CN111377984A - Compounds and conjugates and methods of making and using the same - Google Patents

Compounds and conjugates and methods of making and using the same Download PDF

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CN111377984A
CN111377984A CN201811637314.7A CN201811637314A CN111377984A CN 111377984 A CN111377984 A CN 111377984A CN 201811637314 A CN201811637314 A CN 201811637314A CN 111377984 A CN111377984 A CN 111377984A
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alkyl
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nucleotide
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conjugate
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CN111377984B (en
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张鸿雁
杨志伟
曹力强
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Suzhou Ribo Life Science Co Ltd
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • 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
    • 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

A compound that can form a conjugate with an oligonucleotide, the compound having a structure as shown in formula (101). The present disclosure also provides corresponding oligonucleotide conjugates. The oligonucleotide conjugate disclosed by the invention can specifically target liver cells, so that the problem of in vivo delivery of oligonucleotide drugs is effectively solved, the toxicity is low, and the delivered oligonucleotide has high stability.

Description

Compounds and conjugates and methods of making and using the same
Technical Field
The present application relates to compounds and conjugates for drug delivery and methods for their preparation and use.
Background
The delivery system is one of the key technologies in the development of small nucleic acid drugs, and the delivery system which is the most widely researched delivery system of small nucleic acid worldwide is the targeted conjugation delivery technology.
Disclosure of Invention
In one embodiment, the present disclosure provides a conjugate molecule having a structure represented by formula (101):
Figure BDA0001930334880000011
wherein:
n1is an integer selected from 1-2;
each n is2Independently an integer selected from 1-2;
m1is an integer selected from 1 to 6;
R1is a group capable of binding to an active drug via a covalent bond; (ii) a
Each R2Each independently selected from H, C1-C10Alkyl radical, C1-C10Haloalkyl or C1-C10An alkoxy group;
each L1Is 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 wherein L1Optionally a substituent having any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -SC1-C10Alkyl, -SC1-C10Alkylphenyl, -C1-C10Alkyl radical-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);
each S1Independently is M1Wherein any reactive hydroxyl groups, if any, are protected with a hydroxyl protecting group;
each M1Independently selected from ligands capable of binding to cell surface receptors.
In some embodiments, each L is1A linked combination of one or more independently selected from the group of formula A1-A26:
Figure BDA0001930334880000021
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 alkyl;
each Ra is independently selected from one of the groups of formula A27-A45:
Figure BDA0001930334880000031
Figure BDA0001930334880000041
each Rb is independently a C1-C10 alkyl group;
Figure BDA0001930334880000042
represents the site at which the groups are linked by a covalent bond;
in one embodiment, the present disclosure provides a conjugate having a structure represented by formula (201):
Figure BDA0001930334880000043
wherein:
n1is an integer selected from 1-2;
each n is2Independently selected from integers from 1 to 2;
m1is an integer selected from 1 to 6;
each R2Each independently is H, C1-C10Alkyl radical, C1-C10Haloalkyl or C1-C10An alkoxy group;
R6is an active drug;
R5is a straight chain alkylene group of 1 to 20 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 it isIn, R5May optionally have a substituent of any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -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);
each L1Is 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 wherein L1Optionally a substituent having any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -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);
each M1Selected from ligands capable of binding to cell surface receptors.
In some embodiments, each L is1A linked combination of one or more independently selected from the group of formula A1-A26:
Figure BDA0001930334880000051
Figure BDA0001930334880000061
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 alkyl;
each Ra is independently selected from one of the groups of formula A27-A45:
Figure BDA0001930334880000062
Figure BDA0001930334880000071
each Rb is independently a C1-C10 alkyl group;
Figure BDA0001930334880000072
represents the site at which the groups are linked by a covalent bond;
in some embodiments, there is provided the use of a compound of the present disclosure for the preparation of a medicament for the treatment and/or prevention of a pathological condition or disease caused by the expression of a particular gene.
In some embodiments, the present disclosure provides a method of treating a pathological condition or disease caused by expression of a particular gene in a hepatocyte, comprising providing to a subject an effective amount of a conjugate of the present disclosure.
In some embodiments, the present disclosure provides a method of inhibiting the expression of a particular gene in a hepatocyte, the method comprising contacting with a conjugate of the present disclosure.
In some embodiments, the present disclosure provides a kit comprising a conjugate of the present disclosure.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Is incorporated by reference
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
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 below, capital C, G, U, A represents the base composition of nucleotides, unless otherwise specified; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a 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 a phosphorothioate-based linkage between two nucleotides adjacent to the left and right of the letter s; p1 indicates that the nucleotide adjacent to the right side of P1 is a nucleotide 5 '-phosphate or a nucleotide modified with a 5' -phosphate analog, particularly a nucleotide modified with a vinyl phosphate (VP in the following examples), a nucleotide 5 '-phosphate (P in the following examples), or a nucleotide modified with a 5' -phosphorothioate (Ps in the following examples).
In the present context, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to the skilled person, i.e. in a double stranded nucleic acid molecule, the bases of one strand pair with the bases on the other strand in a complementary manner. In DNA, the purine base adenine (a) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair comprises a purine and a pyrimidine. Two strands are considered to be complementary to each other when adenine on one strand always pairs with thymine (or uracil) on the other strand and guanine always pairs with cytosine, and the sequence of a strand may be deduced from the sequence of its complementary strand. Accordingly, "mismatch" in the art means that in a double-stranded nucleic acid, the bases at the corresponding positions are not paired in a complementary fashion.
In the above and below, essentially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences involved, unless otherwise specified; substantially perfectly reverse complementary means that no more than 1 base mismatch exists between two nucleotide sequences; perfect complementarity means that there is no base mismatch between two nucleotide sequences. In the above and below, the nucleotide difference between one nucleotide sequence and the other nucleotide sequence means that the nucleotide at the same position has a change in the base type as compared with the latter, for example, in the case where one nucleotide base is A in the latter, in the case where the corresponding nucleotide base at the same position is U, C, G or T, it is considered that there is a nucleotide difference between the two nucleotide sequences at that position. In some embodiments, when a nucleotide in situ is replaced with a nucleotide or nucleotide analog without a base, it is also believed that a nucleotide difference is created at that position.
In the above and the following, particularly in describing the preparation method of the conjugate molecule or the preparation method of the siRNA conjugate of the present disclosure, unless otherwise specified, the nucleoside monomer (nucleoside monomer) means that the "unmodified or modified RNA phosphoramidite" is used for so-called solid phase phosphoramidite synthesis, which is a well-known method for synthesizing RNA in the art, respectively, depending on the RNA sequence to be prepared. RNA phosphoramidites are also referred to herein as nucleoside phosphoramidites. Unless otherwise indicated, nucleoside 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-C10An alkyl group is 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,') "Is optionally substitutedThe (optinallly substituted) alkyl group "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 naming 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 obtained by the removal of one molecule of hydrogen from the adjacent carbon atom of the parent alkyl group. The group may be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to: a vinyl group; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (i.e., allyl), prop-2-en-2-yl; butenyl, e.g., but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-2-yl, but-1, 3-dien-1-yl, but-1, 3-dien-2-yl, and the like. In certain embodiments, alkenyl groups have 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to the same residue as alkenyl, but having two points of attachment.
As used herein, "alkynyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond obtained by removing two hydrogen molecules from adjacent carbon atoms of the parent alkyl group. Typical alkynyl groups include, but are not limited to: an ethynyl group; propynyl groups, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl groups such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl and the like. In certain embodiments, alkynyl groups have 2 to 20 carbon atoms, and in other embodiments 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to the same residue as alkynyl, but having two points of attachment.
As used herein, "alkoxy" refers to an alkyl group of the indicated number of carbon atoms attached through an oxygen bridge, 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 attached through an oxygen bridge.
As used herein, "aryl" refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system containing 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 contains a cyclic, delocalized (4n +2) pi-electron system according to H ü kel theory.
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 as defined above wherein the specified number of carbon atoms are 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" means a stable 3-to 18-membered non-aromatic cyclic group containing 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless otherwise indicated in the specification, heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, and may include fused or bridged ring systems. The heteroatoms in the heterocyclic radical may optionally be oxidized. One or more nitrogen atoms (if present) are optionally quaternized. Heterocyclyl groups are partially or fully saturated. The heterocyclic group may be attached to the rest of the molecule through any atom of the ring. Examples of such heterocyclic groups include, but are not limited to: dioxanyl, thienyl [1,3] dithioyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrimidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithioyl, tetrahydropyranyl, thiomorpholinyl, 1-oxathiomorpholinyl, and 1, 1-dioxathiomorpholinyl.
"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, heteroaryl may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., according to H ü ckel theory, heteroaryl comprising a cyclic delocalized (4n +2) pi-electron system includes a fused ring or bridged ring system, heteroaryl is optionally oxidized, one or more nitrogen atoms (if present) are optionally quaternized, the heteroaryl is attached to the remainder of the molecule through any atom in the ring, examples of heteroaryl include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1, 3-benzodioxazolyl, benzofuranyl, benzoxazolyl, 1, 3-benzodioxinyl, 7-5, 7-dihydrooxazolyl, 7-5, 7-H-indazolyl, 7-5, 7-dihydrooxazolyl, 7, 5, 7-dihydrooxazolyl, 7-5, 7-H, 7-oxazolyl, 5-5, 7-dihydrooxazolyl, 7, 5-dihydrooxazolyl, 7-oxazolyl, 5-oxazolyl, 7-oxazolyl, 1, 5-oxazolyl, 5-7, 7-oxazolyl, 5-oxazolyl, 1, 7-5-oxazolyl, 5-dihydrooxazolyl, 7-oxazolyl, 7,1, 7-oxazolyl, 5-7, 5-oxazolyl, 5-dihydrooxazolyl, 7,1, 5-7-dihydrooxazolyl, 5-oxazolyl, 5-7, 7-oxazolyl, 5-oxazolyl, 7-oxazolyl, 5-7-oxazolyl, 1, 5-oxazolyl, 5-7, 5-1, 5-dihydrooxazolyl, 5-7, 5-1, 5-2-7, 5-oxazolyl, 5-1, 5-7, 5-oxazolyl, 5-7, 5-oxazolyl, 7, 5-o [ 7, 5-7-oxazolyl, 5-7, 5-o [ 10-7, 5-dihydrooxazolyl, 5-oxazolyl, 5-7, 5-oxazolyl, 1, 5-oxazolyl, 5-dihydrooxazolyl, 1, 5-oxazolyl, 5-o [ 10-oxazolyl, 1, 5-oxazolyl, 1, 7, 5-oxazolyl, 5-dihydrooxazolyl, 5-oxazolyl, 5-H-oxazolyl, 1, 7,1, 7,1, 5-oxazolyl, 5-o [ 10-oxazolyl, 5-o [ 10-oxazolyl, 1, 5-oxazolyl, 2-oxazolyl, 5-o [ 10-o [ 7.
Various hydroxyl protecting groups may be used in the present disclosure. In general, protecting groups render a chemical functionality insensitive to the particular reaction conditions, and can be added to and removed from 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 2,2ded, 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).
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, "method of treatment," "treatment" to 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.
Conjugated molecules
In one aspect, a conjugate molecule for delivering an active agent or drug is disclosed. 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 are directed specifically to hepatocyte surface receptors, and thus the patent is directed to liver tissue. In some embodiments, the conjugate molecules of the present disclosure are directed specifically to cell surface receptors specific to hepatocytes. In some embodiments, the conjugate molecules of the present disclosure are directed specifically to asialoglycoprotein receptors (ASGPR) on the surface of the liver.
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 delivery to a hepatocyte. Such agents are well known to those skilled in the art and include, but are not limited to, functional nucleotides, such as functional oligonucleotides, particularly those disclosed herein.
In some embodiments, the present disclosure provides a conjugate molecule having a structure represented by formula (101):
Figure BDA0001930334880000111
wherein:
n1is an integer selected from 1-2; each n is2Independently an integer selected from 1-2;
m1is an integer selected from 1 to 6;
R1is a group capable of binding to an active drug via a covalent bond;
each L1Is 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 wherein L1Optionally a substituent having any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -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);
each S1Independently is M1Wherein any reactive hydroxyl groups, if any, are protected with a hydroxyl protecting group;
each M1Independently selected from ligands capable of binding to cell surface receptors.
In some embodiments, m is1May be an integer from 1 to 6, thereby ensuring that S is present in the conjugate molecule1The number of groups is at least 2; in one embodiment, m1Is an integer selected from 2 to 6, such that in the oligonucleotide conjugate formed from the conjugate molecule, M is1The number of ligands is at least 3, such that M1The ligand binds more readily to the liver surface asialoglycoprotein receptor, thereby facilitating entry of the conjugate into cells by endocytosis. Experiments show that when M is 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, therefore, results from ease of synthesis, structure/processA combination of aspects such as cost and delivery efficiency, and in some embodiments, m1Is 2.
In some embodiments, n is1Is an integer selected from 1 to 2, each n2Independently an integer selected from 1-2, such that in an oligonucleotide conjugate formed from the conjugate molecule, a plurality of M' s1Spatial position between ligands is adapted to M1Binding of ligands to hepatic surface asialoglycoprotein receptors in order to make the conjugate molecules provided by the present disclosure simpler, easier to synthesize and/or reduce cost, according to one embodiment of the present disclosure, n1＝n2＝2。
It will be understood by those skilled in the art that when each R is present2Each independently selected from H, C1-C10Alkyl radical, C1-C10Haloalkyl or C1-C10Alkoxy groups, without altering the nature of the conjugate molecules provided by the present disclosure, are all possible to achieve the objectives of the present disclosure. In some embodiments, each R is2Independently selected from H, methyl or ethyl. In some embodiments, each R is2Are all H.
R1Are groups that can be delivered by the conjugate molecules of the present disclosure and bind to an active drug (also referred to as an active agent). In some embodiments, R1To be a group capable of binding to an oligonucleotide, the oligonucleotide will be delivered by a conjugate molecule of the present disclosure. In some embodiments, R1Is a group capable of binding to an oligonucleotide by a covalent bond. In some embodiments, R1Is a group capable of binding to an oligonucleotide via a phosphodiester bond. In some embodiments, R1Are selected to achieve attachment to the N on the nitrogen-containing backbone and to provide suitable reaction sites for synthesis of the oligonucleotide conjugate. In the context of the present disclosure, "nitrogen-containing backbone" means a linkage with R2A chain structure in which the carbon atoms of (b) and N are linked to each other. In some embodiments, R1There may be a group capable of being bonded to the N atom on the nitrogen-containing skeleton in an appropriate manner. In some embodiments, R1Containing links in groups to N on nitrogen-containing skeletonsA linker site and any functional group that may be reacted to conjugate to an oligonucleotide via a phosphodiester bond.
In some embodiments, R1Further comprising a2 nd functional group, said 2 nd functional group being capable of forming a covalent bond with a hydroxyl group or an amino group, or being a solid support attachable by a covalent bond formed with a hydroxyl group or an amino group; in yet another embodiment, the 1 st functional group is a phosphoramidite, hydroxyl, or protected hydroxyl, and in some embodiments, the 2 nd functional group is a phosphoramidite, carboxyl, or carboxylate; in some embodiments, the carboxylate is a carboxylate with a metal cation, an ammonium carboxylate salt, a tertiary amine carboxylate salt, or a quaternary ammonium carboxylate salt; in some embodiments, the carboxylate is triethylamine carboxylate or N, N-diisopropylethylamine carboxylate. In some embodiments, the solid support that can be attached by a covalent bond with a hydroxyl group or an amino group is a solid support that is attached by a phosphate ester bond, a carboxylate ester bond, and/or an amide bond. In some embodiments, the solid support is a resin.
In some embodiments, the 1 st functional group contains a hydroxyl group, -ORkOr a group of formula (C3); and/or the 2 nd functional group has a structure represented by formula (C1), (C2), (C3), (C1 ') or (C3'):
Figure BDA0001930334880000121
Figure BDA0001930334880000131
in the formula, q1Is an integer of 1 to 4, X is O or NH, M+Is a cation, SPS represents a solid support,
Figure BDA0001930334880000132
indicates the site at which the group is covalently attached.
In some embodiments, the 1 st functional group comprises a phosphoramidite functionality, as shown in formula (C3), which can be coupled to a hydroxyl group at any position on a nucleotide, such as the 2 'hydroxyl or the 3' hydroxyl, and oxidized to form a phosphodiester bond, conjugating the conjugation molecule to an oligonucleotide. At this time, the conjugate molecule of the present disclosure can be conjugated to a nucleotide even if the 2 nd functional group is not present. At this point, the conjugate molecules of the present disclosure are adapted to react with hydroxyl groups on the terminal nucleotides in the nucleotide sequence and form phosphodiester linkages during subsequent oxidation, thereby conjugating the conjugate molecules of the present disclosure to the oligonucleotide.
In some embodiments, the 1 st functional group contains a protected hydroxyl group. In some embodiments, the 2 nd functional group contains a group that reacts with a solid support to provide a conjugate molecule containing a solid support. In some embodiments, the 2 nd functional group contains a carboxylic acid functional group, a carboxylate functional group, or a phosphoramidite functional group, as shown in formula (C1), (C2), or (C3), which can undergo an esterification reaction or an amidation reaction with a hydroxyl or amino group on a solid support, e.g., a resin, to form a conjugate molecule containing a carboxylate-linked solid support or an amide-linked solid support. The phosphoramidite functionality can be coupled to a hydroxyl group on a common solid support, such as a resin, and oxidized to form a solid support linked via a phosphodiester linkage. Now, according to one aspect of the invention, there is provided a method of preparing a conjugate of the present disclosure using such a conjugate molecule. In some embodiments, the method comprises first attaching the conjugate molecule to a solid support via a condensation or coupling reaction, and then adding a nucleoside monomer according to the solid phase phosphoramidite synthesis method to provide a conjugate of the present disclosure that conjugates the conjugate molecule of the present disclosure to an oligonucleotide. In some embodiments, deprotection of the 1 st functional group occurs during solid phase phosphoramidite synthesis, followed by coupling to a phosphoramidite group on a nucleotide under coupling reaction conditions.
In some embodiments, R1Contains a1 st functional group and a2 nd functional group, wherein the 1 st functional group contains a hydroxyl group or a protected hydroxyl group; the 2 nd functional group contains a carboxylic ester bond, an amide bond or a phosphodiester bond, or is bound via a carboxyl groupSolid phase carriers connected by acid ester bonds, amido bonds or phosphodiester bonds. In some embodiments, the 2 nd functional group is a group according to formula (C1 ') or (C3'). In some embodiments, when the 2 nd functional group comprises a solid support, the conjugate molecule comprising the solid support facilitates preparation of the conjugates of the present disclosure. Accordingly, in one aspect of the invention, there is provided a method of preparing a conjugate of the present disclosure using the conjugate molecule. In some embodiments, the method comprises reacting a conjugate molecule comprising a solid support with a nucleoside monomer according to a phosphoramidite solid phase synthesis method, thereby conjugating the conjugate molecule of the present disclosure to an oligonucleotide. In some embodiments, the conjugate molecule containing the solid support may be obtained internally by reaction of a conjugate molecule with the solid support, the conjugate molecule reacting with a carboxyl group, a carboxylate salt, or a phosphoramidite. In some embodiments, the conjugate molecule may be commercially available.
In some embodiments, the carboxylate functional group may be represented by-COO-M+Wherein M is+Is a cation, e.g. selected from the group consisting of metal cations, ammonium cations NH4 +One of organic ammonium cations. In some embodiments, the metal ion is selected from one of alkali metal ions, such as K+Or Na+. In view of the solubility enhancement and the ease of reaction, in some embodiments, the organic ammonium ion is an ammonium cation formed from a tertiary amine or a quaternary ammonium cation, such as an ammonium ion formed from triethylamine or an ammonium ion formed from N, N-diisopropylethylamine. In some embodiments, the carboxylate is triethylamine carboxylate or N, N-diisopropylethylamine carboxylate.
In some embodiments of the disclosure, R1Has a structure represented by formula (B9), (B10), (B9 '), (B10'), (B11), (B12), (B11 ') or (B12'):
Figure BDA0001930334880000141
wherein q is1Is an integer of 1 to 4, q2Is an integer of 1 to 10, X is O or NH, M+Is a cation, RkIs a hydroxyl protecting group, SPS represents a solid phase carrier,
Figure BDA0001930334880000142
indicating the site of covalent attachment of the group. In some embodiments, q is1Is 1 or 2. In some embodiments, q is2Is an integer of 1 to 5. In some embodiments, R1Contains a structure represented by the formula (B9) or (B10). In some embodiments, R1Contains a structure represented by the formula (B11) or (B12). In some embodiments, RkIs one or more of Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4 '-bismethoxytrityl), TMTr (4,4' -trimethoxybenzyl). In some embodiments, RkMay be DMTr. L is1Is 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 wherein L1May optionally have a substituent of any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -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, and,Nitro radical, -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). The skilled person will understand that although for convenience L is used1Is defined as a linear alkyl group, but it may not be a linear group or differ in name, for example, by an amine or alkenyl group resulting from the above substitution and/or displacement. For purposes of this disclosure, L1Is the number of atoms in the chain connecting the two attachment points. For this purpose, the ring (e.g., heterocyclylene or heteroarylene) obtained by substituting the carbon atom of the linear alkyl group is counted as one atom.
In some embodiments, L1Has the effect of mixing M1The ligand is linked to the N on the nitrogen-containing backbone, thereby providing liver targeting functionality to the oligonucleotide conjugates of the present disclosure. In some embodiments, L1One or more connecting combinations selected from the group of the formulas A1-A26. In some embodiments, L1A combination of one or more linkages selected from a1, a4, a5, a6, A8, a10, a11, and a 13. In some embodiments, L1A linked combination of at least 2 selected from a1, a4, A8, a10, and a 11. In some embodiments, L1At least 2 connection combinations selected from A1, A8 and A10。
In some embodiments, L1May be 3-25, 3-20, 4-15, or 5-12 atoms in length. In some embodiments, L1Is 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. According to some embodiments of the present disclosure, j1 is an integer from 2 to 10, and in some embodiments, j1 is an integer from 3 to 5. In some embodiments j2 is an integer from 2 to 10, and in some embodiments j2 is an integer from 3 to 5. R 'is a C1-C4 alkyl group, and in some embodiments, R' is one of methyl, ethyl, and isopropyl. Ra is one of a27, a28, a29, a30, and a31, and in some embodiments, Ra is a27 or a 28. Rb is a C1-C5 alkyl group, and in some embodiments Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', Ra, Rb are each selected in formulas A1-A26 to achieve M1Ligands are attached to N on nitrogen-containing backbones and M is1The spatial position between the ligands is more suitable for M1The ligand binds to the hepatic surface asialoglycoprotein receptor.
Each M1Independently selected from ligands capable of binding to cell surface receptors. In some embodiments, at least one M1Are ligands that are capable of binding to receptors on the surface of the liver. In some embodiments, at least one M1Is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, at least one M1Is a ligand capable of binding to human hepatocyte surface receptors. In some embodiments, at least one M1Are ligands capable of binding to the hepatic surface asialoglycoprotein receptor (ASGPR).
In some embodiments, M1May 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 M1Is a saccharide. In some embodiments, each M is1Is sugar. In some embodimentsIn at least one M1Is monosaccharide, disaccharide, trisaccharide or polysaccharide. In some embodiments, each M is1Is monosaccharide, disaccharide, trisaccharide or polysaccharide. In some embodiments, at least one M1Is a modified sugar. In some embodiments, each M is1Is a modified sugar. In some embodiments, each M is1Independently selected from polysaccharides, modified polysaccharides, monosaccharides or monosaccharide derivatives. In some embodiments, each or at least one 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 and maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.
In some embodiments, each or at least one 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, α -D-mannofuranose, β -D-mannofuranose, β 0-D-mannopyranose, β 1-D-mannopyranose, β 2-D-glucopyranose, β 3-D-glucopyranose, α -D-glucofuranose, β -D-glucofuranose, α -D-fructofuranose, α -D-fructopyranose, α -D-galactopyranose, β -D-galactopyranose, α -D-galactofuranose, β -D-galactofuranose, glucosamine, sialic acid, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino- [ (3-O) -1-carboxyethyl-R) -galactosamine]-2-deoxy- β -D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-glycolyl- α -neuraminic acid, 5-thio- β -D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl- α -D-glucopyranoside methyl ester, 4-thio- β -D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio- α -D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allositrile, ribose,D-ribose, D-4-thioribose, L-ribose, L-4-thioribose. In some embodiments, each M is1Are all N-acetylgalactosamine (GalNAc). In some embodiments, ligand selection may be found, for example, in 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 linking group or neutral linking group and 1 or more ligands each selected from the group consisting of polysaccharide, modified polysaccharide, mannose, galactose, mannose derivative, galactose derivative, D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α -D-mannofuranose, β -D-mannofuranose, β 0-D-mannopyranose, β -D-mannopyranose, β -D-glucopyranose, β -D-glucopyranose, β -4-D-glucopyranose, β -D-glucopyranose, β -D-fructofuranose, β -D-fructopyranose, galactopyranose, α -D-glucopyranose, β -D-glucopyranose, galactose-0-D-fructopyranose, 3-D-ribopyranose, acetyl-4-ribopyranose, N-D-ribopyranose, D-464-ribopyranose, L-5-D-mannopyranose, L-D-ribopyranose, L-4-D-ribopyranose, L-466-D-pyranose, D-ribosyl, L-3-D-4-pyranose, L-4-ribosyl, L-D-4-pyranose, L-4-pyranose, D-466-pyranose, D-4-pyranose, D-466, D-pyranose, D-466, D-pyranose, D-4-pyranose, D-466, D-pyranose, D-4-pyranose, D-466, D-pyranose, D-4-pyranose, D-466, D-pyranose, D-466, D.
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 N-acetylgalactosamine (GalNAc) of ASGPR is used as a targeting molecule, and the high affinity ligand has a good effect on liver targeting delivery of nucleic acid drugs. 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, S1Independently is M1. In some embodiments, S1Independently is M1Wherein at least one of the reactive hydroxyl groups is substituted with a hydroxyl protecting group. In some embodiments, S1Independently is M1Wherein all hydroxyl groups are substituted with a hydroxyl protecting group. In some embodiments, any hydroxy protecting group known to those skilled in the art may be used to protect M1The above reactive hydroxyl group. In some embodiments, the protected hydroxy group is represented by the formula YCOO-, wherein each Y is independently selected from C1-C10Alkyl radical, C1-C10Aryl, substituted C1-C10Alkyl or substituted C1-C10And (4) an aryl group. In some embodimentsIn (1), substituted C1-C10Alkyl is selected from the group consisting of alkyl containing one or more halogen substituents and/or one or more C1-C6C of alkyl-substituted radicals1-C10An alkyl group. In some embodiments, substituted C1-C10Aryl is selected from the group consisting of containing one or more halogen substituents and/or one or more C1-C6C of alkyl-substituted radicals1-C10And (4) an aryl group. In some embodiments, 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-C6 alkylphenyl.
In some embodiments, each S is1Each independently one of the groups of formula A46-A54:
Figure BDA0001930334880000171
in some embodiments, S1Is of formula A49 or A50.
In some embodiments, each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and alkylphenyl; for the purpose of simplifying the conjugate molecules of the present disclosure, in some embodiments, Y is methyl.
In some embodiments, the conjugate molecules of the present disclosure have a structure represented by formula (301), (302), (303), (304), or (305):
Figure BDA0001930334880000181
Figure BDA0001930334880000191
in the above formulae (301) to (305), wherein RkIs a hydroxy protecting group, M+Selected from goldBelongs to one of cation, ammonium cation, tertiary amine cation or quaternary ammonium cation. In some embodiments, M+Is composed of
Figure BDA0001930334880000192
In some embodiments, a conjugate molecule of the present disclosure may have a structure represented by formula (501), (502), (503), (504), or (505):
Figure BDA0001930334880000193
Figure BDA0001930334880000201
Figure BDA0001930334880000211
in the above formulas (501) to (505), wherein X is O or NH, RkSPS represents a solid support for a hydroxyl protecting group.
According to some embodiments of the present disclosure, a conjugate molecule of the present disclosure has a structure represented by formula (601), (602), (603), (604), or (605):
Figure BDA0001930334880000212
Figure BDA0001930334880000221
in the above formulas (601) to (605), DMTr represents 4,4' -bismethoxytrityl group, and has a structure
Figure BDA0001930334880000222
Represents the salt of the corresponding carboxylic acid with triethylamine.
In some specific embodiments, the conjugate molecules of the present disclosure may have a structure represented by formula (701), (702), (703), (704), or (705):
Figure BDA0001930334880000231
Figure BDA0001930334880000241
in the above formulae (701) to (705), SPS represents a solid support, and DMTr represents 4,4' -bismethoxytrityl.
Preparation of the conjugate molecules of the present disclosure
One skilled in the art can prepare the conjugate molecules of the present disclosure using any reasonable synthetic route.
In some embodiments of the present disclosure, a method of preparing a conjugate molecule of formula (101) comprises contacting a compound of formula (102) with a cyclic anhydride in an organic solvent under esterification reaction conditions and in the presence of a base and an ester-forming catalyst, ion-exchanging, and isolating to obtain a compound of formula (101):
Figure BDA0001930334880000242
wherein:
R7to provide R in formula (101)1A group of (1). In some embodiments, for example, R7Has a structure represented by formula (A61):
Figure BDA0001930334880000251
n1、n2、m1、R2、L1、S1the respective definitions and alternative ranges are as previously described, RiTo enable connection to N on nitrogen-containing skeletons, to RkO is linked to and is linked to an optional radical of a free hydroxyl group, RkIs a hydroxyl protecting group. In this case, R is obtained1The compound contains a1 st functional group and a2 nd functional group which are used as hydroxyl protecting groups, and the 2 nd functional group contains a compound of a formula (101) shown as a formula (C1) or (C2). In some casesIn the embodiment, R7Is a structure shown as B7 or B8:
Figure BDA0001930334880000252
wherein q is2And RkThe respective definitions are as described above.
The esterification reaction conditions include a reaction temperature of 0-100 ℃ and a reaction time of 8-48 hours, and in one embodiment, the esterification reaction conditions include a reaction temperature of 10-40 ℃ and a reaction time of 20-30 hours.
In some embodiments, the organic solvent is one or more of an epoxy-based solvent, an ether-based solvent, a haloalkane-based solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In one embodiment, the epoxy solvent is dioxane and/or tetrahydrofuran, the ether solvent is diethyl ether and/or methyl tert-butyl ether, and the alkyl halide solvent is one or more of dichloromethane, chloroform and 1, 2-dichloroethane. In one embodiment, the organic solvent is dichloromethane. The organic solvent is used in an amount of 3 to 50L/mol, and in one embodiment 5 to 20L/mol, relative to the compound represented by the formula (102).
In some embodiments, the cyclic anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, or pimelic anhydride, and in one embodiment succinic anhydride. The molar ratio of the cyclic anhydride to the compound of formula (102) is from 1:1 to 10:1, and in one embodiment from 2:1 to 5: 1.
The ester-forming catalyst may be any catalyst that catalyzes the esterification reaction, for example, the catalyst may be 4-dimethylaminopyridine. The molar ratio of the catalyst to the compound of formula (102) is 1:1 to 10:1, and in one embodiment is 2:1 to 5: 1.
In some embodiments, the base can be any inorganic base, organic base, or combination thereof. The base may be, for example, a tertiary amine organic base in view of solubility and product stability. In one embodiment, the tertiary amine organic base is triethylamine or N, N-diisopropylethylamine. The molar ratio of the tertiary amine organic base to the compound of formula (102) is 1:1 to 20:1, and in one embodiment is 3:1 to 10: 1.
The ion exchange is to convert the compound of formula (101) to the desired carboxylic acid or carboxylate salt form, methods of ion exchange are well known to those skilled in the art, and appropriate ion exchange solutions and exchange conditions can be used to obtain the aforementioned cation as M+The conjugate molecule of (3) is not described in detail herein. In one embodiment, the ion exchange reaction is carried out using a triethylamine phosphate solution having a concentration of 0.2 to 0.8M, and in one embodiment 0.4 to 0.6M, in an amount of 3 to 6L/mol, and in one embodiment 4 to 5L/mol, relative to the compound of formula (102).
The compound of formula (101) may be isolated from the reaction mixture using any suitable separation method. In some embodiments, the compound of formula (101) may be isolated by removal of the solvent by evaporation followed by chromatographic methods, e.g., the following chromatographic conditions may be used for isolation: (1) normal phase purification of silica gel: 200-mesh 300-mesh silica gel filler, and performing gradient elution by using dichloromethane containing 1 wt% of triethylamine and methanol at a ratio of 100:18-100: 20; 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 (101) which may be used directly in a subsequent reaction.
In some embodiments, the method for preparing the compound of formula (101) further comprises contacting the product obtained by the above ion exchange reaction with a solid support containing an amino group or a hydroxyl group in an organic solvent in the presence of a condensing agent and a tertiary amine organic base under condensation reaction conditions. In this case, R is obtained1The compound contains a1 st functional group and a2 nd functional group, wherein the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group contains a compound of a formula (101) with a structure shown as a formula (C1').
The solid phase carrier is one of carriers used in solid phase synthesis of siRNA, some of which are well known to those skilled in the art. For example, the solid support may be selected from solid supports containing reactive hydroxyl or amino functional groups, in one embodiment amino resins or hydroxyl resins. In some embodiments, the amino or hydroxyl resin has the following parameters in one embodiment: the particle size is 100-400 meshes (mesh), and the surface amino or hydroxyl loading is 0.2-0.5 mmol/g. The amount ratio of the compound represented by the formula (101) to the solid carrier is 10 to 400. mu. mol of the compound per gram of the solid carrier (. mu. mol/g). In some embodiments, the compound of formula (101) is present in an amount of 50 to 200. mu. mol/g relative to the solid support.
The organic solvent may be any suitable solvent or mixture of solvents known to those skilled in the art. In some embodiments, the organic solvent is one or more of acetonitrile, an epoxy-based solvent, an ether-based solvent, a haloalkane-based solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In some embodiments, the epoxy-based solvent is dioxane and/or tetrahydrofuran, the ether-based solvent is diethyl ether and/or methyl tert-butyl ether, and the haloalkane-based solvent is one or more of dichloromethane, chloroform, and 1, 2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. The organic solvent is used in an amount of 20 to 200L/mol, and in one embodiment 50 to 100L/mol, relative to the compound of formula (102).
In some embodiments, the condensing agent may be benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one, and/or O-benzotriazol-tetramethyluronium hexafluorophosphate, and in one embodiment, the condensing agent is O-benzotriazol-tetramethyluronium hexafluorophosphate. The molar ratio of the condensing agent to the compound of formula (102) is 1:1 to 20:1, and in one embodiment is 1:1 to 5: 1.
In some embodiments, the tertiary amine organic base is triethylamine and/or N, N-diisopropylethylamine, in some embodiments N, N-diisopropylethylamine; the molar ratio of the tertiary amine organic base to the compound of formula (102) is 1:1 to 20:1, and in one embodiment is 1:1 to 5: 1.
In some embodiments, the method for preparing the compound of formula (101) may further comprise contacting the obtained condensation product with a capping reagent and an acylation catalyst in an organic solvent under capping reaction conditions to isolate the compound of formula (101). The capping reaction serves to remove any reactive functional groups that have not reacted to completion to avoid the production of unwanted by-products in subsequent reactions. The capping reaction conditions include a reaction temperature of 0 to 50 deg.C, in some embodiments 15 to 35 deg.C, and a reaction time of 1 to 10 hours, in some embodiments 3 to 6 hours. The capping reagent may be one used in solid phase synthesis of siRNA, and the capping reagent used in solid phase synthesis of siRNA is well known to those skilled in the art.
In some embodiments, the capping reagent consists of capping reagent a (capa) and capping reagent b (capb), wherein capping reagent a is N-methylimidazole, and in some embodiments is provided as a pyridine/acetonitrile mixed solution of N-methylimidazole, wherein the volume ratio of pyridine to acetonitrile is 1:10 to 1: 1. And in some embodiments from 1:3 to 1: 1. In some embodiments, the total volume of pyridine and acetonitrile to the volume of N-methylimidazole is from 1:1 to 10:1, and in some embodiments from 3:1 to 7: 1. In some embodiments, the capping reagent B is acetic anhydride, and in some embodiments, the capping reagent B is provided as an acetonitrile solution of acetic anhydride, wherein the volume of acetic anhydride and acetonitrile is 1:1 to 1:10, and in other embodiments, 1:2 to 1: 6.
In some embodiments, the ratio of the volume of the pyridine/acetonitrile mixed solution of N-methylimidazole to the mass of the compound of formula (102) is 5ml/g to 50ml/g, in some embodiments 15ml/g to 30 ml/g. The ratio of the volume of the solution of acetic anhydride in acetonitrile to the mass of the compound of formula (102) is from 0.5ml/g to 10ml/g, in some embodiments from 1ml/g to 5 ml/g.
In some embodiments, the capping reagent uses equimolar amounts of acetic anhydride and N-methylimidazole. In some embodiments, the organic solvent is one or more of acetonitrile, an epoxy-based solvent, an ether-based solvent, a haloalkane-based solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile. The organic solvent is used in an amount of 10 to 50L/mol, and in some embodiments 5 to 30L/mol, relative to the compound of formula (102).
In some embodiments, the acylation catalyst may be selected from any catalyst useful for ester-forming condensation or amide-forming condensation, such as a basic heterocyclic compound. In some embodiments, the acylation catalyst is 4-dimethylaminopyridine. The mass ratio of the catalyst to the compound of formula (102) is 0.001:1 to 1:1, and in one embodiment 0.01:1 to 0.1: 1.
In some embodiments, the compound of formula (101) may be isolated from the reaction mixture using any suitable separation method. In some embodiments, the compound of formula (101) may be obtained by washing well with an organic solvent selected from acetonitrile, dichloromethane, methanol, in some embodiments acetonitrile, and filtering to remove unreacted reactants, excess capping reagent, and other impurities.
In some embodiments, a method of preparing a conjugate molecule of formula (101) comprises contacting a compound of formula (102) with a phosphoramidite in an organic solvent under coupling reaction conditions and in the presence of a coupling reagent, and isolating the compound of formula (101). In this case, R is obtained1The compound contains a1 st functional group and a2 nd functional group, wherein the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group contains a compound of a formula (101) with a structure shown as a formula (C3).
In some embodiments, the coupling reaction conditions include a temperature of from 0 to 50 ℃, e.g., from 15 to 35 ℃, a molar ratio of the compound of formula (102) to the phosphoramidite of from 1:1 to 1:50, e.g., from 1:5 to 1: 15; the molar ratio of the compound of formula (102) to the coupling reagent is from 1:1 to 1:100, for example from 1:50 to 1: 80; the reaction time is 200-3000 seconds, for example 500-1500 seconds. The phosphorodiamidite may be, for example, bis (diisopropylamino) (2-cyanoethoxy) phosphine, which is commercially available or synthesized according to a method well known in the art. The coupling reagent is one or more selected from 1H-tetrazole, 5-ethylthio 1H-tetrazole, and 5-benzylthio 1H-tetrazole, such as 5-ethylthio 1H-tetrazole. The coupling reaction can be carried out in an organic solvent selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, for example, anhydrous acetonitrile. In some embodiments, the organic solvent is used in an amount of 3 to 50L/mol, for example, 5 to 20L/mol, relative to the compound of formula (102). By carrying out this coupling reaction, the hydroxyl group in the compound of formula (102) reacts with the phosphoramidite to form a phosphoramidite group. In some embodiments, the solvent may be removed directly to provide a crude compound of formula (101) which may be used directly in a subsequent reaction.
In some embodiments, the process for preparing a compound of formula (101) further comprises the steps of: the isolated product is further contacted with a solid support comprising hydroxyl groups under coupling reaction conditions in an organic solvent and in the presence of a coupling reagent. Subsequently, the compound of formula (101) is isolated by capping reaction, oxidation reaction. In this case, R is obtained1The compound contains a1 st functional group and a2 nd functional group, wherein the 1 st functional group contains a hydroxyl protecting group, and the 2 nd functional group has a structure shown as a formula (C3').
In some embodiments, the solid phase support is a solid phase support known in the art and useful for solid phase synthesis of nucleic acids, e.g., a commercially available general-purpose solid phase support after deprotection reaction (c)
Figure BDA0001930334880000271
HLUnyLinkerTM300oligonucleotid Synthesis Support, Kinovate Life Sciences, having the structure shown in formula B80):
Figure BDA0001930334880000281
deprotection reactions are well known to those skilled in the art. In some embodiments, the deprotection conditions include a temperature of 0 to 50 ℃, e.g., 15 to 35 ℃; the reaction time is from 30 to 300 seconds, for example from 50 to 150 seconds. The deprotection agent may be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments, the deprotection agent is dichloroacetic acid. The molar ratio of deprotecting reagent to-DMTr (4,4' -dimethoxytrityl) protecting group on the stationary phase is 2:1 to 100:1, for example 3:1 to 50: 1. By carrying out the deprotection, a free hydroxyl group having reactivity is obtained on the surface of the solid phase carrier, thereby facilitating the next coupling reaction.
The coupling reaction conditions and the choice of coupling reagents are as described above. By carrying out this coupling reaction, the free hydroxyl group formed in the deprotection reaction reacts with the phosphoramidite group to form a phosphite linkage.
In some embodiments, capping reaction conditions include a temperature of 0 to 50 ℃, e.g., 15 to 35 ℃, and a reaction time of 5 to 500 seconds, e.g., 10 to 100 seconds, the capping reaction being carried out in the presence of a capping reagent. The capping reagent is selected and used as described above.
The oxidation reaction conditions include a temperature of from 0 to 50 deg.C, for example, from 15 to 35 deg.C, a reaction time of from 1 to 100 seconds, for example, from 5 to 50 seconds, and an oxidizing agent, for example, iodine (in some embodiments, provided in the form of iodine water). In some embodiments, the molar ratio of oxidizing agent to phosphite groups is from 1:1 to 100:1, and can be, for example, from 5:1 to 50: 1. In some specific embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran, water, and pyridine ═ 3:1:1-1:1: 3.
In some embodiments, the compound of formula (102) may be prepared by the following method: contacting a compound shown as a formula (103) with a compound shown as a formula (104) in an organic solvent in the presence of an amide forming reaction condensing agent and a tertiary amine organic base under condensation reaction conditions, and separating to obtain a compound shown as a formula (102):
Figure BDA0001930334880000282
wherein n is1、n2、m1、R2、R7、L1、S1The respective definitions and alternative ranges are as described above.
Compounds of formula (104) may be prepared using, for example, compounds disclosed in j.am. chem.soc.2014,136,169581-16961, or compounds of formula (104) may be prepared by various methods by those skilled in the art, for example, certain compounds of formula (104) may be prepared by reference to the methods disclosed in US8106022B2, example 1, the entire contents of which are incorporated herein by reference in their entirety.
In some embodiments, the condensation reaction conditions include a reaction temperature of 0 to 100 ℃ and a reaction time of 0.1 to 24 hours, and in one embodiment, a reaction temperature of 10 to 40 ℃ and a reaction time of 0.5 to 16 hours.
The molar ratio of the compound of formula (104) to the compound of formula (103) is from 2:1 to 10:1, and in one embodiment from 2.5:1 to 5: 1.
In some embodiments, the organic solvent is one or more of acetonitrile, an epoxy-based solvent, which is dioxane and/or tetrahydrofuran in one embodiment, an ether-based solvent, which is diethyl ether and/or methyl tert-butyl ether in one embodiment, an ether-based solvent, which is one or more of dichloromethane, chloroform and 1, 2-dichloroethane in one embodiment, an alkyl halide-based solvent, which is acetonitrile in one embodiment, N-diisopropylethylamine, and dimethyl sulfoxide. The organic solvent is used in an amount of 3 to 50L/mol, and in one embodiment 5 to 20L/mol, relative to the compound of formula (103).
In some embodiments, the amide-forming condensation 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 one embodiment 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride. The molar ratio of the amide forming reaction condensing agent to the compound of formula (103) is 2:1 to 10:1, and in one embodiment is 2.5:1 to 5: 1;
the tertiary amine organic base is N-methylmorpholine, triethylamine or N, N-diisopropylethylamine, in one embodiment N-methylmorpholine; the molar ratio of the tertiary amine organic base to the compound of formula (103) is 3:1 to 20:1, and in one embodiment is 5:1 to 10: 1.
Similarly to the above, the compound of formula (102) may be isolated from the reaction mixture using any suitable separation method. In some embodiments, the compound of formula (102) may be isolated by removal of the solvent by evaporation followed by chromatographic methods, e.g., the following chromatographic conditions may be used for isolation: (1) normal phase purification of silica gel: 200-300 mesh silica gel filler, and gradient elution is carried out by using dichloromethane and methanol as 100:5-100: 7; and (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 (102) which may be used directly in a subsequent reaction.
In some embodiments, a compound of formula (103) is reacted with a sufficient amount of one compound of formula (104) at a time to form the desired compound of formula (102), in which case each S is1-L1The portions are identical to each other. In some embodiments, the compound of formula (103) may be batched with a different compound of formula (104), i.e., L, as desired1And/or S1Different compounds of formula (104) are reacted such that the resulting compound of formula (102) contains two or more species of S1And/or L1. For example, for 1eq of a compound of formula (103), it may be contacted first with 2eq of a first compound of formula (104) to attach a first S to the two terminal primary amine groups in the compound of formula (103)1-L1Partially, then, continuing it with (m)1-1) eq of a second compound of formula (104) (m)1Are as defined above) to (m) in the compound of formula (103)1-1) attachment of a second S to a secondary amine group1-L1And (4) partial.
In one embodiment, R7Is one of the groups of formula B7 or B8, in which case the compound of formula (103) can be prepared by: in an organic solvent under amide-forming reaction conditions, and in an amide-forming reaction condensing agent and a tertiary stageContacting a compound represented by the formula (105) with a compound represented by the formula (A-1) or a compound represented by the formula (A-2) in the presence of an amine organic base, and separating to obtain a compound represented by the formula (103):
Figure BDA0001930334880000291
wherein n is1、n2、m1、R2、R7、L1、S1The respective definitions and alternative ranges are as described above.
The amide-forming reaction conditions are a reaction temperature of 0-100 ℃ and a reaction time of 1-48 hours, and in some embodiments, the amide-forming reaction conditions are a reaction temperature of 10-40 ℃ and a reaction time of 2-16 hours.
In some embodiments, the organic solvent is one or more of an alcohol solvent, an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N-dimethylformamide, and N, N-diisopropylethylamine. The alcoholic solvent is in one embodiment one or more of methanol, ethanol, propanol, and in some embodiments ethanol. The epoxy-based solvent is dioxane and/or tetrahydrofuran in some embodiments. The ethereal solvent is, in some embodiments, diethyl ether and/or methyl tert-butyl ether. The haloalkane-based solvent is, in some embodiments, one or more of dichloromethane, trichloromethane and 1, 2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The amount of organic solvent used is 3 to 50L/mol, and in one embodiment 3 to 20L/mol, relative to the compound of formula (105).
In some embodiments, the amide-forming reaction condensing agent is benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1, 2, 3-benzazole-4 (3H) -one, 4- (4, 6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (EEDQ), or O-benzotriazol-tetramethyluronium hexafluorophosphate, in one embodiment 3-diethoxyphosphoryl-1, 2, 3-benzazole-4 (3H) -one. The molar ratio of the amide forming reaction condensing agent to the compound of formula (105) is 1:1 to 10:1, and in one embodiment is 2.5:1 to 5: 1.
In some embodiments, the tertiary amine organic base is triethylamine or N, N-diisopropylethylamine, in one embodiment N, N-diisopropylethylamine. The molar ratio of the tertiary amine organic base to the compound of formula (105) is 3:1 to 20:1, and in one embodiment is 5:1 to 10: 1.
In some embodiments, the compounds of formula (A-1) and formula (A-2) may be prepared by any suitable means. For example, when R iskIn the case of DMTr group, the compound of formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl; similarly, the compound of formula (A-2) may be prepared by first contacting 3-amino-1, 2-propanediol with a cyclic anhydride, which may be a cyclic anhydride having from 4 to 13 carbon atoms, and in one embodiment, from 4 to 8 carbon atoms, and then reacting with DMTrCl. It will be readily understood by those skilled in the art that the selection of the cyclic anhydride corresponds to q in the compound (A-2)2Different values of (A), e.g. when the cyclic anhydride is succinic anhydride, q2When the cyclic anhydride is glutaric anhydride, q is 12And so on for 2.
In some variations, the compound of formula (103) may also be prepared by reacting a compound of formula (105) with the cyclic anhydride, 3-amino-1, 2-propanediol, and DMTrCl in that order. It will be readily understood by those skilled in the art that these modifications do not affect the structure or function of the compound of formula (103), and that these modifications are readily achievable by those skilled in the art based on the methods described above.
Similarly to the above, the compound of formula (103) may be isolated from the reaction mixture using any suitable separation method. In some embodiments, the compound of formula (103) may be isolated by removal of the solvent by evaporation followed by chromatographic methods, e.g., the following chromatographic conditions may be used for isolation: (1) normal phase purification of silica gel: 200-mesh 300-mesh silica gel filler is subjected to gradient elution by using petroleum ether, ethyl acetate, dichloromethane, N-dimethylformamide as the raw materials, wherein the ratio of petroleum ether to ethyl acetate to dichloromethane is 1:1:1:0.5-1:1:1: 0.6; and (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 (103) which may be used directly in a subsequent reaction.
In some embodiments, each R is2Are all the same, and each n2And n1Is equal, in this case, two NH groups in the formula (105)2The groups are chemically equivalent. In some embodiments, a compound of formula (a-1) or (a-2) is reacted with an equimolar amount of a compound of formula (105) followed by isolation to provide a compound of formula (103); in some embodiments, a compound of formula (A-1) or (A-2) is reacted with an excess of a compound of formula (105) and subsequently isolated to yield a compound of formula (103).
The compounds of formula (105) are commercially available or obtained by one skilled in the art using known methods. For example, when m1＝2、n1And each n2Are each 2, and each R2In the case of both H, the compounds of formula (105) are commercially available from the company Afahesar.
Oligonucleotide conjugates
In another aspect, the present disclosure provides an oligonucleotide conjugate having a structure as shown in formula (201):
Figure BDA0001930334880000311
wherein:
n1is an integer selected from 1-2;
each n is2Independently selected from integers from 1 to 2;
m1is an integer selected from 1 to 6;
each R2Each independently is selected from H, C1-C10Alkyl radical, C1-C10Haloalkyl or C1-C10Alkoxy, in some embodiments, R10、R11、R12、R13、R14And R15Each is independently selected from one of H, methyl or ethyl;
R6is an active drug, in some embodiments, R6Including active powerA functional oligonucleotide;
R5is a straight chain alkylene group of 1 to 20 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 wherein R5May optionally have a substituent of any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -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-C10HalogenatedAlkyl), -SO2NH2、-SO2NH(C1-C10Alkyl), -SO2NH (phenyl), -NHSO2(C1-C10Alkyl), -NHSO2(phenyl) and-NHSO2(C1-C10Haloalkyl);
each L1Is 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 wherein L1May optionally have a substituent of any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -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, L1Can be selected from one or more connecting combinations of the groups of the formula A1-A26, A1-A26 are defined as the previous description.
n1、n2、m1、R2、L1、M1The respective definitions and alternative ranges are as described above.
In some embodiments, R5Is R in the compound of formula (101)1The group is reactive linked to the active drug via a group to form a linking group. In some embodiments, R5Is R in the compound of formula (101)1The group is reacted to a functional oligonucleotide to form a linking group. In some embodiments, R5The group contains a connecting site connected with N on the nitrogen-containing skeleton and R6The attachment site to which P in (1) is attached. In some embodiments, R5Wherein the site linked to N on the nitrogen-containing backbone forms an amide bond with N, said amide bond with R6The site of P attachment in (1) forms a phosphoester bond with P. In some embodiments, R5May be B5, B6, B5 'or B6':
Figure BDA0001930334880000321
wherein the content of the first and second substances,
Figure BDA0001930334880000322
denotes the site of covalent bonding of the groups, q2The selection and value ranges of (a) are as described above.
In some embodiments, R6A group of the structure shown as a 59:
Figure BDA0001930334880000323
wherein E is1Is OH, SH or BH2In some embodiments, E1Is OH or SH; nu is an oligonucleotide.
In the context of the present disclosure, unless otherwise indicated, a "conjugate" group or molecule refers to a group or molecule capable of forming a covalent bond with a corresponding ligand, and the conjugate group or molecule and its ligand have specific functions. 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 conjugate moieties having a specific function to an oligonucleotide. In the context of the present disclosure, a "conjugate molecule" is understood to be a specific compound that can be conjugated to an oligonucleotide by a reaction, ultimately forming an oligonucleotide conjugate of the present disclosure. In some embodiments, the oligonucleotide is an siRNA, and when the conjugate of the present disclosure is an siRNA conjugate.
In some embodiments, the conjugates of the present disclosure have a structure represented by formula (401), (402), (403), (404), or (405):
Figure BDA0001930334880000331
Figure BDA0001930334880000341
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 abnormally expressed in the hepatocyte may be, for example, ApoB, ApoC, ANGPTL3, PCSK9, SCD1, FVII, 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, FVII, p53, HBV, HCV, and the like, corresponding to the above-described aberrantly expressed gene. 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.
P in formula A59 can 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 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 a59 can 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 a59 is attached to the end of the single stranded oligonucleotide.
In some embodiments, the functional oligonucleotide in the oligonucleotide conjugates of the present disclosure is a double-stranded oligonucleotide (e.g., siRNA, microRNA, or DNA) comprising a sense strand and an antisense strand. In some embodiments, the P in formula a59 is attached to the end of the sense or antisense strand of the double-stranded oligonucleotide, the end referring to the first 4 nucleotides from one end of the sense or antisense strand, in one embodiment, the P in formula a59 is attached to the end of the sense or antisense strand; in yet another embodiment, P in formula a59 is linked to the 3' end of the sense strand. With P in formula a59 attached to the sense strand of a double-stranded oligonucleotide at the above-described position, upon entry of the oligonucleotide conjugate provided by the present disclosure into a cell, upon unwinding, the individual double-stranded oligonucleotide antisense strand can be released to block the process of translation of the protein by the target mRNA, inhibiting the expression of a particular gene.
P in formula A59 can 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 a59 can 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 a59 is attached to the 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 this case, P in a59 can also be considered as P in the phosphate group contained in the siRNA), or P in formula a59 is attached to the nucleotide by substitution 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 a59 is attached to the nucleotide by substitution 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 only be an siRNA, rather that the oligonucleotide and even the active drug may be the disclosed or other alternative drugs known to those skilled in the art. Based on the detailed description of siRNA conjugates, it is contemplated that other active drugs or 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 is capable of inhibiting at least 55%, 60%, 65%, 70%, 75%, or 80% of HBV gene, ANGPTL3 gene, or APOC3 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, wherein the nucleotide sequence 5 is 1 to 3 nucleotides in length, and is attached to the 3 'end of the antisense strand, thereby constituting a3' overhang of the 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, j.k., g.f.deleavey and m.j.damha, chemical modified siRNA: tools and applications. drug discovery, 2008.13 (19-20): p.842-55, the entire contents of which are incorporated herein by reference).
In the context of the present disclosure, the term "modified nucleotide" is used to refer to a nucleotide or nucleotide 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 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 having a modifying group (or modified phosphate groups and/or modified ribosyl groups). In some embodiments of the disclosure, all of the nucleotides in the sense strand and/or the antisense strand are modified nucleotides.
In some embodiments, each nucleotide in the sense 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 (807).
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 (808). The 2' -substituted alkoxy-modified nucleotide may be, for example, a 2' -O-methoxyethyl-modified nucleotide (2' -MOE), as shown in formula (809). 2 '-amino-modified nucleotide (2' -NH)2) As shown in equation 810. The 2' -Deoxynucleotide (DNA) is represented by the formula (811).
Figure BDA0001930334880000381
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., where LNA is shown as formula (812), ENA is shown as formula (813), and cET BNA is shown as formula (814).
Figure BDA0001930334880000382
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 (815) and GNA is represented by formula (816).
Figure BDA0001930334880000383
Wherein R is selected from H, OH or alkoxy (O-alkyl).
The term "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 a base from the 1' -position to the 2' -position or the 3' -position of a ribose ring, as shown in formula (817) or (818).
Figure BDA0001930334880000391
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 hereinafter, "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" have the same meaning, and refer to a compound having a structure represented by formula (807) 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 (808).
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, a phosphate group modification is in one embodiment a phosphorothioate (phosphothioate) modification as shown below in formula (801) by replacing the non-bridging oxygen atom in the phosphodiester linkage with a sulfur atom, thereby replacing the phosphodiester linkage with a phosphorothioate diester linkage. The modification can stabilize the structure of siRNA and maintain high specificity and high affinity of base pairing.
Figure BDA0001930334880000392
According to some embodiments of the present disclosure, the siRNA wherein the phosphorothioate linkage is present at least one position 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 present 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 (802):
Figure BDA0001930334880000401
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 (803) to (806) disclosed in Anastasia Khvorova and Jonathan K.Watts, The chemical evolution of oligonucleotide therapeutics of clinical utility, Nature Biotechnology,2017,35(3): 238-48:
Figure BDA0001930334880000402
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 one embodiment, the nucleotide modified with a5 '-phosphate or a 5' -phosphate analog is a nucleotide containing a vinyl phosphate (E-VP) represented by formula (803), a nucleotide containing a5 '-phosphate modification represented by formula (802), or a nucleotide containing a 5' -phosphorothioate modification represented by formula (805).
The inventors of the present disclosure have unexpectedly found that the siRNA conjugates of the present disclosure exhibit not significantly reduced silencing activity of target mRNA and excellent gene expression inhibition effect while having significantly improved serum stability. According to one embodiment of the present disclosure, the oligonucleotide conjugate of the present disclosure is an siRNA conjugate comprising an siRNA such as the sirnas shown in tables 1A-4E:
TABLE 1A
Figure BDA0001930334880000403
Figure BDA0001930334880000411
TABLE 1B
Figure BDA0001930334880000412
TABLE 1C
Figure BDA0001930334880000413
TABLE 1D
Figure BDA0001930334880000414
TABLE 1E
Figure BDA0001930334880000415
Figure BDA0001930334880000421
TABLE 2A
Figure BDA0001930334880000422
TABLE 2B
Figure BDA0001930334880000423
Figure BDA0001930334880000431
TABLE 2C
Figure BDA0001930334880000432
TABLE 2D
Figure BDA0001930334880000433
Figure BDA0001930334880000441
TABLE 2E
Figure BDA0001930334880000442
Figure BDA0001930334880000451
TABLE 3A
Figure BDA0001930334880000452
TABLE 3B
Figure BDA0001930334880000453
TABLE 3C
Figure BDA0001930334880000454
Figure BDA0001930334880000461
TABLE 3D
Figure BDA0001930334880000462
TABLE 3E
Figure BDA0001930334880000463
TABLE 4A
Figure BDA0001930334880000464
TABLE 4B
Figure BDA0001930334880000471
TABLE 4C
Figure BDA0001930334880000472
TABLE 4D
Figure BDA0001930334880000473
TABLE 4E
Figure BDA0001930334880000474
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, and in one embodiment is a vinyl phosphate modified nucleotide (indicated by VP in the following examples), a 5' -phosphate modified nucleotide (indicated by P in the following examples), or a phosphorothioate modified nucleotide (indicated by Ps in the following examples).
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, the oligonucleotide conjugates of the present disclosure can be prepared by a method comprising sequentially linking nucleoside monomers in a3 'to 5' direction under the conditions of phosphoramidite solid phase synthesis according to the nucleotide species and order of the oligonucleotide, respectively, the linking of each nucleoside monomer comprising four steps of deprotection, coupling, capping, oxidation, or sulfurization; in some embodiments, the method further comprises contacting the compound of formula (101) with a nucleoside monomer or a nucleotide sequence attached to a solid support under coupling reaction conditions and in the presence of a coupling reagent, such that the compound of formula (101) is attached to the nucleotide sequence via a coupling reaction.
In some embodiments, the method further comprises the steps of deprotecting and cleaving with the solid support, separation and purification, and optionally annealing.
In some embodiments, the oligonucleotide is a double-stranded oligonucleotide and the method of making comprises the steps of: under the coupling reaction conditions andcontacting a compound shown in a formula (101) with a first nucleoside monomer at the 3' end of a sense strand or an antisense strand in the presence of a coupling reagent, connecting the first nucleotide in a connecting sequence to the compound shown in the formula (101), and sequentially connecting the nucleoside monomers in a3' to 5' direction to synthesize a sense strand or an antisense strand of the double-stranded oligonucleotide; wherein the (101) compound is R2The compound contains a1 st functional group and a2 nd functional group, wherein the 1 st functional group contains protected hydroxyl, the 2 nd functional group is a compound shown as a formula (101) shown as a formula (C1 ') or (C3'), and the compound shown as the formula (101) is subjected to deprotection before being connected with a 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: sequentially connecting nucleoside monomers according to the nucleotide types and the sequence of a sense strand or an antisense strand in the double-stranded oligonucleotide and the direction from 3 'to 5' to synthesize the sense strand and the 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 the formula (101) with a sense strand linked to a solid support or an antisense strand linked to a solid support in the presence of a coupling reagent under coupling reaction conditions to thereby link the compound represented by the formula (101) to the sense strand or the antisense strand, wherein the compound represented by the formula (101) is R1A compound of formula (101) containing phosphoramidite as the 1 st functional group; 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 one embodiment, P in formula a59 is attached to the 3' end of the sense strand in the siRNA, and the method of making the siRNA conjugate of the present disclosure comprises:
(1) removal of the hydroxyl protecting group R from the solid support-bound compound of formula (101) (hereinafter also referred to as solid support-bound conjugate molecule)k(ii) a Contacting the 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 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 conjugate 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 protecting group R is removed from the conjugate molecule attached to the solid supportkThe method of (1) comprises contacting a compound of formula (101) 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 (101) is from 10:1 to 1000:1, and in some embodiments from 50:1 to 500: 1.
The coupling reaction conditions and coupling reagents may employ any conditions and reagents capable of effecting the coupling reaction described above. 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 (101) to nucleoside monomer is from 1:1 to 1:50, in some embodiments from 1:2 to 1: 5; the molar ratio of the compound of formula (101) to the coupling reagent is 1:1 to 1:50, in some embodiments 1:3 to 1:10, and the reaction time is 200-3000 seconds, in some embodiments 500-1500 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 compound of formula (101).
In step (2), the sense strand S of the siRNA conjugate is synthesized in the 3'-5' direction by a method of solid phase synthesis of phosphoramidite nucleic acid, starting with the nucleoside monomer attached to the solid support by the conjugate molecule prepared in the above step. At this point, the 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 protecting group YCOO-of the A46-A54 group is converted into a hydroxyl group, S1Conversion of the group to the corresponding M1And (c) a group, thereby producing a conjugate represented by formula (201). 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 molecular weight detection, etc. using methods such as mass spectrometry, etc., to determine that the synthesized siRNA conjugates are the targeted designed siRNA conjugates, and the sequences of the synthesized siRNA are consistent with the sequences of the siRNA to be synthesized, e.g., consistent with the sequences listed in tables 1A-4E above.
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.
In some embodiments, the oligonucleotide conjugates of the present disclosure have excellent liver targeting specificity, and thus are capable of efficiently delivering conjugated functional oligonucleotides to the liver, thereby effectively regulating specific gene expression within hepatocytes. Thus, the oligonucleotide conjugates of the present disclosure have broad application prospects.
According to some embodiments of the present disclosure, there is provided a use of an oligonucleotide conjugate of the present disclosure in the preparation of a medicament for treating and/or preventing a pathological condition or disease caused by the expression of a specific gene in a hepatocyte. The specific 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 specific gene is selected from the group consisting of ApoB, ApoC, ANGPTL3, PCSK9, SCD1, FVII, p53, HBV, HCV, and the like. In some embodiments, the specific 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. In some embodiments, the conjugates provided by the present disclosure may also be used to treat other liver diseases, including diseases characterized by unwanted cellular proliferation, hematologic diseases, metabolic diseases, and diseases characterized by inflammation. The proliferative disease of the liver may be a benign or malignant disease, such as cancer, hepatocellular carcinoma (HCC), liver metastasis or hepatoblastoma. The hematologic or inflammatory disease of the liver may be a disease involving coagulation factors, complement-mediated inflammation, or fibrosis. Metabolic disorders of the liver include dyslipidemia and irregularities in glucose regulation. In some embodiments, liver disease is treated by administering one or more oligonucleotides having high homology to the gene sequences involved in liver disease.
According to another embodiment of the present disclosure, there is provided a method of inhibiting the expression of a specific gene in a hepatocyte, the method comprising contacting the siRNA conjugate of the present disclosure with the hepatocyte.
By administering the oligonucleotide conjugates of the present disclosure to a patient in need thereof, the prevention and/or treatment of pathological conditions or diseases caused by the expression of specific genes in hepatocytes can be achieved through a mechanism that regulates gene expression. Thus, the oligonucleotide conjugates of the present disclosure may be used for the prevention and/or treatment of said pathological condition or disease, or for the manufacture of a medicament for the prevention and/or treatment of said pathological condition or disease.
The term "administering" as used herein refers to placing a conjugate into a patient by a method or route that allows for at least partial positioning of the conjugate, such as an oligonucleotide conjugate, at a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include, but are not limited to, local and systemic administration. In general, topical administration results in delivery of more oligonucleotide conjugate to a particular site than to the entire body of the patient; whereas systemic administration results in delivery of the oligonucleotide conjugate to substantially the entire body of the patient. In view of the present disclosure aimed at providing a means of preventing and/or treating pathological conditions or diseases caused by the expression of specific genes in hepatocytes, in some embodiments, an administration mode capable of delivering drugs to the liver.
Administration to a patient may be by any suitable route known in the art, including but not limited to: oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration. The frequency of administration may be 1 or more times per day, week, month, or year.
The oligonucleotide conjugates described in the present disclosure can be used in dosages that are conventional in the art, and which can be determined according to various parameters, particularly the age, weight, and sex of the patient. Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD50 (the dose lethal to 50% of the population) and ED50 (the dose that gives rise to 50% of the maximal response intensity in a quantitative response, and in a qualitative response, the dose that gives rise to a positive response in 50% of the subjects). The range of human doses can be derived based on data obtained from cell culture analysis and animal studies.
In administering the conjugates of the present disclosure, for example, for male or female, 6-12 week old, C57BL/6J or C3H/HeNCrlVr mice weighing 18-25g, the ratio of the amount of oligonucleotide in the oligonucleotide conjugate: for oligonucleotide conjugates of a functional oligonucleotide and a conjugate molecule, the amount of oligonucleotide delivered by the conjugate can be from 0.001 to 100mg/kg body weight, in some embodiments from 0.01 to 50mg/kg body weight, in some embodiments from 0.05 to 20mg/kg body weight, and in one specific embodiment from 0.1 to 10mg/kg body weight. Reference may be made to the amounts described above in administering the oligonucleotide conjugates described in the present disclosure.
In addition, by introducing the oligonucleotide conjugate of the present disclosure into hepatocytes in which a specific gene is abnormally expressed, the purpose of suppressing the expression of the specific gene in the hepatocytes can also be achieved by a mechanism of gene expression regulation. In some embodiments, the hepatocyte is a hepatitis cell, in some embodiments a hepg2.2.15 cell. In some embodiments, the hepatocyte may be selected from Hep3B, HepG2, Huh7 and like hepatoma cell lines or isolated primary hepatoma cells, in some embodiments Huh7 hepatoma cells.
The method provided by the present disclosure is used to inhibit the expression of a particular gene in hepatocytes and the amount of functional oligonucleotide in the oligonucleotide conjugate provided is readily determined by one skilled in the art based on the effect desired to be obtained. For example, in some embodiments, the oligonucleotide conjugate is an siRNA conjugate, and the amount of siRNA in the siRNA conjugate provided is an amount that: it is sufficient to reduce the expression of the target gene and results in an extracellular concentration of 1pM to 1 μ M, or 0.01nM to 100nM, or 0.05nM to 50nM or to about 5 nM. The amount required to achieve this local concentration will vary depending on a variety of factors including the method of delivery, the site of delivery, the number of cell layers between the delivery site and the target cell or tissue, whether the delivery is local or systemic, and the like. The concentration at the delivery site may be significantly higher than the concentration at the surface of the target cell or tissue.
Advantageous effects
In some embodiments, a double-stranded oligonucleotide, composition, or oligonucleotide conjugate provided by the present disclosure may have greater stability, lower toxicity, and/or greater activity in vivo. In some embodiments, the double-stranded oligonucleotide provided by the present disclosure is a saRNA. In some embodiments, the saRNA, saRNA composition or saRNA conjugate provided by the present disclosure exhibits an increase in expression of a target gene in vivo of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. In some embodiments, a double-stranded oligonucleotide provided by the present disclosure is an siRNA. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits a rate of inhibition of expression of the target gene of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits an HBV gene expression inhibition rate of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits an inhibitory rate of HBV gene expression in vivo of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in liver. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits an inhibitory rate of HBV gene expression in vivo in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the animal model. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits an HBV surface antigen expression inhibition rate of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition of ANGPTL3 gene expression in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition of intrahepatic ANGPTL3 gene expression in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits an inhibition rate of intrahepatic ANGPTL3 gene expression in vivo in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of animal models. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits an inhibition rate of ANGPTL3 gene expression in the liver in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of human subjects in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% inhibition of APOC3 gene expression in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits an inhibition rate of intrahepatic APOC3 gene expression of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits an inhibition rate of intrahepatic APOC3 gene expression in vivo in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of animal models. In some embodiments, the siRNA, siRNA composition or siRNA conjugate provided by the present disclosure exhibits an inhibition rate of intrahepatic APOC3 gene expression in human subjects of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% in vivo. In some embodiments, the double-stranded oligonucleotide, composition, or oligonucleotide conjugate provided by the present disclosure does not exhibit a significant off-target effect. The off-target effect can be, for example, inhibition of normal expression of a gene other than the target gene. It is believed that off-target effects are not significant if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20% or 10% compared to the effect on the target gene.
According to one embodiment of the present disclosure, when the oligonucleotide is an siRNA that inhibits expression of a Hepatitis B Virus (HBV) gene, the siRNA conjugate provided by the present disclosure can effectively deliver the siRNA to the liver and exhibit excellent properties of inhibiting expression of the HBV gene: while having low off-target effect, the compound can inhibit 81.54 to 83.8 percent of HBV gene expression in the liver of a hepatitis B model mouse at the dose of 1 mg/kg. Meanwhile, the siRNA conjugate disclosed by the invention can also effectively reduce the HBV surface antigen expression in a hepatitis B model mouse, and can reach the HBV surface antigen expression inhibition rate of 92.2% and the HBV DNA inhibition rate of 89.2% under the dosage of 3 mg/kg. In particular, the specific siRNA conjugates formed by the specific modified siRNA and the specific conjugate molecule provided by the present disclosure can continuously exhibit excellent inhibitory effects on HBV expression for up to 140 days of experiment while having a low administration dose, compared to conjugates formed by conjugate molecules provided by the prior art.
According to one embodiment of the present disclosure, when the oligonucleotide is an siRNA that inhibits hepatitis b virus gene expression, the siRNA conjugate provided by the present disclosure can effectively deliver siRNA to the liver and exhibit excellent properties of inhibiting HBV gene expression: the HBV gene expression in the liver of a hepatitis B model mouse can be inhibited by more than 75 percent under the dosage of single administration of 1mg/kg while the low off-target effect is achieved. Meanwhile, the siRNA conjugate disclosed by the invention can also effectively reduce the expression of HBV surface antigen in a hepatitis B model mouse, and can reach the HBV surface antigen expression inhibition rate of 95.2% and the HBV DNA inhibition rate of 91.6% under the dosage of 3 mg/kg. In particular, the specific siRNA conjugates formed by the specific modified siRNA and the specific conjugate molecule provided by the present disclosure can continuously exhibit excellent inhibitory effects on HBV expression for up to 84 days of experiment while having a low administration dose, compared to conjugates formed by conjugate molecules provided by the prior art.
According to one embodiment of the present disclosure, when the oligonucleotide is an siRNA that inhibits the expression of an angiopoietin-like protein 3(ANGPTL3) gene, the siRNA conjugate provided by the present disclosure can effectively deliver the siRNA to the liver and exhibit excellent properties of inhibiting the expression of an ANGPTL3 gene: inhibiting the expression of at least 48.9% of ANGPTL3 gene in liver of a high fat model mouse at a dose of 1 mg/kg; under the dosage of 3mg/kg, the gene inhibition rate is as high as 80.8%. In particular, the specific siRNA conjugates formed by the specific modified siRNA and the specific conjugate molecule provided by the present disclosure show superior gene suppression rate compared to conjugates formed by conjugate molecules provided by the prior art; moreover, the specific siRNA conjugate provided by the disclosure can continuously show excellent ANGPTL3 expression inhibition and blood fat reduction effects in the condition of low administration dose and low administration frequency within the experiment time of 49 days.
According to one embodiment of the present disclosure, when the oligonucleotide is an siRNA that inhibits expression of apolipoprotein C3(ApoC3) gene, the siRNA conjugate provided by the present disclosure can effectively deliver siRNA to liver and exhibit excellent characteristics of inhibiting expression of ApoC3 gene: at least 68.2% of APOC3 gene expression in the liver of a high-fat model mouse is inhibited at a dose of 3 mg/kg. In particular, the specific siRNA conjugates formed by the specific modified siRNA and the specific conjugate molecule provided by the present disclosure show superior gene suppression rate compared to conjugates formed by conjugate molecules provided by the prior art; also, the specific siRNA conjugates provided by the present disclosure can continuously exhibit excellent blood lipid inhibitory effects for an experimental period of up to 65 days with a low administration dose and a low administration frequency.
In certain embodiments, the siRNA conjugates of the disclosure also exhibit low animal level toxicity and good safety, e.g., in some embodiments, no significant toxic response is observed for the conjugates of the disclosure even when administered up to 100-fold the onset concentration (3 mg/kg as onset concentration) in C57BL/6J mice.
The above examples illustrate that the oligonucleotide conjugates provided by the present disclosure are capable of effectively delivering functional oligonucleotides to the liver and remain active in vivo for a long period of time, thereby effectively treating and/or preventing pathological conditions and diseases caused by the expression of specific genes in hepatocytes.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Reagent kit
In another aspect, provided herein is a kit comprising a conjugate as described above.
In some embodiments, the kits provided herein comprise a container containing the conjugate. In some embodiments, the kits provided herein comprise a container of pharmaceutically acceptable excipients. In some embodiments, the kits provided herein further comprise a pharmaceutically acceptable excipient, such as a stabilizer or preservative. In some embodiments, the kits provided herein comprise at least one additional therapeutic agent. In some embodiments, the kit comprises at least one additional therapeutic agent in a container different from the conjugate described in the present disclosure. In some embodiments, the kit can include instructions for mixing the conjugate with a pharmaceutically acceptable excipient (for those with excipients) or other ingredients.
In the kits of the present disclosure, the conjugate and optional pharmaceutically acceptable excipients may be provided in any form, such as a liquid form, a dried form, or a lyophilized form. In some embodiments, the oligonucleotide conjugate and optional pharmaceutically acceptable excipients are substantially pure and/or sterile. In some embodiments, sterile water is provided in the kits of the present disclosure.
Examples
The present disclosure will be described in detail below by way of examples. Unless otherwise specified, reagents and media used in the following examples are commercially available, and the procedures for nucleic acid electrophoresis, real-time PCR and the like used therein are performed by the method described in molecular cloning (Cold Spring Harbor LBlaboratory Press (1989)).
HEK239A cells were supplied by the institute of molecular medicine, university of beijing, nucleic acid technology laboratory, and cultured in DMEM complete medium (Hyclone) containing 20% fetal bovine serum (FBS, Hyclone), 0.2 v% blueberry antibiotic (penicillin-streptomycin, Gibco, Invitrogen). Cultured at 37 ℃ in an incubator containing 5% CO 2/95% air.
Huh7 cells were purchased from ATCC and cultured in DMEM complete medium (Hyclone) containing 10% fetal bovine serum (FBS, Hyclone), 1% non-essential amino acids (NEAA, Corning) at 37 ℃ in an incubator containing 5% CO 2/95% air.
Unless otherwise stated, when cells were transfected with the siRNA conjugates synthesized in preparation examples 7 to 9 below, Lipofectamine TM2000(Invitrogen) was used as a transfection reagent, and the detailed procedures were performed according to the manufacturer's instructions.
Unless otherwise stated, the reagent ratios provided below are calculated as volume ratios (v/v).
Unless otherwise stated, the animal models used are as follows:
c57BL/6J mice: purchased from Beijing Wittiulihua laboratory animal technology, Inc.;
HBV transgenic mice C57BL/6J-Tg (Alb1HBV)44 Bri/J: purchased from the laboratory animal department of medicine of Beijing university. Selecting mice with S/CoV > 10 before experiment;
AAV-HBV transgenic mice: AAV-HBV models were prepared according to literature methods (Doudi, et al, Chin J Biotech 2010, May 25; 26(5): 679-. rAAV8-13HBV, type D (ayw) Virus (available from Acanthopanax beijing and molecular medicine research institute, Inc., 1 × 1012viral genome (v.g.)/mL, lot No. 2016123011) was diluted to 5 × 10 with sterile PBS11v.g./mL, 200. mu.L of diluted rAAV8-1.3HBV per mouse (i.e., 1 × 10 HBV per mouse)10v.g). On day 28 post virus injection, all mice were tested for HBsAg and HBV DNA by orbital bleeding (approximately 100 μ L) for serum collection;
BALB/c mice: 6-8 weeks old, purchased from Beijing Wittiulihua laboratory animal technology Co., Ltd;
human APOC3 transgenic mice: b6; CBA-Tg (APOC3)3707Bres/J, available from Jackson Lab;
preparative example 1B-2 preparation of conjugate molecule (conjugate molecule 1)
In this preparation example, a compound of conjugate molecule 1 (hereinafter, also referred to as B-2 conjugate molecule) was synthesized as follows:
(1-1) Synthesis of conjugated end segment GAL-5 (end molecule of B-2 conjugated molecule)
Figure BDA0001930334880000551
Synthesis of (1-1a) GAL-2
100.0g GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS number: 1772-03-8, available from Ningbo Honghong Biochemical company, 463.8mmol) was dissolved in 1000ml of anhydrous pyridine, 540ml of acetic anhydride (available from Enox company, 5565.6mmol) was added under ice-water bath, and the reaction was stirred at room temperature for 1.5 hours. Pouring the reaction solution into 10L of ice water, carrying out suction filtration under reduced pressure, washing a filter cake with 2L of ice water, adding an acetonitrile/toluene mixed solvent (volume ratio of acetonitrile to toluene is 1:1) until the acetonitrile/toluene mixed solvent is completely dissolved, and evaporating the solvent to dryness to obtain a white solid product GAL-2130.0 g.
(1-1b) Synthesis of GAL-3
GAL-2(35.1g, 90.0mmol) obtained in step (1-1a) was dissolved in 213ml of anhydrous 1, 2-dichloroethane, and 24.0g of TMSOTf (CAS number: 27607-77-8, available from Michael corporation, 108.0mmol) was added under ice-water bath and nitrogen protection, and reacted at room temperature overnight.
The reaction solution was diluted with 400ml of dichloromethane, filtered through celite, and then 1L of saturated aqueous sodium bicarbonate was added, stirred well, the organic phase was separated, the aqueous phase was extracted twice with 300ml of dichloroethane, the organic phases were combined, washed with 300ml of saturated aqueous sodium bicarbonate and 300ml of saturated brine, respectively, the organic phase was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to obtain light yellow viscous syrup product GAL-326.9 g.
(1-1c) Synthesis of GAL-4
GAL-3(26.9g, 81.7mmol) obtained in step (1-1b) was dissolved in 136ml of anhydrous 1, 2-dichloroethane, and dried
Figure BDA0001930334880000552
30g of molecular sieve powder was added, 9.0g of 5-hexen-1-ol (CAS number: 821-41-0, available from Adamas-beta, 89.9mmol) was added, and the mixture was stirred at room temperature for 30 minutes, and 9.08g of TMSOTf (40.9mmol) was added under ice bath and nitrogen protection, and the reaction was stirred at room temperature overnight. Filtering to remove
Figure BDA0001930334880000553
Molecular sieve powder, adding 300ml dichloroethane into the filtrate for dilution, filtering with diatomite, adding 500ml saturated sodium bicarbonate aqueous solution, stirring for 10 minutes for washing, separating an organic phase, extracting the aqueous phase once with 300ml dichloroethane, combining the organic phases, washing with 300ml saturated sodium bicarbonate aqueous solution and 300ml saturated saline solution respectively, separating the organic phase, drying with anhydrous sodium sulfate, evaporating the solvent under reduced pressure to obtain a yellow syrup product GAL-441.3g, and directly carrying out the next oxidation reaction without purification.
Synthesis of (1-1d) GAL-5
GAL-4(14.9g, 34.7mmol) obtained by the method described in step (1-1c) was dissolved in a mixed solvent of 77ml of methylene chloride and 77ml of acetonitrile, 103ml of deionized water and 29.7g of sodium periodate (CAS number: 7790-28-5, available from Aladdin company, 138.8mmol), respectively, were added thereto, stirred for 10 minutes in an ice-water bath, ruthenium trichloride (CAS number: 14898-67-0, available from Annona Gico., 238mg, 1.145mmol) was added thereto, and reacted at room temperature overnight. Adding 300ml water into the reaction solution, diluting and stirring, adding saturated sodium bicarbonate to adjust the pH value to be about 7.5,the organic phase was separated off and discarded, and the aqueous phase was extracted three times with 200ml of dichloromethane each time, and the organic phase was discarded. Adjusting pH of the water phase with citric acid solid to about 3, extracting with dichloromethane three times (200 ml each time), combining organic phases, drying with anhydrous sodium sulfate, and evaporating the solvent under reduced pressure to obtain white foamy solid product GAL-56.5 g.1H NMR(400MHz,DMSO)δ12.01(br,1H),7.83(d,J＝9.2Hz,1H),5.21(d,J＝3.2Hz,1H),4.96(dd,J＝11.2,3.2Hz,1H),4.49(d,J＝8.4Hz,1H),4.07–3.95(m,3H),3.92–3.85(m,1H),3.74–3.67(m,1H),3.48–3.39(m,1H),2.20(t,J＝6.8Hz,2H),2.11(s,3H),2.00(s,3H),1.90(s,3H),1.77(s,3H),1.55–1.45(m,4H).
(1-2) Synthesis of A-1:
Figure BDA0001930334880000561
dissolving DMTrCl (4,4' -bis (methoxytrityl chloride, 38.12g, 112.5mmol) in 450ml of anhydrous pyridine, adding DL-calcium glycerate hydrate (12.88g, 45.0mmol), reacting at 45 ℃ for 22h, filtering the reaction solution, leaching the filter cake with 200ml of DCM, concentrating the filtrate under reduced pressure to dryness, redissolving the residue with 500ml of dichloromethane, washing with 0.5M triethylamine phosphate (pH 7-8) for 2 times, 200ml each time, extracting the aqueous phase with dichloromethane for 2 times, 200ml each time, combining the organic phases, drying with anhydrous sodium sulfate, filtering, evaporating the solvent under reduced pressure, purifying with 200-mesh 300-mesh normal-phase silica gel column, eluting with a gradient of petroleum ether, ethyl acetate, dichloromethane, methanol, 1:1:1:0.35-1:1:1:0.55, collecting the product, evaporating the solvent under reduced pressure, redissolving 500ml of dichloromethane, washing with 200ml of 0.5M triethylamine phosphate for 1 time, extracting the water phase with dichloromethane for 2 times (200 ml each time), mixing organic phases, drying with anhydrous sodium sulfate, filtering, evaporating the solvent under reduced pressure, and vacuum-pumping with vacuum oil pump until it is dried overnight to obtain white solid product A-120.7 g.1H NMR(400MHz,DMSO-d6)δ7.46(ddd,J＝6.5,2.3,1.1Hz,1H),7.40–7.28(m,7H),6.89–6.81(m,4H),4.84(d,J＝5.0Hz,1H),4.36–4.24(m,1H),4.29(s,6H),3.92(dd,J＝12.4,7.0Hz,1H),3.67(dd,J＝12.3,7.0Hz,1H),2.52(q,J＝6.3Hz,6H),1.03(t,J＝6.3Hz,9H).MS m/z：C24H23O6，[M-H]-, theory: 407.15, actually measuring: 406.92.
(1-3) Synthesis of A-2:
Figure BDA0001930334880000562
a-1(5.100g, 10mmol) obtained in step (1-2), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 10.410g, 20mmol), 1-hydroxybenzotriazole (HOBt, 2.700g, 20mmol), diisopropylethylamine (DIEA, 6.460g, 50mmol) were dissolved in 50ml of dichloromethane, and the reaction mixture was stirred at room temperature for 30 minutes, poured into a solution of triethylenetetramine (TETA, 11.700g, 80mmol) in 50ml of dichloromethane, and stirred at 25 ℃ for 21 hours. Washing with 100ml of saturated saline solution for 1 time, extracting the aqueous phase with 100ml of dichloromethane for 2 times, combining organic phases, drying with anhydrous sodium sulfate, filtering, evaporating the solvent under reduced pressure, purifying with a normal phase silica gel column (dichloromethane: methanol: ammonia water: 100:40:10-100:40:14 to elute the product), collecting the product eluate, and evaporating the solvent under reduced pressure to obtain 4.181g of the product.1H NMR(400MHz,DMSO-d6)δ7.97(t,J＝4.4Hz,1H),7.57–7.49(m,4H),7.51–7.41(m,2H),7.35–7.24(m,3H),6.81–6.73(m,4H),5.86(d,J＝7.0Hz,1H),5.08–4.98(m,2H),4.13(dt,J＝6.9,6.3Hz,1H),3.92(dd,J＝11.3,6.3Hz,1H),3.81(s,5H),3.67(dd,J＝11.4,6.3Hz,1H),3.39(dtd,J＝15.1,5.4,4.4Hz,1H),3.00(dtd,J＝15.1,5.4,4.2Hz,1H),2.88–2.73(m,2H),2.72–2.57(m,3H),2.58–2.32(m,5H),1.33(p,J＝4.2Hz,2H).MS m/z：C30H41N4O5，[M+H]Theory: 537.68, actually measuring: 537.53.
the B-2 conjugate molecule was synthesized using the GAL-5 compound obtained as described above, by the following process scheme:
Figure BDA0001930334880000571
(1-4) Synthesis of GAL-C6-1
GAL-5(4.5g, 10.0mmol), tert-butyl 6-aminocaproate hydrochloride (2.2g, 12.0mmol), O-benzotriazole-tetramethylurea hexafluorophosphate (5.7g, 15.0mmol) and diisopropylethylamine (3.9g, 30.0mmol) were added to 40ml of N, N-dimethylformamide, and the reaction was stirred at room temperature for 4 hours. 100ml of saturated aqueous sodium bicarbonate solution is slowly added into the reaction solution, extraction is carried out for 3 times by 100ml of ethyl acetate, organic phases are combined, 100ml of saturated saline solution is washed once, the organic phase is separated, anhydrous sodium sulfate is dried, the solvent is evaporated under reduced pressure and is pumped by an oil pump to obtain 10.5g of crude oily matter which is directly subjected to the next reaction.
(1-5) Synthesis of GAL-C6-2
The crude GAL-C6-1 (10.5g, 10mmol) obtained in step (1-4) was dissolved in 60ml of formic acid, and the reaction was stirred at room temperature for 16 hours. And (3) spin-drying the reaction solution, purifying and collecting the target product by column chromatography (200-300 meshes of normal phase silica gel, and performing gradient elution with dichloromethane: methanol: 100:18-100: 20), and concentrating to obtain 5.2g of the target product. 1H NMR (400MHz, DMSO-d6) δ 7.87(s,0H),7.46(s,0H), 6.05-5.94 (m,0H),5.17(t, J ═ 7.0Hz,0H),4.54(dd, J ═ 12.4,6.9Hz,0H),4.33(q, J ═ 7.0Hz,0H),3.88(t, J ═ 7.0Hz,0H), 3.75-3.58 (m,0H),3.38(td, J ═ 12.3,2.1Hz,0H), 3.17-3.07 (m,0H), 2.57-2.46 (m,0H), 2.50-2.35 (m,0H),2.28(ddd, J ═ 12.3,4.5,2.1, 0.76, 2.27H), 1, 1.90H, 1H, 1.09 (m,1H), 1-1H, 1.1H, 1H: C25H39N2O12, [ M-H ] -, theory: 559.25, actually measuring: 559.32.
(1-6) Synthesis of B-1:
GAL-C6-2(2.018g, 3.6mmol) obtained in step (1-5), 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (DEPBT, 1.496g, 5.0mmol) and diisopropylethylamine (DIEA, 1.292g, 10.0mmol) were dissolved in 10ml of dichloromethane, and the reaction was stirred at room temperature for 5 minutes, then A-2(0.537g, 1.0mmol) was added, and the reaction was stirred at 25 ℃ for 24 hours. The reaction solution was washed with 20ml of saturated sodium bicarbonate 1 time, the aqueous phase was extracted with 20ml of dichloromethane 3 times, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure, purified by a normal phase silica gel column (1% triethylamine neutralizes the silica gel acidity, and the product was eluted with petroleum ether, ethyl acetate, dichloromethane, and methanol at 1:1:1: 0.2-0.25), and the eluting solvent was evaporated under reduced pressure to give 1.79g of a pure product.1H NMR(400MHz,DMSO-d6)δ8.01–7.87(m,2H),7.75(td,J＝4.4,0.9Hz,1H),7.51–7.42(m,1H),7.35–7.25(m,1H),7.17–7.09(m,1H),6.81–6.73(m,1H),5.26(ddd,J＝6.4,5.2,1.0Hz,1H),5.09(ddd,J＝6.2,5.1,1.0Hz,1H),4.46(dd,J＝6.3,0.9Hz,1H),4.34(ddd,J＝12.1,2.3,1.0Hz,1H),4.14–4.03(m,2H),3.96(dddd,J＝6.2,4.0,2.5,1.3Hz,1H),3.95–3.76(m,1H),3.81(s,2H),3.80–3.71(m,2H),3.72–3.57(m,1H),3.61–3.46(m,1H),3.47(dd,J＝5.9,1.6Hz,1H),3.35(dt,J＝11.8,6.0Hz,1H),3.11(dtd,J＝14.3,6.2,4.4Hz,0H),2.99(tdd,J＝6.2,4.3,1.6Hz,1H),2.87(dtd,J＝14.2,6.2,4.3Hz,1H),2.39(dt,J＝14.0,6.9Hz,1H),2.31–2.08(m,2H),2.09(dd,J＝6.9,1.6Hz,1H),2.08–1.93(m,6H),1.97–1.88(m,3H),1.83–1.78(m,3H),1.73–1.08(m,10H).MS m/z：C105H155N10O38，[M+H]Theory: 2165.43, actually measuring: 2165.62.
(1-7) Synthesis of B-2:
b-1(2.727g, 1.26mmol, obtained by combining two products) obtained by the method described in step (1-6), succinic anhydride (0.378g, 3.78mmol) and 4-dimethylaminopyridine (DMAP, 0.462g, 3.78mmol) were dissolved in 13ml of dichloromethane, DIEA (0.814g, 6.30mmol) was added, and the reaction was stirred at 25 ℃ for 24 hours. 5ml of 0.5M triethylamine phosphate washes the reaction solution, the aqueous phase is extracted 3 times with 5ml of dichloromethane each time, the combined organic phases are evaporated to dryness under reduced pressure to obtain a crude product. The column purification uses 60g of 200-mesh 300-mesh normal phase silica gel, 1% of triethylamine neutralizes the acidity of the silica gel, the dichloromethane balances the column, the product is eluted by dichloromethane containing 1 ‰ of triethylamine and methanol in a ratio of 100: 18-20, and the solvent is evaporated to dryness under reduced pressure to obtain 2.719g of pure product.1H NMR(400MHz,DMSO-d6)δ8.01–7.87(m,2H),7.75(td,J＝4.4,0.9Hz,1H),7.51–7.42(m,1H),7.34–7.25(m,1H),7.17–7.09(m,1H),6.81–6.73(m,1H),5.81(t,J＝5.8Hz,1H),5.26(ddd,J＝6.4,5.1,0.9Hz,1H),5.09(ddd,J＝5.5,5.0,1.0Hz,1H),4.46(dd,J＝6.3,0.9Hz,1H),4.34(ddd,J＝12.2,2.3,1.0Hz,1H),4.14–4.04(m,2H),4.08–3.81(m,2H),3.85–3.71(m,5H),3.69–3.54(m,1H),3.59–3.46(m,1H),3.47(dd,J＝5.9,1.6Hz,1H),3.35(dt,J＝11.8,6.0Hz,0H),3.11(dtd,J＝14.3,6.2,4.4Hz,1H),2.99(tdd,J＝6.2,4.3,1.6Hz,1H),2.93–2.72(m,1H),2.71–2.47(m,3H),2.46–2.33(m,1H),2.31–2.05(m,3H),2.08–1.99(m,6H),2.03–1.88(m,4H),1.83–1.78(m,3H),1.71–1.56(m,1H),1.60–1.41(m,5H),1.46–1.38(m,1H),1.43–1.35(m,1H),1.40–1.32(m,1H),1.32(ddt,J＝6.9,2.0,1.0Hz,1H),1.32–1.08(m,1H),1.03(t,J＝7.2Hz,3H).MS m/z：C109H159N10O41，[M+H]Theory: 2265.50, actually measuring: 2265.38. the structure of the obtained B-2 conjugated molecule is shown as a formula (601).
Preparative example 2 preparation of D-6 conjugate molecule (conjugate molecule 2)
In the present preparation, a compound of conjugate molecule 2 (hereinafter, also referred to as D-6 conjugate molecule) was synthesized as follows:
Figure BDA0001930334880000591
(2-1) Synthesis of D-5:
Figure BDA0001930334880000601
GAL-5(1.611g, 3.6mmol), 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (DEPBT, 1.496g, 5.0mmol), diisopropylethylamine (DIEA, 1.292g, 10.0mmol) were dissolved in 10ml of dichloromethane, the reaction was stirred at room temperature for 5 minutes, A-2(0.537g, 1.0mmol) was added, and the reaction was stirred at 25 ℃ for 24 hours. The reaction solution was washed with 20ml of saturated sodium bicarbonate 1 time, the aqueous phase was extracted with dichloromethane 3 times, 20ml each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the solvent was evaporated under reduced pressure, purified by a normal phase silica gel column (1% triethylamine neutralized silica gel acidity, product was eluted with petroleum ether: ethyl acetate: dichloromethane: methanol: 1:1:0.2-1:1:1: 0.25), and the eluting solvent was evaporated under reduced pressure to give a pure product 1.35 g.1H NMR(400MHz,DMSO-d6)δ8.01–7.87(m,2H),7.51–7.41(m,1H),7.35–7.25(m,1H),7.17–7.09(m,1H),6.81–6.73(m,1H),5.86(d,J＝7.0Hz,0H),5.26(ddd,J＝6.4,5.2,1.0Hz,1H),5.09(ddd,J＝5.5,5.0,1.0Hz,1H),4.74(dt,J＝7.0,6.3Hz,0H),4.46(dd,J＝6.3,0.9Hz,1H),4.34(ddd,J＝12.2,2.3,1.0Hz,1H),4.14–4.03(m,2H),4.00–3.90(m,1H),3.94–3.76(m,1H),3.81(s,2H),3.80–3.71(m,2H),3.72–3.57(m,1H),3.61–3.46(m,1H),3.47(dd,J＝5.9,1.6Hz,1H),3.35(dt,J＝11.8,6.0Hz,0H),2.39(dt,J＝14.0,6.9Hz,0H),2.31–2.09(m,1H),2.06–1.95(m,6H),1.99–1.90(m,3H),1.83–1.78(m,3H),1.68–1.22(m,4H).MS m/z：C87H122N7O35，[M+H]Theory: 1825.95, actually measuring: 1825.77.
(2-2) Synthesis of D-6:
Figure BDA0001930334880000602
d-5(2.299g, 1.26mmol, obtained by combining the products from the plural batches) obtained according to the method described in step (2-1) and succinic anhydride (0.378g, 3.78mmol) and 4-dimethylaminopyridine (DMAP, 0.462g, 3.78mmol) were dissolved in 13ml of dichloromethane, DIEA (0.814g, 6.30mmol) was added, and the reaction was stirred at 25 ℃ for 24 hours. The reaction solution was washed with 5ml of 0.5M triethylamine phosphate, the aqueous phase was extracted 3 times with 5ml of dichloromethane each time, the combined organic phases were evaporated to dryness under reduced pressure to give a crude product. The column purification uses 60g of 200-mesh 300-mesh normal phase silica gel column, the silica gel acidity is neutralized by 1% triethylamine, the column is balanced by dichloromethane, the product is eluted by dichloromethane containing 1 ‰ triethylamine and methanol at the ratio of 100:18-100:20, and the solvent is evaporated under reduced pressure to obtain 1.836g of a pure product.1H NMR(400MHz,DMSO-d6)δ8.01–7.87(m,2H),7.50–7.41(m,1H),7.35–7.25(m,1H),7.17–7.09(m,1H),6.81–6.73(m,1H),5.81(t,J＝5.8Hz,0H),5.26(ddd,J＝6.5,5.2,1.0Hz,1H),5.09(ddd,J＝5.5,5.0,1.0Hz,1H),4.46(dd,J＝6.3,0.9Hz,1H),4.34(ddd,J＝12.2,2.3,1.0Hz,1H),4.14–4.07(m,1H),4.08(dq,J＝2.4,1.7,1.2Hz,1H),4.08–3.94(m,1H),3.99–3.82(m,1H),3.86–3.71(m,5H),3.69–3.54(m,1H),3.59–3.46(m,1H),3.47(dd,J＝5.9,1.6Hz,1H),3.35(dt,J＝11.8,6.0Hz,1H),2.78(dt,J＝14.6,8.5Hz,0H),2.71–2.47(m,3H),2.46–2.33(m,1H),2.31–2.09(m,1H),2.06–1.96(m,6H),2.00–1.90(m,3H),1.83–1.78(m,3H),1.68–1.22(m,4H),1.03(t,J＝7.2Hz,3H).MS m/z：C91H126N7O38，[M+H]Theory: 1926.02, actually measuring: 1926.15. the structure of the resulting D-6 conjugate molecule is shown in formula (602).
PREPARATION EXAMPLE 3C-2 preparation of conjugate molecule (conjugate molecule 3)
In this preparation example, a compound of conjugate molecule 3 (hereinafter, also referred to as C-2 conjugate molecule) was synthesized as follows:
Figure BDA0001930334880000611
(3-1) Synthesis of GAL-C2-1
GAL-5(4.5g, 10.0mmol), glycine tert-butyl ester hydrochloride (2.0g, 12.0mmol), O-benzotriazole-tetramethylurea hexafluorophosphate (5.7g, 15.0mmol), and diisopropylethylamine (3.9g, 30.0mmol) were added to 40ml of N, N-dimethylformamide, and the reaction was stirred at room temperature for 4 hours. 100ml of saturated aqueous sodium bicarbonate solution was added to the reaction solution, extraction was performed 3 times with 100ml of ethyl acetate, the organic phases were combined, 100ml of saturated saline solution was washed once, the organic phase was separated, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure and dried by an oil pump to obtain 10.1g of crude oily substance, which was directly subjected to the next reaction.
(3-2) Synthesis of GAL-C2-2
The crude GAL-C2-1 (10.1g, 10mmol) was dissolved in 60ml of formic acid and the reaction was stirred at room temperature for 16 hours. And (3) spin-drying the reaction solution, purifying and collecting a target product by column chromatography (200-300 meshes of normal phase silica gel, and performing gradient elution by using dichloromethane/methanol), and concentrating to obtain 5.0g of the target product. 1H NMR (400MHz, DMSO-d6) δ 8.49(s,1H),7.46(s,1H), 6.05-5.94 (m,2H), 5.01-4.90 (m,1H), 4.52-4.33 (m,3H), 3.79-3.61 (m,3H),3.14(td, J ═ 12.2,3.2Hz,1H),2.68(td, J ═ 12.2,3.3Hz,1H),2.27(td, J ═ 12.6,2.8Hz,1H),2.15(td, J ═ 12.6,2.9Hz,1H), 2.07-1.95 (m,12H),1.82 (J ═ 12.9,2.8Hz,1H),1.54 (t, 12.6, 1H), 1.81 (m, 0.97H), 1.7-1H, 1.7 (m, 0.1H), 1.7 (m: C21H31N2O12, [ M-H ] -, theory: 503.19, actually measuring: 503.26.
(3-3) Synthesis of C-1:
GAL-C2-2(1.816g, 3.6mmol), 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (DEPBT, 1.496g, 5.0mmol), diisopropylethylamine (DIEA, 1.292g, 10.0mmol) were dissolved in 10ml of dichloromethane, stirred at room temperature for 5 minutes, then A-2(0.537g, 1.0mmol) was added, and stirred at 25 ℃ for 24 hours. Washing the reaction solution with 20ml saturated sodium bicarbonate for 1 time, extracting the water phase with dichloromethane for 3 times (20 ml each time), mixing the organic phases, drying with anhydrous sodium sulfate, filtering, evaporating the solvent under reduced pressure, and purifying with normal phase silica gel column (1% triethylamine neutralizes the acidity of the silica gel, petroleum ether: ethyl acetate: dichloromethane: methanol: 1:1:0.2-0.25 to obtain the productMaterial), the eluting solvent was evaporated under reduced pressure to dryness to obtain a pure product of 1.74 g.1H NMR(400MHz,DMSO-d6)δ8.14(td,J＝5.8,1.0Hz,1H),8.01–7.87(m,1H),7.87–7.79(m,0H),7.51–7.42(m,1H),7.35–7.25(m,1H),7.17–7.09(m,1H),6.81–6.73(m,1H),5.86(d,J＝7.0Hz,0H),5.26(ddd,J＝6.4,5.2,1.0Hz,1H),5.09(ddd,J＝5.6,5.0,1.0Hz,1H),4.74(dt,J＝7.0,6.3Hz,0H),4.46(dd,J＝6.3,0.9Hz,1H),4.34(ddd,J＝12.2,2.3,1.0Hz,1H),4.14–4.03(m,2H),4.02–3.56(m,9H),3.60–3.46(m,1H),3.47(dd,J＝5.9,1.6Hz,1H),3.35(dt,J＝11.8,6.0Hz,1H),2.21(dt,J＝15.1,6.9Hz,0H),2.14–2.05(m,1H),2.10–1.98(m,6H),2.01–1.90(m,3H),1.83–1.78(m,3H),1.68–1.22(m,4H).MS m/z：C93H131N10O38，[M+H]Theory: 1997.10, actually measuring: 1997.32.
(3-4) Synthesis of C-2:
c-1(2.515g, 1.26mmol, obtained by combining two products) obtained by the method described in step (3-3), succinic anhydride (0.378g, 3.78mmol) and 4-dimethylaminopyridine (DMAP, 0.462g, 3.78mmol) were dissolved in 13ml of dichloromethane, DIEA (0.814g, 6.30mmol) was added, and the reaction was stirred at 25 ℃ for 24 hours. 5ml of 0.5M triethylamine phosphate washes the reaction solution, the aqueous phase is extracted 3 times with 5ml of dichloromethane each time, the combined organic phases are evaporated to dryness under reduced pressure to obtain a crude product. The column purification uses 60g of 200-mesh 300-mesh normal phase silica gel, 1% of triethylamine neutralizes the acidity of the silica gel, the dichloromethane balances the column, the product is eluted by dichloromethane containing 1 ‰ of triethylamine and methanol in a ratio of 100: 18-20, and the solvent is evaporated to dryness under reduced pressure to obtain 2.469g of a pure product.1H NMR(400MHz,DMSO-d6)δ8.14(td,J＝5.8,1.0Hz,1H),8.01–7.87(m,1H),7.87–7.79(m,0H),7.51–7.41(m,1H),7.35–7.25(m,1H),7.17–7.09(m,1H),6.81–6.73(m,1H),5.81(t,J＝5.8Hz,0H),5.26(ddd,J＝6.5,5.2,1.0Hz,1H),5.09(ddd,J＝5.5,5.1,1.0Hz,1H),4.46(dd,J＝6.3,1.0Hz,1H),4.34(ddd,J＝12.2,2.3,1.0Hz,1H),4.10(ddd,J＝6.9,2.5,0.9Hz,1H),4.10–3.80(m,4H),3.84–3.68(m,5H),3.69–3.43(m,3H),3.35(dt,J＝11.8,6.0Hz,0H),2.78(dt,J＝14.6,8.5Hz,0H),2.71–2.47(m,3H),2.40(dt,J＝14.9,8.6Hz,0H),2.21(dt,J＝15.1,6.9Hz,1H),2.14–2.05(m,1H),2.10–1.99(m,6H),2.03–1.90(m,3H),1.83–1.78(m,3H),1.68–1.22(m,4H),1.03(t,J＝7.2Hz,3H).MS m/z：C97H135N10O41，[M+H]Theory: 2097.18, actually measuring: 2097.25. the structure of the obtained C-2 conjugated molecule is shown as a formula (603).
PREPARATION EXAMPLE 4 preparation of P-2 conjugation molecule (conjugation molecule 4)
In this preparation example, a compound of conjugate molecule 4 (hereinafter, also referred to as P-2 conjugate molecule) was synthesized as follows:
Figure BDA0001930334880000631
(4-1) Synthesis of GAL5-C4-1
GAL-5(4.5g, 10.0mmol), 4-amino-acid tert-butyl ester hydrochloride (1.9g, 12.0mmol), O-benzotriazole-tetramethylurea hexafluorophosphate (5.7g, 15.0mmol), and diisopropylethylamine (3.9g, 30.0mmol) were added to 40ml of N, N-dimethylformamide, and the reaction was stirred at room temperature for 4 hours. 100ml of saturated aqueous sodium bicarbonate solution was slowly added to the reaction solution, extraction was performed 3 times with 100ml of ethyl acetate, the organic phases were combined, 100ml of saturated saline solution was washed once, the organic phase was separated, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure and dried by an oil pump to obtain 10.3g of crude oily matter which was directly subjected to the next reaction.
(4-2) Synthesis of GAL5-C4-2
The crude GAL5-C4-1 (10.3g, 10mmol) was dissolved in 60ml of formic acid and the reaction was stirred at room temperature for 16 hours. And (3) spin-drying the reaction solution, purifying and collecting the target product by column chromatography (200-300 meshes of normal phase silica gel, and performing gradient elution with dichloromethane: methanol being 100:18-100: 20), and concentrating to obtain 5.1g of the target product.1H NMR(400MHz,DMSO-d6)δ7.89(s,1H),7.46(s,1H),6.05–5.94(m,2H),5.25(t,J＝7.0Hz,1H),4.53–4.35(m,2H),4.14(t,J＝7.0Hz,1H),3.81(dd,J＝12.1,6.8Hz,1H),3.46(td,J＝12.1,3.3Hz,1H),3.30(td,J＝12.4,3.0Hz,1H),3.01(td,J＝12.1,2.8Hz,1H),2.75(td,J＝12.4,3.0Hz,1H),2.45–2.08(m,6H),2.07–1.95(m,12H),1.81–1.49(m,3H),1.35–1.20(m,1H).MS m/z：C23H35N2O12，[M-H]-, theory: 531.22, actually measuring: 531.15.
(4-3) Synthesis of P-1:
GAL5-C4-2(1.917g, 3.6mmol), 3-diethoxyphosphoryl-1, 2, 3-benzole 4: (3H) -ketone (DEPBT, 1.496g, 5.0mmol), diisopropylethylamine (DIEA, 1.292g, 10.0mmol) were dissolved in 10ml dichloromethane, stirred at room temperature for 5 min, then A-2(0.537g, 1.0mmol) was added, and stirred at 25 ℃ for 24 h. The reaction solution was washed with 20ml of saturated sodium bicarbonate 1 time, the aqueous phase was extracted with 20ml of dichloromethane 3 times, each time 20ml, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the solvent was evaporated under reduced pressure, the product was purified by normal phase silica gel column (1% triethylamine neutralization silica gel acidity, petroleum ether: ethyl acetate: dichloromethane: methanol: 1:1:0.2-0.25 elution), and the elution solvent was evaporated under reduced pressure to give 1.74g of a pure product.1H NMR(400MHz,DMSO-d6)δ8.01–7.87(m,2H),7.75(td,J＝4.4,0.9Hz,1H),7.51–7.41(m,1H),7.35–7.25(m,1H),7.17–7.09(m,1H),6.81–6.73(m,1H),5.86(d,J＝7.0Hz,0H),5.26(ddd,J＝6.4,5.2,1.0Hz,1H),5.09(ddd,J＝5.6,5.0,1.0Hz,1H),4.74(dt,J＝7.0,6.3Hz,0H),4.46(dd,J＝6.3,0.9Hz,1H),4.34(ddd,J＝12.2,2.3,1.0Hz,1H),4.14–4.03(m,2H),4.00–3.71(m,6H),3.72–3.57(m,1H),3.61–3.46(m,1H),3.47(dd,J＝5.9,1.6Hz,1H),3.35(dt,J＝11.8,6.0Hz,0H),3.14(dtd,J＝14.2,6.3,4.4Hz,0H),3.02(tdd,J＝6.2,4.4,1.6Hz,1H),2.90(dtd,J＝14.3,6.3,4.4Hz,0H),2.50(dt,J＝14.2,7.0Hz,1H),2.43–2.25(m,1H),2.28–2.05(m,2H),2.10–1.99(m,5H),2.01(s,1H),2.01–1.90(m,3H),1.83–1.78(m,3H),1.77–1.22(m,6H).MS m/z：C99H143N10O38，[M+H]Theory: 2081.27, actually measuring: 2081.09.
(4-4) Synthesis of P-2:
p-1(2.621g, 1.26mmol, obtained by combining two products) obtained by the method described in step (4-3), succinic anhydride (0.378g, 3.78mmol) and 4-dimethylaminopyridine (DMAP, 0.462g, 3.78mmol) were dissolved in 13ml of dichloromethane, DIEA (0.814g, 6.30mmol) was added, and the reaction was stirred at 25 ℃ for 24 hours. The reaction solution was washed with 5ml of 0.5M triethylamine phosphate, the aqueous phase was extracted 3 times with 5ml of dichloromethane each time, the combined organic phases were evaporated to dryness under reduced pressure to give a crude product. The column purification uses 60g of 200-mesh 300-mesh normal phase silica gel, 1% of triethylamine neutralizes the acidity of the silica gel, the dichloromethane balances the column, the product is eluted by dichloromethane containing 1 ‰ of triethylamine and methanol in a ratio of 100:18-100:20, and the solvent is evaporated to dryness under reduced pressure to obtain 2.654g of pure product.1H NMR(400MHz,DMSO-d6)δ8.01–7.87(m,2H),7.75(td,J＝4.4,0.9Hz,1H),7.51–7.41(m,1H),7.34–7.25(m,1H),7.17–7.09(m,1H),6.81–6.73(m,1H),5.81(t,J＝5.8Hz,0H),5.26(ddd,J＝6.5,5.2,1.0Hz,1H),5.09(ddd,J＝5.5,5.0,1.0Hz,1H),4.46(dd,J＝6.3,1.0Hz,1H),4.34(ddd,J＝12.2,2.3,1.0Hz,1H),4.14–4.04(m,2H),4.08–3.81(m,2H),3.85–3.71(m,5H),3.69–3.54(m,1H),3.59–3.46(m,1H),3.47(dd,J＝5.9,1.6Hz,1H),3.35(dt,J＝11.8,6.0Hz,1H),3.14(dtd,J＝14.2,6.3,4.4Hz,0H),3.02(tdd,J＝6.2,4.4,1.6Hz,1H),2.90(dtd,J＝14.3,6.3,4.4Hz,0H),2.78(dt,J＝14.6,8.5Hz,0H),2.71–2.44(m,3H),2.46–2.35(m,1H),2.40–2.19(m,1H),2.22–2.04(m,2H),2.06–1.96(m,6H),2.00–1.85(m,3H),1.83–1.78(m,3H),1.77–1.22(m,7H),1.03(t,J＝7.2Hz,3H).MS m/z：C103H147N10O41，[M+H]Theory: 2181.34, actually measuring: 2181.48. the structure of the obtained P-2 conjugated molecule is shown as a formula (604).
PREPARATION EXAMPLE 5 preparation of X-3 conjugation molecule (conjugation molecule 5)
In this preparation example, a compound of the conjugate molecule 5 (hereinafter, also referred to as X-3 conjugate molecule) was synthesized as follows:
Figure BDA0001930334880000651
(5-1) Synthesis of X-1:
a-1(5.100g, 10mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 10.410g, 20mmol), 1-hydroxybenzotriazole (HOBt, 2.700g, 20mmol) and diisopropylethylamine (DIEA, 6.460g, 50mmol) were dissolved in 50ml of dichloromethane, and the reaction mixture was stirred at room temperature for 30 minutes, poured into a solution of tetraethylenepentamine (15.145g, 80mmol) in 50ml of dichloromethane, and stirred at 25 ℃ for 21 hours. Washing with 100ml of saturated saline solution for 1 time, extracting the aqueous phase with dichloromethane for 2 times, each time 100ml, combining the organic phases, drying with anhydrous sodium sulfate, filtering, evaporating the solvent under reduced pressure, purifying with normal phase silica gel column (dichloromethane: methanol: ammonia water: 100:40:10-100:40:14 eluting the product), collecting the product eluate, and evaporating the solvent under reduced pressure to obtain 3.761g of the product.1H NMR(400MHz,DMSO-d6)δ7.97(t,J＝4.4Hz,1H),7.60–7.52(m,4H),7.51–7.41(m,2H),7.35–7.24(m,3H),6.81–6.73(m,4H),5.86(d,J＝7.0Hz,1H),5.11–5.00(m,2H),4.13(dt,J＝6.9,6.2Hz,1H),3.92(dd,J＝11.3,6.3Hz,1H),3.81(s,5H),3.67(dd,J＝11.4,6.3Hz,1H),3.37(dtd,J＝15.2,5.4,4.4Hz,1H),3.04(dtd,J＝15.1,5.4,4.4Hz,1H),2.99–2.77(m,2H),2.71–2.35(m,12H),1.33(m,3H).MS m/z：C32H45N5O5，[M+H]Theory: 579.74, actually measuring: 579.59.
(5-2) Synthesis of X-2:
GAL-5(2.238g, 5.0mmol), 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (DEPBT, 1.795g, 6.0mmol), diisopropylethylamine (DIEA, 1.550g, 12.0mmol) were dissolved in 10ml of dichloromethane, and the reaction was stirred at room temperature for 5 minutes, then X-1(0.580g, 1.0mmol) was added, and the reaction was stirred at 25 ℃ for 24 hours. The reaction solution was washed with 20ml of saturated sodium bicarbonate 1 time, the aqueous phase was extracted with dichloromethane 2 times, 20ml each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the solvent was evaporated under reduced pressure, the product was purified by normal phase silica gel column (1% triethylamine neutralization silica gel acidity, petroleum ether: ethyl acetate: dichloromethane: methanol ═ 1:1:0.2-0.25 elution), and the elution solvent was evaporated under reduced pressure to give a pure product 1.73 g.1H NMR(400MHz,DMSO-d6)δ8.01–7.87(m,2H),7.50–7.42(m,1H),7.35–7.25(m,1H),7.17–7.09(m,1H),6.81–6.73(m,1H),5.31–5.22(m,1H),5.13–5.05(m,1H),4.50–4.43(m,1H),4.39–4.30(m,1H),4.14–4.03(m,2H),4.00–3.81(m,2H),3.85–3.71(m,5H),3.72–3.43(m,3H),2.32–2.08(m,2H),2.06–1.90(m,10H),1.81(dd,J＝4.1,1.7Hz,3H),1.68–1.22(m,5H).MS m/z：C108H154N9O45，[M+H]Theory: 2298.44, actually measuring: 2298.31.
(5-3) Synthesis of X-3:
x-2(2.895g, 1.26mmol, obtained by combining two products) obtained by the method described in step (5-2), succinic anhydride (0.378g, 3.78mmol) and 4-dimethylaminopyridine (DMAP, 0.462g, 3.78mmol) were mixed and dissolved in 13ml of dichloromethane, DIEA (0.814g, 6.30mmol) was added thereto, and the reaction was stirred at 25 ℃ for 24 hours. Washing the reaction solution with 5ml0.5M triethylamine phosphate, extracting the aqueous phase with dichloromethane for 3 times, each time 5ml, combining the organic phases, evaporating to dryness under reduced pressure to obtain a crude product. The column purification uses 60g of 200-mesh 300-mesh normal phase silica gel, 1% of triethylamine neutralizes the acidity of the silica gel, the dichloromethane balances the column, the product is eluted by dichloromethane containing 1 ‰ of triethylamine and methanol in a ratio of 100:18-100:20, and the solvent is evaporated to dryness under reduced pressure to obtain 2.826g of pure product.1H NMR(400MHz,DMSO-d6)δ8.01–7.87(m,2H),7.50–7.42(m,1H),7.35–7.25(m,1H),7.17–7.09(m,1H),6.81–6.73(m,1H),5.31–5.22(m,1H),5.13–5.05(m,1H),4.50–4.43(m,1H),4.39–4.30(m,1H),4.14–4.05(m,2H),4.09–3.82(m,2H),3.86–3.71(m,5H),3.69–3.52(m,1H),3.56–3.43(m,2H),2.71–2.47(m,2H),2.46–2.33(m,1H),2.32–2.06(m,2H),2.06–1.90(m,10H),1.81(dd,J＝4.1,1.7Hz,3H),1.68–1.22(m,5H),1.03(t,J＝7.2Hz,2H).MS m/z：C112H158N9O48，[M+H]Theory: 2398.51, actually measuring: 2398.66. the structure of the obtained X-3 conjugated molecule is shown as a formula (605).
Preparation example 6 preparation of K-3 conjugate molecule (comparative conjugate molecule 1)
In this preparation example, a compound of comparative conjugate molecule 1 (hereinafter, also referred to as K-3 conjugate molecule) was synthesized in the following manner.
Figure BDA0001930334880000671
(6-1) Synthesis of K-1:
a-1(3.0g, 6.0mmol), PyBOP (6.2g, 12.0mmol), HOBt (1.6g, 2.0mmol) and diisopropylethylamine (DIPEA, 3.9g, 30.0mmol) obtained according to the method described in step (1-2) were added to 60ml of dichloromethane, and the reaction was stirred at room temperature for 10 minutes, followed by addition of the above solution to K-0(5.6g, 30.0mmol) and reaction at room temperature for 1 hour 50 minutes. The reaction mixture is poured into 30ml of saturated sodium bicarbonate solution, the aqueous phase is extracted 3 times with 30ml of dichloromethane each time, the organic phases are combined and washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated by filtration. Purifying by a 200-mesh 300-mesh normal-phase silica gel column, performing gradient elution by using dichloromethane, methanol and ammonia water (25wt percent) to obtain 10:2:0.1-4:4:1, collecting product eluent, concentrating to remove the solvent, and performing foaming drying by using a vacuum oil pump to obtain a white solid product K-12.2 g.1H NMR(400MHz,DMSO-d6)δ8.02(s,1H),7.43(d,J＝7.8Hz,2H),7.34–7.17(m,7H),6.87(d,J＝8.6Hz,4H),4.05(d,J＝5.2Hz,1H),3.74(s,6H),3.20–3.01(m,5H),2.60–2.38(m,12H),1.60–1.39(m,8H),1.24(s,1H).MSm/z：C33H47N4O5，[M+H]Theory: 579.35, actually measuring: 579.26.
(6-2) Synthesis of K-2:
GAL-5(483mg, 1.08mmol), 3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -one (359mg, 1.2mmol), diisopropylethylamine (DIPEA, 310mg, 2.4mmol) obtained by the method described in (1-1) was added to 3ml of dichloromethane, stirred at room temperature for 30 minutes, followed by addition of K-1(174mg, 0.3mmol) and reacted at room temperature for 16 hours. The reaction mixture is poured into 10ml of saturated sodium bicarbonate solution, the aqueous phase is extracted 3 times with 10ml each time of dichloromethane, the organic phases are combined and washed with 10ml of saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated by filtration. Purifying with 200-mesh 300-mesh normal phase silica gel column, eluting with dichloromethane and methanol at a ratio of 20:1, collecting product eluate, concentrating to remove solvent, and vacuum oil pump drying and foaming to obtain yellow solid product K-2205 mg.
(6-3) Synthesis of K-3:
k-2(205mg, 0.11mmol), succinic anhydride (22mg, 0.22mmol), 4-dimethylaminopyridine (DMAP, 27mg, 0.22mmol) and diisopropylethylamine (DIPEA, 71mg, 0.55mmol) were added to 1.1ml of dichloromethane, and the reaction was stirred at room temperature overnight. The reaction solution was washed 3 times with 0.5M triethylamine phosphate solution, 0.5ml each time, and the aqueous phase was back-extracted once with 0.5ml dichloromethane each time, dried over anhydrous sodium sulfate, concentrated to remove the solvent, dried by vacuum oil pump and foamed to give 218mg of K-3 conjugate molecule (comparative conjugate molecule 1) as a pale yellow solid product.
Preparation of 7B3-siHBa1 conjugate (conjugate 6)
In this preparation example, starting from the B-2 conjugate molecule (conjugate molecule 1), a B3-siHBa1 conjugate (hereinafter, also referred to as conjugate 6) was prepared in the following manner
(7-1) Synthesis of B-3 Compound:
Figure BDA0001930334880000681
in this step, the B-3 compound is prepared by attaching the B-2 conjugate molecule to a solid support.
Mixing the B-2 conjugated molecule (0.456g, 0.22mmol) obtained in the step (1-7), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 0.125g, 0.33mmol) and diisopropylethylamine (DIEA, 0.057g, 0.44mmol) and dissolving in 18ml acetonitrile, stirring at room temperature for 5 minutes to obtain a uniform solution, adding aminomethyl resin (1.76g, 100 mesh, 200 mu mol/g of amino loading, 400 mu mol/g of amino loading, purchased from Nankai Kaishika) into the reaction solution, starting shaking table reaction at 25 ℃, rotating speed 150 revolutions per minute, filtering after 18h of reaction, leaching the filter cake with DCM for 2 times, 50ml each time, leaching acetonitrile for 3 times, 50ml each time, leaching with diethyl ether for 1 time, vacuum drying for 2h with an oil pump to obtain a solid phase carrier connected with D-6, and then carrying out capping reaction according to the charge ratio shown in Table 5.
TABLE 5 Cap reaction feed ratio
Raw materials Weight (D) Specification of Batch number Manufacturer of the product
Cap1 40ml —— —— Self-made
Cap2 4.5ml —— —— Self-made
DMAP 0.022g Analytical purity I1422139 Aladdin
Acetonitrile 4.5ml Pure spectrum O15161001 Shanghai xing can
Wherein, Cap1 and Cap2 are capping reagent solutions, Cap1 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; cap2 is a 20% by volume acetic anhydride solution in acetonitrile;
adding Cap1, Cap2, 4-Dimethylaminopyridine (DMAP) and acetonitrile into the solid phase carrier connected with the D-6, starting table reaction at 25 ℃, rotating at 200 r/min for 5h, filtering the reaction solution, leaching the filter cake for 3 times by using acetonitrile, each time 50ml, filtering to dryness, drying overnight by using a vacuum oil pump, and obtaining 2.127g of a B-3 compound (namely, D-6 conjugated molecules connected with the solid phase carrier) with the loading of 106.59 mu mol/g. The structure of the B-3 compound is shown as a formula (701).
(7-2) Synthesis of sense Strand of B3-siHBa1 conjugate
In this step, the siRNA of the siRNA conjugate is sequence No. siHBa 1:
siHBa1
sense strand: 5'-CCUUGAGGCAUACUUCAAA-3' (SEQ ID NO:1),
antisense strand: 5'-UUUGAAGUAUGCCUCAAGGUU-3' (SEQ ID NO: 2);
the nucleoside monomers are linked one by one in the 3'-5' direction in the above sequence order using the initial cycle of the B-3 compound prepared in the above procedure by the method of solid phase synthesis of phosphoramidite nucleic acid. 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 Cap1 and Cap2 with a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence attached to the solid phase carrier is acetic anhydride, 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.
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. After the ammonia treatment, the product was dissolved with 0.4 ml/. mu.mol of N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, followed by addition of 0.3 ml/. mu.mol of triethylamine and 0.6 ml/. mu.mol of triethylamine trihydrofluoride to remove the protection of 2' -TBDMS on ribose. 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 92.4% as determined by ion exchange chromatography (IEX-HPLC); molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS) with theoretical value 7253.96, found value 7253.12.
Thus, in this step the B-2 conjugate molecule was ligated to the 3 'end of the resulting sense strand, resulting in siRNA sense strand S with the B-3 conjugate molecule conjugated to the end of siRNA 3'.
(7-3) Synthesis of antisense chain
In this step, a general solid phase carrier (UnyLinker) is usedTMloaded
Figure BDA0001930334880000691
HL solid supports, Kinovate Life Sciences), synthesized the antisense strand AS of the B3-siHBa1 conjugate. 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 synthesized sense strand, so that the siRNA 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). Theoretical 6675.04, found 6674.50.
(7-4) Synthesis of B3-siHBa1 conjugate
Mixing S chain and AS chain in equal molar ratio, dissolving in water for injection, heating to 95 deg.C, cooling at room temperature, and allowing them to form double chain structure via hydrogen bond.
After completion of the above synthesis, the conjugate was diluted to a concentration of 0.2mg/mL with ultrapure water (resistivity 18.2 M.OMEGA.. multidot.cm (25 ℃ C.)) which was manufactured by Milli-Q ultrapure water meter. Molecular weight determination was carried out using a LC-MS (Liquid Chromatography-Mass spectrometer, model: LCT Premier, available from Waters, Inc.). As a result, theoretical value S: 7253.96, AS: 6675.04, found S: 7253.24, AS: 6674.61, found to be consistent with the theoretical values, thereby confirming that the synthesized conjugate is the target designed double-stranded nucleic acid sequence with the D-6 conjugate molecule. The structure of the B3-siHBa1 conjugate (conjugate 6) is shown in formula (401).
Preparation example 8 preparation of siRNA conjugates of conjugates 7-27, 156-157, 32-95, 158-159, 100-129, 160-161, 134-151 and 162-163
The title respective siRNA conjugates were prepared using the same method as preparation example 7, except that: 1) the conjugated siRNA had the sequences corresponding to conjugates 7-27, 156-157, 32-95, 158-159, 100-129, 160-161, 134-151 and 162-163 shown in tables 6A-6D; 2) when the two nucleotides in the target sequence are connected by phosphorothioate, replacing the oxidation reaction step in the connection of the latter nucleotide in the two nucleotides by the following sulfurization reaction step; the conditions of each step of sulfuration reaction are the same, including the temperature of 25 ℃, the reaction time of 300 seconds, and the sulfuration reagent of hydrogenated flavonol. The molar ratio of the sulfurizing reagent to the nucleic acid sequence attached to the solid support in the coupling step is 120: 1. The reaction is carried out in a mixed solvent of acetonitrile and pyridine in a ratio of 1: 1; and 3) when the 2 '-position of all nucleotides in the target sequence is modified hydroxyl group, the cutting and deprotection condition does not include the step of removing the 2' -TBDMS protection on ribose. Thus, siRNA conjugates of conjugates 7-27, 156-157, 32-95, 158-159, 100-129, 160-161, 134-151 and 162-163 of the present disclosure were prepared and numbered according to tables 6A-6D, respectively. The molecular weight is detected by a liquid chromatograph-mass spectrometer, the measured value of the molecular weight of the conjugate is consistent with a theoretical value, and the structures of the conjugate and the conjugate are shown as a formula (401).
TABLE 6A siRNA conjugates
Figure BDA0001930334880000701
Figure BDA0001930334880000711
TABLE 6B
Figure BDA0001930334880000721
Figure BDA0001930334880000731
Figure BDA0001930334880000741
Figure BDA0001930334880000751
Figure BDA0001930334880000761
TABLE 6C
Figure BDA0001930334880000762
Figure BDA0001930334880000771
Figure BDA0001930334880000781
TABLE 6D
Figure BDA0001930334880000782
Figure BDA0001930334880000791
S: a sense strand; AS: antisense strand
Note: capital C, G, U, A indicates the base composition of the nucleotide; 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 to the right of the letter VP is a vinyl phosphate modified nucleotide; p represents that one nucleotide to the right of the letter P is a phosphate modified nucleotide; ps means that one nucleotide to the right of the letter Ps is a phosphorothioate modified nucleotide.
Wherein, the 2' -methoxyl modified uridine monomer (VP-Um) modified by vinyl phosphate is synthesized according to the following method:
Figure BDA0001930334880000801
(8-1) Synthesis of VP-U-2
The VP-U-2 molecule was synthesized as follows:
Figure BDA0001930334880000802
2 '-methoxy-modified uracil nucleotide (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 dichloromethane and washed with 300ml saturated sodium bicarbonate, the aqueous phase was extracted 3 times with 300ml each time of Dichloromethane (DCM), the organic phases were combined, washed with 5% oxalic acid until the pH of the aqueous phase was <5, and the crude VP-U-1 was obtained after evaporation of the solvent to dryness and used directly for the subsequent synthesis of VP-U-2.
After dissolving the VP-U-1 crude product with 100ml dichloromethane, stirring in an ice bath for 10 minutes, adding 450ml of 2% p-toluenesulfonic acid solution (the solvent is a methanol-dichloromethane mixed solvent with the volume ratio of 3: 7) refrigerated in a refrigerator at 4 ℃ in advance, and reacting for 10 minutes. The reaction was quenched with an additional 200ml of saturated sodium bicarbonate solution, and the organic phase was washed with a saturated aqueous solution of sodium bicarbonate to pH 8. The aqueous phases are combined, extracted 2 times with 200ml of dichloromethane each time, the organic phases are combined, washed once more with 200ml of saturated brine and the solvent is evaporated to dryness. Purifying by a 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 40.00g of a pure product VP-U-2. 1H NMR (400MHz, DMSO-d6) δ 7.96(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,1 ddh), 3.68(d, J ═ 11.8,7.0,4.6, 1H),3.96(t, J ═ 4.7Hz,1 ddh), 3.68 (m, 39H, 1H), MS (m, 8, 1H: C26H33N2O6Si, [ M + H ] +, theory: 497.21, actually measuring: 497.45.
(8-2) Synthesis of VP-U-4:
Figure BDA0001930334880000811
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. And dissolving tetraethyl methylenediphosphonate (21.44g, 74.4mmol) in 120ml of THF, cooling in an ice bath, adding t-BuOK (11.36g, 101.2mmol) at the ice bath temperature, reacting at the ice bath temperature for 10min, heating to room temperature, reacting for 0.5h, adding into the reaction solution, completing the addition for about 1h, reacting at the ice bath temperature for 1h, and heating to room temperature, and reacting 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 brine and the solvent is evaporated to dryness. Purifying with 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 with vacuum oil pump to obtain pure product VP-U-4(14.00 g). 1H NMR (400MHz, DMSO-d6) δ 7.96(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(t, J ═ 9.7,8.5,6.9Hz,2H), MS (t, J ═ 8.8, 1H), 1H, 1.05 (t, J ═ 4.8, 1H): C31H42N2O8PSi, [ M + H ] +, theory: 629.24, actually measuring: 629.51.
(8-3) Synthesis of VP-U-5:
Figure BDA0001930334880000812
VP-U-4(14.00g, 22.29mmol) was dissolved in 100ml tetrahydrofuran, triethylamine trihydrofluoric acid (17.96g, 111.45mmol) was added, and the reaction was stirred at room temperature for 20h to complete the reaction. The solvent was evaporated directly to dryness, dissolved in dichloromethane and evaporated to dryness 2 times using 50ml of dichloromethane each time to give the crude product. Purifying with 200-mesh 300-mesh normal phase silica gel column, loading petroleum ether into the column, performing gradient elution with petroleum ether, ethyl acetate, dichloromethane and methanol at a ratio of 1:1:1:0.05-1:1:1:0.25, collecting product eluent, evaporating the solvent under reduced pressure, and performing vacuum oil pump foaming and drying to obtain 6.70g of pure product VP-U-5. 1H NMR (400MHz, DMSO-d6) δ 7.96(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 (MS, J ═ 6.9,0.6, 6H, 1H/z: C15H24N2O8P, [ M + H ] +, theory: 391.13, actually measuring: 391.38.
(8-4) Synthesis of VP-U-6:
Figure BDA0001930334880000821
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) delta 150.34,150.29,17.07,15.50.MS m/z: C24H41N4O9P2, [ M + H ] +, theory: 591.23, actually measuring: 591.55. it shows that VP-U-6 is a target product VP-Um and participates in RNA strand synthesis as a nucleoside monomer.
The 5 '-phosphate modification was attached to the 5' end of the antisense strand using the following method:
the starting material was a phosphorylated structural monomer having the structure of formula CPR-I, supplied by suma, Cat # 13-2601-XX:
Figure BDA0001930334880000822
after all nucleoside monomers of the antisense chain are connected, according to the method of phosphoramidite nucleic acid solid phase synthesis, the CPR-I monomer is connected to the 5' terminal of the antisense chain through four steps of deprotection, coupling, capping and oxidation. Cleavage and deprotection were then carried out according to the following conditions to obtain the antisense strand:
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. After the ammonia treatment, the product was dissolved with 0.4 ml/. mu.mol of N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, followed by addition of 0.3 ml/. mu.mol of triethylamine and 0.6 ml/. mu.mol of triethylamine trihydrofluoride to remove the protection of 2' -TBDMS on ribose. 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.
In the case where the target product has a 5' -phosphorothioate modification, the same procedure as described above is used except that the sulfurization reaction is carried out under sulfurization reaction conditions instead of the oxidation reaction conditions described above at the time of ligation.
For the sense strand and the antisense strand synthesized as described above, purity was checked using ion exchange chromatography (IEX-HPLC), and molecular weight was analyzed by liquid mass spectrometry (LC-MS), confirming that the synthesized nucleic acid sequences were sirnas corresponding to each conjugate in tables 6A to 6D and the conjugate of comparative example.
Preparation example 9 Synthesis of siRNA conjugates of conjugates 28-31, 96-99, 130-133 and 152-155 and comparative conjugates 2-5
siRNA conjugates of conjugates 28-31, 96-99, 130-133 and 152-155 and comparative conjugates 2-5 were prepared in the same manner as in preparation example 7, except that: 1) the conjugate molecules of the conjugates obtained in preparative examples 2-6 and the comparative conjugates described above were substituted for the B-2 conjugate molecules (e.g., when the D-6 conjugate molecule of conjugate 2 was substituted for the B-2 conjugate molecule, the D7 conjugates of conjugates 28, 96, 130 and 152 were obtained, when the C-2 conjugate molecule of conjugate 3 was substituted for the D-6 conjugate molecule, the C3 conjugates of conjugates 29, 97, 131 and 153 were obtained, and so on); 2) the conjugated siRNA had the sequences shown in tables 6A-6D corresponding to conjugates 28-31, 96-99, 130-155 and 152-155 and comparative conjugates 2-5; 3) when phosphorothioate linkage is present between two nucleotides in the target sequence, the step of the sulfurization reaction described in preparation example 8 is used instead of the step of the oxidation reaction in the linkage of the latter of the two nucleotides; and 4) when the 2 '-position of all nucleotides in the target sequence is modified hydroxyl group, the cutting and deprotection condition does not include the step of removing the 2' -TBDMS protection on ribose. Thus, siRNA conjugates giving conjugates 28-31, 96-99, 130-133 and 152-155 of the present disclosure and comparative conjugates 2-5 were prepared and numbered as per tables 6A-6D, respectively. Subsequently, the molecular weights of the single strand and the double strand are respectively detected by a liquid chromatograph-mass spectrometer, the measured value is consistent with the theoretical value, the structures of the synthesized conjugate siRNA conjugates are respectively shown as formulas (402), (403), (404) and (405), and the structures of the comparative conjugates are respectively shown as formula (901):
Figure BDA0001930334880000831
experimental example 1 this experiment illustrates the animal level toxicity of the siRNA conjugates of the present disclosure.
On C57BL/6J mice (purchased from Beijing Wintolite laboratory animal technology Co., Ltd.), 300mg/kg (calculated as siRNA) of the siRNA conjugates 21-31, 156-157, 83, 120 and 148 were subcutaneously and singly administered to each mouse, respectively, and no animal death occurred or clinical symptoms associated with adverse drug reactions were observed for 14 days continuously, and no abnormality was found in gross dissection. Thus, the above results indicate that the siRNA conjugates of the present disclosure have lower toxicity at an animal level.
In the following experimental examples 2 to 5, the properties and effects of the siRNA conjugates of tables 6A to 6D were experimentally verified according to the siRNA target positions and sequence associations, respectively.
Experimental example 2 Effect experiment of siRNA conjugates of Table 6A
Experimental example 2-1 this experiment demonstrates the inhibitory efficiency of the siRNA conjugates of table 6A on HBV mRNA expression levels in vitro (in vitro).
HepG2.2.15 cells were transfected in vitro with siRNA conjugates of conjugates 7-8, 13-15, 21-22, 24-31, 156-157 and control conjugate 2 at final siRNA concentrations of 50nM, 10nM and 1nM, respectively. Each concentration was 3 replicates, and at least 3 experiments were repeated. 48 hours after transfection, the expression level of HBV mRNA in the cells harvested above was determined using real-time fluorescent quantitative PCR (real-time fluorescent qPCR), specifically: total RNA was extracted using RNeasy Mini Kit (QIAGEN, cat.74106) according to the instructions, and the extracted total RNA was reverse-transcribed into cDNA, followed by measuring the inhibitory efficiency of siRNA against HBV mRNA expression of HepG2.2.15 cells by the fluorescent quantitative PCR method.
In the fluorescent quantitative PCR method, HBV and GAPDH were detected using a primer for HBV and a primer for GAPDH with the GAPDH gene as an internal reference gene, respectively, and the primer sequences are shown in table 7A:
TABLE 7A sequence of detection primers
Figure BDA0001930334880000841
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 GAPDH in test group)/(copy number of HBV gene in mock group/copy number of GAPDH in mock group) × 100%,
the inhibition rate of the siRNA to mRNA was then calculated according to the following formula:
the mRNA inhibition rate (1-HBV gene expression residual) × 100%,
wherein each test group was HepG2.2.15 cells treated with the siRNA conjugates listed in Table 6A, respectively, including conjugates 7-8, 13-15, 21-22, 24-31, 156-157 and comparative conjugate 2, and the mock group was HepG2.2.15 cells without any siRNA treatment.
The following table 8A shows the results of measuring the inhibitory activity of the siRNA conjugates listed in table 6A and the comparative conjugates on HBV mRNA expression in hepg2.2.15 cells.
Table 8A siRNA conjugates in vitro activity assay
Figure BDA0001930334880000842
Figure BDA0001930334880000851
As can be seen from the results of table 8A, each siRNA conjugate in table 6A showed excellent HBV gene expression inhibitory activity at a cellular level.
Experimental examples 2-2 this experiment demonstrates the stability of the siRNA conjugates of Table 6A in human plasma in vitro
Conjugates 13-14 and 21-25 (each 0.9% NS solution, siRNA concentration 20. mu.M, 12. mu.l) were mixed with 108. mu.L of 90% Human plasma (diluted with PBS), incubated at 37 ℃ constant temperature, 10. mu.L of each sample was taken at 0, 8, 24, and 48 hours, immediately frozen in a freezer with liquid nitrogen, and after sampling at each time point, 10. mu.L of each sample was diluted 5-fold with 1 × PBS (pH7.4), and at the same time, equimolar amounts of siRNA conjugates (siRNA concentration 2. mu.M, 2. mu.L) were mixed with 8. mu.L of 1 × PBS (pH7.4) to prepare 10. mu.L of samples not treated with Human plasma, designated "untreated". 20 wt.% of non-denatured polyacrylamide gel, which was mixed with 4. mu.L of loading buffer (20mM EDTA, 36 wt.% glycerol, 0.06 wt.% of bromophenol blue), loaded, and stained with 80mA, stained with Sybrene dye after electrophoresis for 52 minutes, stained with a constant current electrophoresis (Invitrogen) for 52 minutes, and stained with a staining time point 5 minutes, indicated by Invitrogen (Invitrogen) and electrophoresis time point).
Table 9A shows the results of semi-quantitative determination of the stability of the test siRNA conjugates and the control siRNA conjugates listed in table 6A in human plasma in vitro. The results are expressed as the ratio of the longest fragment remaining after incubation of the test and control siRNA conjugates with human plasma to the longest fragment of untreated siRNA (RL).
Table 9A plasma stability quantification of siRNA conjugates
Figure BDA0001930334880000852
As can be seen from the results of table 9A, each siRNA conjugate has excellent stability in plasma.
Experimental examples 2-3 this experiment demonstrates the inhibitory efficiency of siRNA conjugates of Table 6A on the expression level of HBV Mrna in vivo (in vivo)
In this experimental example, the siRNA conjugates of examples 13-14, 21-22 and 156-157 and comparative conjugate 2 were examined for the inhibition efficiency of the expression level of HBV mRNA in HBV transgenic mouse C57BL/6J-Tg (Alb1HBV)44 Bri/J.
HBV transgenic mouse C57BL/6J-Tg (Alb1HBV)44Bri/J used in this experimental example was purchased from the laboratory animal sciences of the university of Beijing.
First, C57BL/6J-Tg (Alb1HBV)44Bri/J mice (both female) were numbered for each group of 5 mice according to the siRNA conjugates in Table 3A, and PBS controls were added. All animals were dosed by weight in a single dose (subcutaneous dose) of 1mg/kg and 5ml/kg volume. Animals were sacrificed on day 14 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.
The expression level of HBV mRNA in liver tissue is detected by real-time fluorescent quantitative PCR, specifically, the extracted total RNA is reverse transcribed into cDNA by using ImProm-IITM reverse transcription kit (Promega corporation) according to the instruction, and then the inhibition efficiency of siRNA to HBV mRNA expression in liver tissue is detected by using fluorescent quantitative PCR kit (Beijing kang, century Biotechnology Co., Ltd.) in the fluorescent quantitative PCR method, β -actin (β -actin) gene is used as an internal reference gene, and HBV and β -actin are detected by using a primer for HBV and a primer for β -actin respectively.
See table 10A for sequences of detection primers.
TABLE 10A sequence of detection primers
Figure BDA0001930334880000861
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 β -actin in test group)/(copy number of HBV gene in control group/copy number of β -actin in control group) is × 100%,
the mRNA inhibition rate was then calculated according to the following formula:
the mRNA inhibition rate (1-HBV gene expression residual) × 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 11A below.
TABLE 11A inhibition of HBV mRNA expression in mouse liver by siRNA conjugates
Figure BDA0001930334880000862
As can be seen from the above results, on the one hand, the conjugates of the various embodiments of the present disclosure all showed high HBV mRNA inhibitory activity in mice; on the other hand, although the results of Table 8A indicate that the conjugate of comparative conjugate 2 showed similar in vitro HBV gene inhibitory activity as the conjugate of the present disclosure, it can be seen from the results of Table 11A that the siRNA conjugates of examples 21 and 156-157 showed significantly higher inhibitory rate of HBV gene mRNA in vivo experiments in hepatitis B mouse liver tissue than the comparative conjugate 2 having different conjugate groups under the same nucleic acid sequence and the same base modification scheme. This also demonstrates that the siRNA conjugates of the present disclosure have good in vivo delivery efficiency.
Experimental examples 2-4 this experiment demonstrates the time-dependent measurement of serum HBsAg expression level and inhibitory efficiency of HBV DNA by siRNA of siRNA conjugates of Table 6A in HBV transgenic mice
AAV-HBV model, rAAV8-1.3HBV, type D (ayw), was prepared according to literature methods (Dong Xiao rock et al, Chin J Biotech 2010, May 25; 26(5):679-686), available from Acanthopanax beijing and molecular medicine research institute, Inc., 1 × 1012viral genome (v.g.)/mL, lot No. 2016123011 dilution with sterile PBS to 5 × 10 before experiment11v.g./mL. 200. mu.L/mouse, i.e. 1 × 10/mouse11v.g. On day 28 after virus injection, all mice were tested for HBsAg and HBV DNA by orbital bleeding (approximately 100 μ L) for serum collection. After successful animal modeling, animals were randomized into groups (5 per group) based on serum HBsAg content and were given siRNA conjugates of conjugates 13-14, 21-22, 24 and 156, respectively, along with a PBS blank. All animals were dosed as single subcutaneous doses of 3mg/kg and 5ml/kg volume based on body weight. Mice were bled from the orbital venous plexus on days 7, 14, 21, 28, 56, 84, 112, 140 before and after dosing, and serum HBsAg levels were measured at each time point.
The blood is taken from orbit about 100 μ l each time, and the blood serum is not less than 20 μ l after centrifugation. Detecting the expression level of the HBsAg in serum by using an HBsAg CLIA kit (AnTurkey, CL 0310); serum DNA was extracted with reference to QIAamp 96DNA Blood Kit instructions, and quantitative PCR was performed to detect the expression level of HBV DNA.
The HBsAg inhibition rate is calculated by the following equation:
the HBsAg inhibition rate (1-HBsAg content after administration/HBsAg content before administration) × 100% where HBsAg content is expressed in terms of how many equivalents (UI) of HBsAg per milliliter (ml) of serum.
The HBV DNA inhibition rate is calculated as follows:
HBV DNA inhibition rate (1-HBV DNA content after administration/HBV DNA content before administration) × 100%.
Wherein, HBV DNA content is expressed as how many copies of HBV DNA are contained per milliliter (ml) of serum.
The results are shown in tables 12A and 13A below.
TABLE 12 inhibition of HBsAg expression in mouse serum by siRNA conjugates
Figure BDA0001930334880000871
As can be seen from the results in table 12A, the PBS negative control group did not show any inhibition at various time points after administration; in contrast, the siRNA conjugates of each example exhibited excellent HBsAg inhibitory effects at different time points after administration. In particular, conjugates 21 and 156 continued to exhibit a high serum HBsAg inhibition rate for a period of up to 140 days, indicating that they were able to stably and efficiently inhibit the expression of HBV genes for a long period of time.
TABLE 13 inhibition of HBV DNA expression in mouse sera by siRNA conjugates
Figure BDA0001930334880000872
As can be seen from the results of table 13A, the siRNA conjugates of the examples also showed high-efficiency HBV DNA expression inhibition, and maintained high inhibition rate for as long as 84 days.
Experimental example 3 Effect experiment of siRNA conjugates of Table 6B
Experimental example 3-1 this experiment demonstrates the inhibitory efficiency of the siRNA conjugates of table 6B on HBV mRNA expression levels in vitro (in vitro).
HepG2.2.15 cells were transfected in vitro with siRNA conjugates 53-56, 81-92, 158-159 and control conjugate 3 at final siRNA concentrations of 50nM, 10nM and 1nM, respectively. Each concentration was 3 replicates, and at least 3 experiments were repeated. 48 hours after transfection, the expression level of HBV mRNA in the cells harvested above was determined using real-time fluorescent quantitative PCR (real-time fluorescent qPCR), specifically: total RNA was extracted using RNeasy Mini Kit (QIAGEN, cat.74106) according to the instructions, and the extracted total RNA was reverse-transcribed into cDNA, followed by measuring the inhibitory efficiency of siRNA against HBV mRNA expression of HepG2.2.15 cells by the fluorescent quantitative PCR method.
In the fluorescent quantitative PCR method, HBV and GAPDH were detected using a primer for HBV and a primer for GAPDH with the GAPDH gene as an internal reference gene, respectively, and the primer sequences are shown in table 7B:
TABLE 7B sequence of detection primers
Figure BDA0001930334880000881
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 GAPDH in test group)/(copy number of HBV gene in mock group/copy number of GAPDH in mock group) × 100%,
the inhibition rate of the siRNA to mRNA was then calculated according to the following formula:
the mRNA inhibition rate (1-HBV gene expression residual) × 100%,
wherein each test group was HepG2.2.15 cells treated with the siRNA conjugates listed in Table 6B, respectively, including the siRNA conjugates of conjugates 53-56, 81-92, 158-159 and the control siRNA conjugate of comparative conjugate 3; mock groups were hepg2.2.15 cells without any siRNA treatment.
Table 8B below shows the results of measuring the inhibitory activity of the test siRNA conjugates listed in table 6B and the control siRNA conjugates on HBV mRNA expression in hepg2.2.15 cells.
Table 8B siRNA conjugates in vitro activity assay
Figure BDA0001930334880000882
Figure BDA0001930334880000891
As can be seen from the results of table 8B, each siRNA conjugate in table 6B showed excellent HBV gene expression inhibitory activity at a cellular level.
Experimental examples 3-2 this experiment demonstrates the stability of the siRNA conjugates of Table 6B in human plasma in vitro
The siRNA conjugates of conjugates 53 to 58 and 83 to 88 and comparative conjugate 3 (siRNA concentrations were 20. mu.M and 12. mu.l each) were mixed with 108. mu.L of 90% Human plasma (diluted with PBS), incubated at 37 ℃ at constant temperature, 10. mu.L of each sample was taken out at 0, 8, 24 and 48 hours, immediately frozen in a freezer at-80 ℃ with liquid nitrogen, 10. mu.L of each sample was taken after diluting 1 × PBS (pH7.4) 5-fold at each time point, 20 wt% of a non-denatured polyacrylamide gel was prepared, and the above samples were mixed with 4. mu.L of a loading buffer (20mM EDTA, 36 wt% glycerol, 0.06 wt% bromophenol blue), loaded, subjected to electrophoresis at a constant current of 80mA for about 60 minutes, and after the electrophoresis, stained with 1 × Sybr Gold dye (Invitrogen, Cat.11494) for 15 minutes and imaged, with the results shown in Table 9B.
Table 9B shows the results of semi-quantitative determination of the stability of the siRNA conjugates listed in table 6B versus the comparative conjugates in human plasma in vitro. The results are expressed as the Ratio (RL) of the longest fragment remaining after incubation of the siRNA conjugate and the comparative siRNA conjugate with human plasma to the longest fragment of the untreated siRNA.
Table 9B plasma stability quantification of siRNA conjugates
Figure BDA0001930334880000892
As can be seen from the results of table 9B, each siRNA conjugate has excellent stability in plasma.
Experimental examples 3-3 this experiment demonstrates the inhibitory efficiency of siRNA conjugates of Table 6B on HBV mRNA expression levels in vivo (in vivo)
In this experimental example, the siRNA conjugates of conjugates 53-54, 57-58, 81-88 and comparative conjugate 3 were examined for the inhibitory efficiency of the expression amount of HBV mRNA in HBV transgenic mouse C57BL/6J-Tg (Alb1HBV)44 Bri/J.
HBV transgenic mouse C57BL/6J-Tg (Alb1HBV)44Bri/J used in this experimental example was purchased from the laboratory animal sciences of the university of Beijing.
First, C57BL/6J-Tg (Alb1HBV)44Bri/J mice were randomly grouped by serum HbsAg content (both female), 5 mice per group were numbered according to the siRNA conjugates in Table 6B, and PBS controls were added. All animals were dosed by weight in a single dose (subcutaneous dose) of 1mg/kg and 5ml/kg volume. Animals were sacrificed on day 14 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.
The expression level of HBV mRNA in liver tissue is detected by real-time fluorescent quantitative PCR, specifically, the extracted total RNA is reverse transcribed into cDNA by using ImProm-IITM reverse transcription kit (Promega corporation) according to the instruction, and then the inhibition efficiency of siRNA to HBV mRNA expression in liver tissue is detected by using fluorescent quantitative PCR kit (Beijing kang, century Biotechnology Co., Ltd.) in the fluorescent quantitative PCR method, β -actin (β -actin) gene is used as an internal reference gene, and HBV and β -actin are detected by using a primer for HBV and a primer for β -actin respectively.
See table 10B for sequences of detection primers.
TABLE 10B sequence of detection primers
Figure BDA0001930334880000901
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 β -actin in test group)/(copy number of HBV gene in control group/copy number of β -actin in control group) is × 100%,
the mRNA inhibition rate was then calculated according to the following formula:
the mRNA inhibition rate (1-HBV gene expression residual) × 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 11B below.
TABLE 11B inhibition of HBV mRNA expression in mouse liver by siRNA conjugates
Figure BDA0001930334880000902
As can be seen from the above results, on the one hand, the conjugates of the various embodiments of the present disclosure all showed high HBV mRNA inhibitory activity in mice; on the other hand, although the results of table 8B indicate that comparative conjugate 3 shows similar in vitro HBV gene inhibitory activity to the conjugate of the present disclosure, it can be seen from the results of table 11B that conjugate 83 shows significantly higher inhibitory rate of HBV gene mRNA in vivo experiment in hepatitis B mouse liver tissue compared to comparative conjugate 3 with different conjugate group under the same nucleic acid sequence and the same base modification scheme. This also demonstrates that the siRNA conjugates of the present disclosure have good in vivo delivery efficiency.
Experimental examples 3-4 this experiment demonstrates the time-dependent measurement of serum HBsAg expression level and inhibitory efficiency of HBV DNA by siRNA of siRNA conjugates of Table 6B in HBV transgenic mice
AAV-HBV model, rAAV8-1.3HBV, type D (ayw), was prepared according to literature methods (Dong Xiao rock et al, Chin J Biotech 2010, May 25; 26(5):679-686), available from Acanthopanax beijing and molecular medicine research institute, Inc., 1 × 1012viral genome (v.g.)/mL, lot No. 2016123011 dilution with sterile PBS to 5 × 10 before experiment11v.g./mL. Each mouse was injected with 200. mu.L,i.e. 1 × 10 per mouse11v.g. On day 28 after virus injection, all mice were tested for HBsAg and HBV DNA by orbital bleeding (approximately 100 μ L) for serum collection. After successful animal modeling, the animals were randomized into groups (5 per group) based on serum HBsAg levels and were given siRNA conjugates 87-88 and 158-159, respectively, and PBS blanks. All animals were dosed as single subcutaneous doses of 3mg/kg and 5ml/kg volume based on body weight. Mice were bled from the orbital venous plexus on days 7, 14, 21, 28, 56, and 84 before and after dosing, and serum HBsAg levels were measured at each time point.
The blood is taken from orbit about 100 μ l each time, and the blood serum is not less than 20 μ l after centrifugation. Detecting the expression level of the HBsAg in serum by using an HBsAg CLIA kit (AnTurkey, CL 0310); serum DNA was extracted with reference to QIAamp 96DNA Blood Kit instructions, and quantitative PCR was performed to detect the expression level of HBV DNA.
The HBsAg inhibition rate is calculated by the following equation:
the HBsAg inhibition rate (1-HBsAg content after administration/HBsAg content before administration) × 100% where HBsAg content is expressed in terms of how many equivalents (UI) of HBsAg per milliliter (ml) of serum.
The HBV DNA inhibition rate is calculated as follows:
HBV DNA inhibition rate (1-HBV DNA content after administration/HBV DNA content before administration) × 100%.
Wherein, HBV DNA content is expressed as how many copies of HBV DNA are contained per milliliter (ml) of serum.
The results are shown in tables 12B and 13B below.
TABLE 12 inhibition of HBsAg expression in mouse serum by siRNA conjugates
Figure BDA0001930334880000911
As can be seen from the results in table 12B, the PBS negative control group did not show any inhibition at various time points after administration; in contrast, the siRNA conjugates of each conjugate exhibited excellent HBsAg inhibitory effects at different time points after administration.
TABLE 13B inhibition of HBV DNA expression in mouse sera by siRNA conjugates
Figure BDA0001930334880000912
As can be seen from table 13B, the siRNA conjugates of the respective examples also showed highly efficient HBV DNA expression inhibition, similarly to the HBsAg inhibitory effect, and the inhibition rate remained substantially stable for as long as 84 days.
Experimental example 4 Effect test of siRNA conjugates of Table 6C
Experimental example 4-1 this experiment demonstrates the efficiency of the inhibition of ANGPTL3mRNA expression levels in vitro (in vitro) by siRNA conjugates of table 6C.
The human hepatoma cell line Huh7 was transfected in vitro with siRNA conjugates of conjugates 100-107, 116-117, 124-133 and 160-161 and comparative conjugate 4, the final concentrations (in terms of siRNA) of the siRNA conjugates were 5nM, 0.5nM and 0.05nM, respectively, and 3 replicates of each concentration were performed for at least 3 replicates.
Specifically, Huh7 was seeded at a density of 4 × 10 onto 24-well plates in DMEM complete medium containing 10% fetal bovine serum5Cells/well, 0.5mL of medium per well, incubated overnight at 37 ℃.
The cell culture medium in the 24-well plate was aspirated away, and 0.5mL of Opti-MEM serum-free medium was added to each well. mu.L of siRNA conjugates at a concentration (by siRNA amount) of 0.02. mu.M, 0.2. mu.M and 2. mu.M, respectively, were diluted with 50. mu.L of Opti-MEM serum-free medium; mu.L of LipofectamineTM2000(Invitrogen corporation) in 50. mu.L of Opti-MEM serum-free medium, mixed and incubated at room temperature for 5 minutes; mixing diluted siRNA conjugates and diluted LipofectamineTM2000, gently mixed and left to stand at room temperature for 20 minutes to allow formation of the transfection complex. The above final mixed solution was added to a 24-well plate seeded with Huh7 cells at 100. mu.L per well. The final concentrations of siRNA conjugates were 0.05nM, 0.5nM, 5 nM. The cells were cultured at 37 ℃ for 4 hours, and 1mL of DMEM complete medium containing 10% fetal bovine serum was added to each well, and the culture was continued overnight at 37 ℃.
The expression level of ANGPTL3mRNA in Huh7 cells transfected with each siRNA conjugate was measured by Real-Time Quantitative PCR (Quantitative Real-Time PCR). The specific procedures were that after culturing the transfected cells for 24 hours, total RNA in the cells was extracted using RNAVzol (Vigorous, cat # N002), 1. mu.g of total RNA was reverse-transcribed according to the method of use of reverse transcription kit (Promega, cat # A3500) to obtain cDNA, 2 × Ultra SYBR mix (with ROX) (Beijing Kangkang is Biotech Co., Ltd., cat # CW0956) kit was used, and the expression level of ANGPTL3mRNA was measured using cDNA as a template according to the procedures described in the instructions.A PCR primer for amplifying ANGPTL3 and β -actin as an internal reference gene is shown in Table 7C.
TABLE 7C sequence of detection primers
Figure BDA0001930334880000921
The inhibition ratio of siRNA to the expression level of ANGPTL3mRNA was calculated by the following equation, [1- (test group ANGPTL3mRNA expression amount/test group β -Actin mRNA expression amount)/(mock group ANGPTL3mRNA expression amount/mock group β -Actin mRNA expression amount) ] ×%. wherein each test group was Huh7 cells treated with siRNA conjugates listed in Table 3E, respectively, the siRNA conjugates comprising the siRNA conjugates of conjugates 100-.
Table 8C siRNA conjugates in vitro activity assay
Figure BDA0001930334880000922
Figure BDA0001930334880000931
From the results in table 8C, it can be seen that at each concentration, each siRNA conjugate in table 6C showed excellent inhibitory activity for ANGPTL3mRNA expression at the cellular level, and at 5nM, the inhibition rate of the siRNA conjugate reached more than 50%, and some conjugates reached more than 70%.
Experimental examples 4-2 this experiment demonstrates the stability of the siRNA conjugates of Table 6C in human plasma in vitro
The siRNA conjugates of the conjugate 124-125 and the comparative conjugate 4 (20. mu.M, 12. mu.l in terms of siRNA concentration) were mixed with 108. mu.L of 90% Human plasma (diluted with PBS), incubated at 37 ℃ at constant temperature, 10. mu.L of each sample was taken out at 0, 8, 24, and 48 hours, immediately frozen in a freezer at-80 ℃ with liquid nitrogen, 10. mu.L of each sample was taken after diluting 1 × PBS (pH7.4) 5-fold at each time point, 20 wt% of a native polyacrylamide gel was prepared, and the above samples were mixed with 4. mu.L of a loading buffer (20mM EDTA, 36 wt% glycerol, 0.06 wt% bromophenol blue), loaded, and electrophoresed under a constant current of 80mA for about 60 minutes, and after the electrophoresis, stained with 1 × Sybr Gold dye (Invitrogen, Cat.11494) for 15 minutes, and the results are shown in Table 9C.
Table 9C shows the results of semi-quantitative determination of the stability of the siRNA conjugates listed in table 6C versus the comparative siRNA conjugates in human plasma in vitro. The results are expressed as the Ratio (RL) of the longest fragment remaining after incubation of siRNA conjugate and comparative siRNA conjugate with human plasma to the longest fragment of untreated siRNA.
Table 9C quantification of plasma stability of siRNA conjugates
Figure BDA0001930334880000932
As can be seen from the results of table 9C, the siRNA conjugates of the present disclosure have excellent stability in plasma with little degradation within 48 hours.
Experimental examples 4-3 this experiment demonstrates the efficiency of inhibition of the expression level of ANGPTL3mRNA by siRNA conjugates of Table 6C in vivo (in vivo)
In this experimental example, the siRNA conjugates of conjugates 120, 123, 124, 160-161 and comparative conjugate 4 were examined for their inhibition of the expression level of ANGPTL3 in liver tissues in normal BALB/c mice.
Normal BALB/c mice (purchased from Experimental animals and technologies, Inc., Viton, Beijing) 6-8 weeks old were randomly grouped into 6 mice (males and females) per group, and siRNA conjugates of conjugates 120, 123, 124, 160, 161 and control conjugate 4, and PBS were administered to each group of mice, respectively. All animals were dosed by weight and given a single subcutaneous injection, with a siRNA conjugate dose (based on the amount of siRNA) of 3mg/kg and a dosing volume of 10 mL/kg. Mice were sacrificed 14 days after administration, livers were collected and stored with RNA later (Sigma Aldrich company); liver tissue was homogenized using a tissue homogenizer and total RNA was extracted using Trizol (Thermo Fisher Co.) according to standard procedures for total RNA extraction.
Detecting the expression level of ANGPTL3mRNA in the liver tissue by adopting real-time fluorescent quantitative PCR, specifically: using ImProm-IITMIn the quantitative fluorescence PCR method, an β -actin (β -actin) gene was used as an internal reference gene, and ANGPTL3 and ANGPTL β -actin were detected using a primer for ANGPTL3 and a primer for β -actin, respectively.
See table 10C for sequences of detection primers.
TABLE 10C sequence of detection primers
Figure BDA0001930334880000941
The inhibition rate of siRNA on the expression level of ANGPTL3mRNA was calculated according to the following equation of [1- (expression amount of test group ANGPTL3 mRNA/expression amount of test group β -Actin mRNA)/(expression amount of control group ANGPTL3 mRNA/expression amount of control group β -Actin mRNA) ] × 100% where the control group is the control group mice to which PBS was applied in the present experiment, and each test group is the administration group mice to which different siRNA conjugates were applied, respectively, and the results are shown in table 11C below.
TABLE 11C inhibition of ANGPTL3mRNA expression in mouse liver by siRNA conjugates
Figure BDA0001930334880000942
As can be seen from the results in Table 11C, on the one hand, the siRNA conjugates of the conjugates 120, 123, 124, 160-161 of the present disclosure all showed extremely high inhibitory activity of ANGPTL3mRNA compared to PBS; on the other hand, in the case of the same nucleic acid sequence and the same modification scheme, the siRNA conjugates represented by the conjugates 124 and 160-161 showed higher mRNA inhibition rate in vivo experiments compared to the comparative conjugate 4 having a different conjugate group, which also indicates that the siRNA conjugates of the present disclosure have good in vivo delivery efficiency.
Experimental examples 4-4 this experiment demonstrates the efficiency of inhibition of the expression level of ANGPTL3mRNA and the effect on blood lipids by siRNA conjugates of Table 6C in vivo (in vivo)
In this experimental example, the siRNA conjugates of example 120(B3-siAN1M1SP) and example 124(B3-siAN1M3SP) were examined for their inhibitory rate on the expression level of ANGPTL3 in liver tissues and their influence on the total Cholesterol (CHO), Triglyceride (TG) and low-density lipoprotein (LDL-c) contents in serum in ob/ob model mice.
6-8 week old ob/ob female mice (purchased from Kyowa Kavens laboratory animals Co., Ltd.) were randomly divided into 5 groups of 5 mice each, grouped as follows: (1) a PBS control group; (2) conjugate 1203 mg/kg group; (3) conjugate 1243 mg/kg group; (4) conjugate 1201 mg/kg group; (5) conjugate 1241 mg/kg group. All animals were dosed by weight in a single subcutaneous dose of 10 mL/kg.
Orbital bleeds (approximately 100 μ L) were taken 2 days before dosing (noted-2 days), and 7, 14, 21, 28, 35, 42, 49 days after dosing, respectively, for blood lipid levels.
Mice were sacrificed on day 49, livers were collected and stored with RNA laters (Sigma Aldrich); liver tissue was homogenized using a tissue homogenizer and total RNA was extracted using Trizol (Thermo Fisher Co.) according to standard procedures for total RNA extraction.
The expression level of ANGPTL3mRNA in liver tissue was measured by real-time fluorescent quantitative PCR in the same manner as in Experimental example 4-3. The results are shown in table 12C below.
TABLE 12 inhibition of ANGPTL3mRNA expression in mouse liver by siRNA conjugates
Figure BDA0001930334880000951
Blood collected from the orbit was centrifuged to obtain serum, and the serum was further measured for the contents of total Cholesterol (CHO), Triglyceride (TG) and low-density lipoprotein (LDL-C) using a PM1P000/3 full-automatic serum biochemical analyzer (SABA, italy), and the blood lipid results were standardized, and the inhibition ratio of the blood lipid level was calculated according to the following equation (1-blood lipid content in test group after administration/blood lipid content in test group before administration) × 100%. blood lipid means total cholesterol, triglyceride or low-density lipoprotein, and the measurement results are shown in tables 13C, 14C and 15C below.
TABLE 13C Effect of siRNA conjugates on Total Cholesterol expression levels in mouse serum
Figure BDA0001930334880000952
TABLE 14C Effect of siRNA conjugates on triglyceride expression levels in mouse serum
Figure BDA0001930334880000961
TABLE 15 influence of siRNA conjugates on the expression level of low density lipoprotein in mouse serum
Figure BDA0001930334880000962
As can be seen from the results in tables 13C, 14C, and 15C above, the siRNA conjugates of conjugate 120 and conjugate 124 at different doses were able to significantly inhibit the expression of ANGPTL3 in mouse liver tissues, and there was a significant dose response; the siRNA conjugate of conjugate 120(B3-siAN1M1SP) has 48.9% inhibition rate on the expression of ANGPTL3 gene at a low dose of 1 mg/kg; under the high dose of 3mg/kg, the inhibition rate of the gene expression of ANGPTL3 is as high as 80.8%; the results of monitoring the content of CHO, TG and LDL-c in the serum of the mice show that the content of CHO, TG and LDL-c in the serum of the mice treated by the siRNA conjugate of the conjugate 120 or the conjugate 124 is obviously reduced, and the serum still shows higher blood fat reduction effect at least at 49 days.
Experimental example 5 Effect test of siRNA conjugates of Table 6D
Experimental example 5-1 this experiment demonstrates the efficiency of the siRNA conjugates of table 6D in inhibiting APOC3mRNA expression levels in vitro (in vitro).
The human hepatoma cell line Huh7 was transfected in vitro with the siRNA conjugates of conjugates 134-, 146-, 151-, 162-163 and comparative conjugate 5 at final concentrations (based on the amount of siRNA) of 5nM, 0.5nM and 0.05nM, respectively, and 3 replicates per concentration, and the assay was repeated at least 3 times.
Specifically, Huh7 was seeded at a density of 4 × 10 onto 24-well plates in DMEM complete medium containing 10% fetal bovine serum5Cells/well, 0.5mL of medium per well, incubated overnight at 37 ℃.
The cell culture medium in the 24-well plate was aspirated away, and 0.5mL of Opti-MEM serum-free medium was added to each well. mu.L of siRNA conjugates at a concentration (by siRNA amount) of 0.02. mu.M, 0.2. mu.M and 2. mu.M, respectively, were diluted with 50. mu.L of Opti-MEM serum-free medium; mu.L of LipofectamineTM2000(Invitrogen corporation) in 50. mu.L of Opti-MEM serum-free medium, mixed and incubated at room temperature for 5 minutes; mixing diluted siRNA conjugates and diluted LipofectamineTM2000, gently mixed and left to stand at room temperature for 20 minutes to allow formation of the transfection complex. The above final mixed solution was added to a 24-well plate seeded with Huh7 cells at 100. mu.L per well. The final concentrations of siRNA conjugates were 0.05nM, 0.5nM, 5 nM. The cells were cultured at 37 ℃ for 4 hours, and 1mL of DMEM complete medium containing 10% fetal bovine serum was added to each well, and the culture was continued overnight at 37 ℃.
The expression level of APOC3mRNA in Huh7 cells transfected with each siRNA conjugate was determined by Real-Time Quantitative PCR (Quantitative Real-Time PCR). The specific procedures were that after culturing the transfected cells for 24 hours, total RNA in the cells was extracted using RNAVzol (Vigorous, cat # N002), 1. mu.g of total RNA was reverse-transcribed according to the method of use of reverse transcription kit (Promega, cat # A3500) to obtain cDNA, 2 × Ultra SYBR mix (with ROX) (Beijing Kangji-century Biotech Co., Ltd., cat # CW0956) was used to detect the expression level of APOC3mRNA using cDNA as a template according to the procedures described in the specification, and PCR primers for amplifying APOC3 and β -actin as an internal reference gene are shown in Table 7D.
TABLE 7D sequence of detection primers
Figure BDA0001930334880000971
The inhibition rate of siRNA on the expression level of APOC3mRNA was calculated by the following equation, [1- (test group ANGPTL3mRNA expression/test group β -Actin mRNA expression/mock group APOC3mRNA expression/mock group β -Actin mRNA expression) ] × 100%, wherein each test group was Huh7 cells treated with siRNA conjugates listed in Table 6D, respectively, including siRNA conjugates of conjugates 134, 146, 151, 162, 163 and control siRNA conjugate of comparative conjugate 5, and mock group was Huh7 cells not treated with any siRNA conjugate, the results of the measurement of the inhibition activity of APOC3mRNA expression in Huh7 cells by siRNA conjugates of examples listed in Table 6D and siRNA conjugates of comparative examples are shown in Table 8D below.
Table 8D siRNA conjugates in vitro activity assay
Figure BDA0001930334880000972
As can be seen from the results of table 8D, each siRNA conjugate in table 6D showed excellent APOC3mRNA expression-inhibiting activity at the cellular level at each concentration.
Experimental examples 5-2 this experiment demonstrates the stability of the siRNA conjugates of Table 6D in human plasma in vitro
The siRNA conjugates 150-155, 162-163 and comparative conjugate 5 (20. mu.M, 12. mu.l in terms of siRNA concentration) were mixed with 108. mu.L of 90% Human plasma (diluted with PBS), incubated at 37 ℃ at constant temperature, 10. mu.L of each sample was taken out at 0, 8, 24 and 48 hours, immediately frozen in a freezer at-80 ℃ with liquid nitrogen, 10. mu.L of each sample was taken after diluting 1 × PBS (pH7.4) 5-fold at each time point, 20% by weight of native polyacrylamide gel was prepared, and the above samples were mixed with 4. mu.L of loading buffer (20mM EDTA, 36% by weight of glycerol, 0.06% by weight of bromophenol blue), loaded, electrophoresed under a constant current of 80mA for about 60 minutes, and then, stained with 1 × br Gold dye (Syitrogen, Cat.11494) for 15 minutes, and the results are shown in Table 9D.
Table 9D shows the results of semi-quantitative determination of the stability of the example siRNA conjugates and the comparative example siRNA conjugates listed in table 6D in human plasma in vitro. The results are expressed as the Ratio (RL) of the longest fragment remaining after incubation of the example and comparative siRNA conjugates with human plasma to the longest fragment of untreated siRNA.
Table 9 plasma stability quantification of siRNA conjugates
Figure BDA0001930334880000981
As can be seen from the results of table 9D, the siRNA conjugates of the present disclosure have excellent stability in plasma with little degradation within 48 hours.
Experimental examples 5-3 this experiment demonstrates the inhibitory efficiency of siRNA conjugates of Table 6D on the expression level of APOC3mRNA in vivo (in vivo)
In this experimental example, the siRNA conjugates of conjugates 147, 148, 150, 162-163 and comparative conjugate 5 were examined for their inhibition of the level of APOC3 expression in liver tissue in human APOC3 transgenic mice (B6; CBA-Tg (APOC3)3707Bres/J, purchased from Jackson Lab).
6-8 week old human APOC3 transgenic mice were randomly grouped into 6 mice per group (hermaphroditic halves), and the siRNA conjugates of conjugate 147, 148, 150, 162-163 and control conjugate 5, and PBS were administered to each group of mice, respectively. All animals were dosed by weight and given a single subcutaneous injection, with a siRNA conjugate dose (based on the amount of siRNA) of 3mg/kg and a dosing volume of 10 mL/kg. Mice were sacrificed 28 days after administration, livers were collected and preserved with RNAlater (Sigma Aldrich); liver tissue was homogenized using a tissue homogenizer and total RNA was extracted using Trizol (Thermo Fisher Co.) according to standard procedures for total RNA extraction.
Detecting the expression level of APOC3mRNA in the liver tissue by adopting real-time fluorescent quantitative PCR, specifically: using ImProm-IITMThe extracted total RNA was reverse transcribed into cDNA using a reverse transcription kit (Promega corporation) according to the instructions thereof, and then the inhibition efficiency of siRNA against the expression of APOC3mRNA in liver tissue was examined using a fluorescent quantitative PCR kit (Beijing kang, century Biotechnology Co., Ltd.) in this fluorescent quantitative PCR method, the gene β -actin (β -actin) was used as an internal reference gene, and primers for APOC3 and β -actin were used to examine APOC3 and β -actin, respectively.
See table 10D for sequences of detection primers.
TABLE 10D sequence of detection primers
Figure BDA0001930334880000982
The inhibition rate of siRNA against the expression level of APOC3mRNA was calculated according to the following equation of 1- (test group APOC3mRNA expression amount/test group β -Actin mRNA expression amount)/(control group APOC3mRNA expression amount/control group β -Actin mRNA expression amount) ] × 100% wherein the control group was control group mice to which PBS was applied in the present experiment, and each test group was administration group mice to which different siRNA conjugates were applied, respectively, and the results are shown in table 11D below.
TABLE 11 inhibition of APOC3mRNA expression in mouse liver by D siRNA conjugates
Figure BDA0001930334880000991
As can be seen from the results of table 11D, on the one hand, the conjugates 147, 148, 150 and 162-163 of the present disclosure all showed superior inhibitory activity of APOC3mRNA compared to PBS; on the other hand, in the case of the same nucleic acid sequence and the same modification scheme, the siRNA conjugates represented by the conjugates 150 and 162-163 exhibited higher mRNA inhibition rates in vivo experiments than the comparative conjugate 5, which is different in the conjugate group, and this also indicates that the siRNA conjugates of the present disclosure had good in vivo delivery efficiency.
Experimental examples 5-4 this experiment demonstrates the effect of siRNA conjugates of conjugate 150 on blood lipid levels in vivo (in vivo)
In this experimental example, the effect of the siRNA conjugate of conjugate 150 (B3-siAP1M2SP) on total Cholesterol (CHO) and Triglyceride (TG) levels in serum was examined in human APOC3 transgenic mice (B6; CBA-Tg (APOC3)3707Bres/J, purchased from Jackson Lab).
Human APOC3 transgenic mice 6-8 weeks old were randomly divided into 3 groups of 6 mice (male and female halves) each, as follows: (1) a PBS control group; (2) conjugate 1503 mg/kg group; (3) conjugate 1501 mg/kg group. All animals were dosed by weight in a single subcutaneous dose with a siRNA conjugate dose volume of 10 mL/kg.
Orbital blood collection (about 100 μ L) was performed on each of day 1 before administration (referred to as-1 day) and days 7, 14, 21, 28, 35, 42, 49, and 65 after administration, serum was obtained by centrifugation, and the serum was further subjected to measurement of the total Cholesterol (CHO) and Triglyceride (TG) content using a PM1P000/3 full-automatic serum biochemical analyzer (SABA, italy), and the blood lipid results were normalized, and the inhibition ratio of the blood lipid level was calculated according to the equation of inhibition ratio (1-post-administration test group blood lipid content/pre-administration test group blood lipid content) × 100% blood lipid means total cholesterol or triglyceride, and the measurement results are shown in table 12D below.
TABLE 12 Effect of D siRNA conjugates on Total Cholesterol and triglyceride expression levels in mouse serum
Figure BDA0001930334880000992
As can be seen from table 12D, the siRNA conjugate shown by conjugate 150 significantly down-regulated the total cholesterol and triglyceride levels in the serum of mice and still showed a higher blood lipid lowering effect at least at 65 days.
While the present disclosure has been described in detail with reference to the specific embodiments, the present disclosure is not limited to the details of the embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical spirit of the present disclosure, and the simple modifications are within the scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.

Claims (31)

1. A compound having a structure represented by formula (101):
Figure FDA0001930334870000011
wherein:
n1is an integer selected from 1-2;
each n is2Independently an integer selected from 1-2;
m1is an integer selected from 1 to 6;
R1is a group capable of binding to an active drug via a covalent bond;
each R2Each independently selected from H, C1-C10Alkyl radical, C1-C10Haloalkyl or C1-C10An alkoxy group;
each L1Is a linear alkylene group of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally selected fromSubstituted with one or more of 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 wherein L1Optionally a substituent having any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -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);
each S1Independently is M1Wherein any reactive hydroxyl groups, if any, are protected with a hydroxyl protecting group;
each M1Independently selected from ligands capable of binding to cell surface receptors.
2. The compound of claim 1, wherein each L1A linked combination of one or more independently selected from the group of formula A1-A26:
Figure FDA0001930334870000012
Figure FDA0001930334870000021
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 alkyl;
each Ra is independently selected from one of the groups of formula A27-A45:
Figure FDA0001930334870000022
Figure FDA0001930334870000031
each Rb is independently a C1-C10 alkyl group;
Figure FDA0001930334870000032
represents the site at which the groups are linked by a covalent bond;
alternatively, L1A combination of one or more selected from A1, A4, A5, A6, A8, A10, A11 and A13;
alternatively, L1A linked combination of at least 2 selected from a1, a4, A8, a10, and a 11;
alternatively, L1A combination of at least 2 linkages selected from a1, A8, a 10;
alternatively, L1Is 3-25 atoms in length, said L1The length of (A) means from the atom bonded to the N atom in the nitrogen-containing skeleton to the atom bonded to S1The number of chain-forming atoms on the longest atom chain formed by the connecting atoms;
alternatively, L1Is 4-15 atoms in length.
3. The compound of claim 2, wherein j1 is an integer from 2 to 10, j2 is an integer from 2 to 10, R' is C1-C4 alkyl, Ra is one of a27, a28, a29, a30, and a31, Rb is C1-C5 alkyl;
alternatively, j1 is an integer from 3 to 5, j2 is an integer from 3 to 5, R' is one of methyl, ethyl and isopropyl, Ra is A27 or A28, and Rb is one of methyl, ethyl, isopropyl and butyl.
4. The compound of claim 1, wherein n1And each n2Are each 2, or n1And each n2Are all 1.
5. The compound of claim 1, wherein m1Is an integer from 2 to 6;
alternatively, m1Is an integer of 2 to 4.
6. The compound of claim 1, wherein the protected hydroxyl group has the formula YCOO-, and each Y is independently selected from the group consisting of: c1-C10Alkyl and C1-C10Aryl, or C1-C10Alkyl and C1-C10The hydrogen in the aryl group being optionally substituted by one or more groups including halogen, C1-C6An alkyl-substituted group;
alternatively,each Y is independently selected from the group consisting of: methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halogen-substituted phenyl and C1-C6An alkyl phenyl group.
7. 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 a modified sugar;
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, α -D-mannofuranose, β -D-mannofuranose, β 0-D-mannopyranose, β 1-D-mannopyranose, β 2-D-glucopyranose, β 3-D-glucopyranose, α -D-glucofuranose, β -D-glucofuranose, α -D-fructofuranose, α -D-fructopyranose, α -D-galactopyranose, β -D-galactopyranose, α -D-galactofuranose, β -D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-carboxyethyl-1-carboxyethyl-R-1-carboxyethyl-1-D-fructosyl]-2-deoxy- β -D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-glycolyl- α -neuraminic acid, 5-thio- β -D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl- α -D-glucopyranoside methyl ester, 4-thio- β -D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio- α -D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allosonitrile, ribose, D-4-thioribose, L-ribose or L-4-thio, preferably at least one M of these, preferably M1Is N-acetylgalactosamine (GalNAc);
alternatively,each M1Are all N-acetylgalactosamine (GalNAc).
8. The compound of claim 1, wherein each S1Independently selected from one of the groups of formula A46-A54:
Figure FDA0001930334870000041
Figure FDA0001930334870000051
each Y is independently selected from the group consisting of: methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halogen-substituted phenyl and C1-C6An alkyl phenyl group; preferably, S1Is of formula A49 or A50, and Y is methyl.
9. The compound of claim 1, wherein each R2Independently H, methyl or ethyl.
10. The compound of claim 1, wherein R1Is a group capable of attachment to an active drug via a covalent bond;
alternatively, R1Is a group linked to an oligonucleotide by a phosphodiester bond;
alternatively, R1Comprising a linear alkylene group of 1 to 20 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 wherein R5May optionally have any one or more of the group consisting ofThe substituent (b): c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -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);
alternatively, R1Contains a1 st functional group, said 1 st functional group being reactive with a group on an oligonucleotide or nucleotide to form a phosphate linkage;
alternatively, R1Further containing a2 nd functional group, said 2 nd functional group being capable of reacting with a hydroxyl group or ammoniaThe radical groups form covalent bonds or are solid phase carriers that can be attached by covalent bonds with hydroxyl groups or amino groups; alternatively, the 1 st functional group is a phosphoramidite, hydroxyl, or protected hydroxyl, and the 2 nd functional group is a phosphoramidite, carboxyl, or carboxylate; optionally, the carboxylate is a carboxylate with a metal cation, an ammonium carboxylate salt, a tertiary amine carboxylate salt, or a quaternary ammonium carboxylate salt; optionally, the carboxylate is triethylamine carboxylate or N, N-diisopropylethylamine carboxylate;
alternatively, the solid support that can be linked by a covalent bond formed with a hydroxyl group or an amino group is a solid support that is linked by a phosphate ester bond, a carboxylate ester bond, and/or an amide bond; optionally, the solid support is a resin;
alternatively, R1Containing hydroxy groups, -ORkOr a group represented by formula (C3):
Figure FDA0001930334870000061
wherein R iskIs a hydroxyl-protecting group, and is a hydroxyl-protecting group,
Figure FDA0001930334870000062
represents the site at which the groups are linked by a covalent bond;
alternatively, R1Contains a structure represented by formula (C1), (C2), (C3), (C1 ') or (C3'):
Figure FDA0001930334870000063
in the formula, q1Is an integer of 1 to 4, X is O or NH, M+Is a cation, SPS represents a solid support,
Figure FDA0001930334870000064
represents the site at which the groups are linked by a covalent bond;
alternatively, R1Containing a structure represented by formula (B9), (B10), (B11), (B12), (B9 '), (B10'), (B11 ') or (B12')：
Figure FDA0001930334870000065
Figure FDA0001930334870000071
Wherein q is1Is an integer of 1 to 4, q2Is an integer of 1 to 10, X is O or NH, M+Being a cation, SPS represents a solid support, RkIs a hydroxyl-protecting group, and is a hydroxyl-protecting group,
Figure FDA0001930334870000072
represents the site at which the groups are linked by a covalent bond; alternatively, q1Is an integer of 1 to 5; q. q.s2Is 1 or 2.
11. A compound according to any one of claims 1 to 10, wherein the compound has a structure according to formula (301), (302), (303), (304), (305), (501), (502), (503), (504) or (505):
Figure FDA0001930334870000073
Figure FDA0001930334870000081
Figure FDA0001930334870000091
Figure FDA0001930334870000101
wherein X is O or NH, M+Is a cation, RkIs a hydroxyl protecting group, SPS represents a solid phase carrier;
alternatively, M+Is alkali metal cation, ammonium cationOne of an ion, a tertiary amine cation and a quaternary ammonium cation, RkOne selected from trityl, 4-methoxytrityl, 4 ' -bismethoxytrityl and 4,4 ', 4 ' -trimethoxybenzyl, and SPS is resin.
12. The compound of claim 1, wherein the receptor is a hepatocyte surface receptor;
optionally, the receptor is a receptor on the surface of a mammalian cell;
optionally, the receptor is an asialoglycoprotein receptor on human hepatocytes.
13. A conjugate having a structure represented by formula (201):
Figure FDA0001930334870000111
wherein:
n1is an integer selected from 1-2;
each n is2Independently selected from integers from 1 to 2;
m1is an integer selected from 1 to 6;
each R2Each independently is H, C1-C10Alkyl radical, C1-C10Haloalkyl or C1-C10An alkoxy group;
R6is an active drug;
R5is a straight chain alkylene group of 1 to 20 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 wherein R5May optionally have a substituent of any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -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);
each L1Is 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 group,C6-C10Arylene radical, C3-C18Heterocyclylene and C5-C10A heteroarylene group; and wherein L1Optionally a substituent having any one or more of the group consisting of: c1-C10Alkyl radical, C6-C10Aryl radical, C5-C10Heteroaryl group, C1-C10Haloalkyl, -OC1-C10Alkyl, -OC1-C10Alkylphenyl, -C1-C10alkyl-OH, -OC1-C10Haloalkyl, -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);
each M1Selected from the group consisting of the ability to interact with cell surface receptorsA bound ligand.
14. The conjugate of claim 13, wherein each L is1A linked combination of one or more independently selected from the group of formula A1-A26:
Figure FDA0001930334870000121
wherein j1 is an integer from 1 to 20; j2 is an integer from 1 to 20;
r' is C1-C10 alkyl;
ra is selected from one of the groups of the formula A27-A45:
Figure FDA0001930334870000131
rb is C1-C10 alkyl;
Figure FDA0001930334870000141
represents the site of covalent attachment of the group;
alternatively, L1A combination of one or more selected from A1, A4, A5, A6, A8, A10, A11 and A13;
alternatively, L1A linked combination of at least 2 selected from a1, a4, A8, a10, and a 11;
alternatively, L1A combination of at least 2 linkages selected from a1, A8, a 10;
alternatively, L1Is 3-25 atoms in length, said L1Length of (b) means the atom bound to the N atom of the nitrogen-containing skeleton to M1The number of chain-forming atoms on the longest atom chain formed by the connecting atoms;
alternatively, L1Is 4-15 atoms in length.
15. The conjugate of claim 14, wherein j1 is an integer from 2 to 10, j2 is an integer from 2 to 10, R' is a C1-C4 alkyl group, Ra is one of a27, a28, a29, a30, and a31, Rb is a C1-C5 alkyl group;
alternatively, j1 is an integer from 3 to 5, j2 is an integer from 3 to 5, R' is one of methyl, ethyl and isopropyl, Ra is A27 or A28, and Rb is one of methyl, ethyl, isopropyl and butyl.
16. The conjugate of claim 13, wherein n is1And each n2Are each 2, or n1And each n2Are all 1.
17. The conjugate of claim 13, wherein m is1Is an integer from 2 to 6;
alternatively, m1Is an integer of 2 to 4.
18. The conjugate of claim 13, wherein each M is1Independently a sugar;
optionally, each M1Independently a monosaccharide, disaccharide, trisaccharide or polysaccharide;
optionally, at least one M1Is a modified sugar;
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, α -D-mannofuranose, β -D-mannofuranose, β 0-D-mannopyranose, β 1-D-mannopyranose, β 2-D-glucopyranose, β 3-D-glucopyranose, α -D-glucofuranose, β -D-glucofuranose, α -D-fructofuranose, α -D-fructopyranose, α -D-galactopyranose, β -D-galactopyranose, α -D-galactofuranose, β -D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2-amino-3-O-carboxyethyl-1-carboxyethyl-R-1-carboxyethyl-1-D-fructosyl]-2-deoxy- β -D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4, 6-dideoxy-4-carboxamido-2, 3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-ethylglucopyranoseOne of alkanoyl- α -neuraminic acid, 5-thio- β -D-glucopyranose, 2,3, 4-tri-O-acetyl-1-thio-6-O-trityl- α -D-glucopyranoside methyl ester, 4-thio- β -D-galactopyranose, 3,4,6, 7-tetra-O-acetyl-2-deoxy-1, 5-dithio- α -D-glucopyranoside ethyl ester, 2, 5-anhydro-D-allositrile, ribose, D-4-thioribose, L-ribose or L-4-thioribose, preferably at least one M, preferably1Is N-acetylgalactosamine (GalNAc);
optionally, each M1Are all N-acetylgalactosamine (GalNAc).
19. The conjugate of claim 13, wherein each R is2Independently H, methyl or ethyl.
20. The conjugate of claim 13, wherein R6Contains functional oligonucleotides.
21. The conjugate of claim 20, wherein R6Is a group of formula A59:
Figure FDA0001930334870000142
wherein E is1Is OH or SH, Nu is functional oligonucleotide;
alternatively, R5And R6The P atom in (1) forms a phosphate ester bond;
alternatively, R5Selected from B5, B6, B5 'or B6':
Figure FDA0001930334870000151
wherein the content of the first and second substances,
Figure FDA0001930334870000152
denotes the site of linkage by covalent bond, q2Is an integer of 1 to 10.
22. The conjugate according to claim 1 or 21, wherein the conjugate has a structure represented by formula (401), (402), (403), (404) or (405):
Figure FDA0001930334870000153
Figure FDA0001930334870000161
Figure FDA0001930334870000171
wherein Nu is a functional oligonucleotide.
23. The conjugate of any one of claims 20-22, wherein the functional oligonucleotide is selected from one of small interfering RNA, microrna, anti-microrna, microrna antagonists, microrna mimetics, decoy oligonucleotides, immunostimulatory substances, G-quadrupoles, variable spliceosomes, single stranded RNA, antisense nucleic acids, aptamers, stem-loop RNA, mRNA fragments, activating RNA, or 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 a59 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 a59 is attached to the end of the single stranded oligonucleotide; alternatively, P in formula a59 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 a59 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 a59 is attached to the end of the sense strand or the antisense strand; preferably, P in formula a59 is attached to the 3' end of the sense strand; preferably, P in formula a59 is linked to the 2', 3' or 5' position of a nucleotide in the oligonucleotide conjugate by forming a phosphodiester bond.
24. The conjugate of claims 20-23, 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 mRNA of hepatitis b virus, the mRNA expressed by the angiopoietin-like protein 3 gene, or the mRNA expressed by the apolipoprotein C3 gene;
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;
optionally, 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 target mRNA in the direction from the 5 'end to the 3' end;
optionally, the nucleotide sequence is selected from one of table 1A, 2A, 3A or 4A.
25. The conjugate of claim 24, 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 refers to a nucleotide in which the 2' -hydroxyl group of the ribose group of the nucleotide is substituted with a methoxy group;
optionally, the nucleotide sequence is selected from one of tables 1B, 2B, 3B or 4B.
26. The conjugate according to claim 25, 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 modifying group is a thiophosphate group with a structure as shown in formula (801):
Figure FDA0001930334870000191
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
between the 2 nd and 3 rd nucleotides at the 3' terminal end of the antisense strand;
optionally, the nucleotide sequence is selected from one of table 1C, 2C, 3C or 4C.
27. The conjugate of any one of claims 24-26, 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 having one of the following formulae (802) to (806):
Figure FDA0001930334870000192
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 (802), formula (803), or formula (805);
optionally, the nucleotide sequence is selected from one of table 1D, 2D, 3D or 4D;
optionally, the nucleotide sequence is selected from one of tables 1E, 2E, 3E or 4E.
28. Use of a conjugate according to any one of claims 13 to 27 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 liver cell;
optionally, the specific gene is selected from a hepatitis b virus gene, an angiopoietin-like protein 3 gene, or an apolipoprotein C3 gene.
29. The use according to claim 28, 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.
30. 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 11 to 25;
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.
31. A kit comprising the conjugate of any one of claims 11-25.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112142761A (en) * 2020-11-08 2020-12-29 江西师范大学 Synthesis method of tetrahydropyrano [3, 2-d ] oxazole ring compound
WO2022068923A1 (en) * 2020-09-30 2022-04-07 纳肽得(青岛)生物医药有限公司 Sirna of angiopoietin-like 3 (angptl3) and use thereof

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WO2015006740A2 (en) * 2013-07-11 2015-01-15 Alnylam Pharmaceuticals, Inc. Oligonucleotide-ligand conjugates and process for their preparation

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Publication number Priority date Publication date Assignee Title
WO2015006740A2 (en) * 2013-07-11 2015-01-15 Alnylam Pharmaceuticals, Inc. Oligonucleotide-ligand conjugates and process for their preparation

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022068923A1 (en) * 2020-09-30 2022-04-07 纳肽得(青岛)生物医药有限公司 Sirna of angiopoietin-like 3 (angptl3) and use thereof
CN112142761A (en) * 2020-11-08 2020-12-29 江西师范大学 Synthesis method of tetrahydropyrano [3, 2-d ] oxazole ring compound
CN112142761B (en) * 2020-11-08 2022-03-22 江西师范大学 Synthesis method of tetrahydropyrano [3, 2-d ] oxazole ring compound

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