises contacting the hepatocyte with an effective amount of a conjugate according to any one of claims 8 to 18;
optionally, the specific gene is selected from one of the following genes: ApoB, ApoC, ANGPTL3, PCSK9, SCD1, FVII, p53, HBV, HCV;
optionally, the specific gene is selected from a hepatitis b virus gene, an angiopoietin-like protein 3 gene, or an apolipoprotein C3 gene.
22. A kit comprising the conjugate of any one of claims 8-18.
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