CN109957566A

CN109957566A - The compound of the oligonucleotides of modification and the oligonucleotides that can be used for synthetic modification

Info

Publication number: CN109957566A
Application number: CN201711431610.7A
Authority: CN
Inventors: 张必良; 王玮; 叶奕栋
Original assignee: Guangzhou Ribobio Co ltd
Current assignee: Guangzhou Ribobio Co ltd
Priority date: 2017-12-26
Filing date: 2017-12-26
Publication date: 2019-07-02
Anticipated expiration: 2037-12-26
Also published as: CN109957566B

Abstract

The invention belongs to biomedicine technical fields, and in particular to the compound of the oligonucleotides of modification and the oligonucleotides that can be used for synthetic modification, and to the method that oligonucleotides is modified.The invention further relates to the purposes of the oligonucleotides of the modification, it is used to prevent and/or treat disease relevant to liver in subject.

Description

The compound of the oligonucleotides of modification and the oligonucleotides that can be used for synthetic modification

Technical field

The invention belongs to biomedicine technical fields, and in particular to the oligonucleotides of modification and the widow that can be used for synthetic modification The compound of nucleotide, and to the method that oligonucleotides is modified.

Background technique

Asialoglycoprotein receptor (asialoglycoprotein receptor, ASGPR) is quantity one kind abundant The endocytosis receptor of heteoro-oligomers is primarily present in liver parenchymal cell towards the cell membrane surface of sinusoid side, has To the specific of sugar since various glycoprotein are after removing terminal sialic acid with enzyme hydrolysis or with acidolysis, the secondary end that exposes Galactose residue, so the sugared binding specificity of ASGPR is actually galactosyl, therefore also known as gala sugar specificity by Body.ASGPR is distributed mainly on liver parenchymal cell, other cell contents are low, therefore becomes the best receptor of liver orientation transhipment.

End is that the galactolipin (Gal) of irreducibility or the glycoprotein of N- acetylgalactosamine (GalNAc) residue can be with It is identified by ASGPR, compatibility ratio Gal high about 50 times (Iobst S T et al, J Biol of the GalNAc in conjunction with ASGPR Chem,1996,271(12):6686-6693).Experiment in vitro show cluster saccharide residue can by and meanwhile occupy receptor Binding site and make saccharide residue of its compatibility much higher than not cluster, the order of affinity are as follows: four feelers > tri- feelers > > bis- feelers > > single-tap angle galactoside (Lee Y C, et al, J Biol Chem, 1983,258 (1): 199-202).

The receptor-mediated Liver targeting oligonucleotides of ASGPR is the new breakthrough of nucleic acid field of innovative medicine research.2012, beauty The three same siRNAs of feeler GalNAc structure (siRNA) that forefathers study in Alnylam drugmaker, state are covalently attached, and are realized The Liver targeting delivering of internal siRNA.Using this technology, researcher has carried out amyloid lesion, hemophilia, high cholesterol The drug development of the diseases such as blood trouble, liver porphyrin, hepatitis B, multiple drug candidates come into clinical research (http: // www.alnylam.com/product-pipeline/).2014, U.S.'s ISIS pharmacy was by three feeler GalNAc and antisense core Acid be covalently attached, realize animal body in liver targeted drug delivery, after connection the activity of antisense nucleic acid improve 10 times (Prakash, T.P.et al, Nucleic Acids Res.42,8796-807.).

Summary of the invention

The present inventor has obtained a kind of compound with ASGPR ligand by in-depth study and creative labor, It can be used for modifying oligonucleotides, result in a kind of oligonucleotides of modification comprising conjugate group.

Therefore, in one aspect, this application provides a kind of compound comprising oligonucleotides and conjugate group leads to Formula isWherein, PN is oligonucleotides, and Y is selected from the integer between 1-10, and X is selected from whole between 0-10 Number, M_TSelected from the conjugate group as shown in formula (1), formula (2), formula (3) and formula (4), when X is not 0, X M is each independently Selected from the conjugate group as shown in formula (1 '), formula (2 ') and formula (3 '),

Wherein, A_xFor ligand, linker is linking arm, and Q is hydroxyl or modifier.

In certain embodiments, formula (1)-formula (4), in conjugate group shown in formula (1 ')-formula (3 '), A_xRespectively solely It is on the spot the ligand of people's asialoglycoprotein receptor (ASGPR).

In certain embodiments, formula (1)-formula (4), in conjugate group shown in formula (1 ')-formula (3 '), A_xFor gala Sugar, acetylamino galactosamine, the polysaccharide containing galactolipin, the polysaccharide containing acetylamino galactosamine, galactose derivative (such as gala The ester of sugar, such as galactolipin acetic acid esters) or acetylamino galactosamine derivative (such as the ester of acetylamino galactosamine, such as second Acylamino- galactolipin acetic acid esters).Optionally, A_xOn also each independently have modification group, such as carbonylic alkyl or ester group alkane Base, the alkyl are preferably C_1-6Alkyl or C_6-12Alkyl.

In certain embodiments, A_xIt is selected from:

In certain embodiments, formula (1)-formula (4), in conjugate group shown in formula (1 ')-formula (3 '), linker's Structure is each independently as shown in formula (i), formula (ii), formula (iii), formula (iv) or formula (v).

Wherein, n is selected from the integer between 1-10.In certain embodiments, n is 1 or 6.

Wherein, n₁And n₂The integer being each independently selected between 1-10.In certain embodiments, n₁It is 1.Certain In embodiment, n₂It is 4.In certain embodiments, n₁For 1 and n₂It is 4.

Wherein, n₁、n₂、n₃The integer being each independently selected between 1-10.In certain embodiments, n₁It is 1.At certain In a little embodiments, n₂It is 3.In certain embodiments, n₃It is 4.In certain embodiments, n₁It is 1, n₂For 3 and n₃It is 4.

Wherein, n is selected from the integer between 1-10.In certain embodiments, 1 n.

Wherein, n is selected from the integer between 1-10.In certain embodiments, 4 n.

In certain embodiments, in conjugate group shown in formula (1) or formula (1 '), A_xIt is each independently selected from A₁、 A₂、A₃、A₁’、A₂' or A₃', shown in the structure of linker such as formula (i).In certain embodiments, n is 1 or 6.

In certain embodiments, in conjugate group shown in formula (1) or formula (1 '), A_xIt is each independently selected from A₁Or A₁', shown in the structure of linker such as formula (ii).In certain embodiments, n₁It is 1, and n₂It is 4.

In certain embodiments, in conjugate group shown in formula (1) or formula (1 '), A_xIt is each independently selected from A₁Or A₁', shown in the structure of linker such as formula (iii).In certain embodiments, n₁It is 1, n₂It is 3, and n₃It is 4.

In certain embodiments, in conjugate group shown in formula (1) or formula (1 '), A_xIt is each independently selected from A₁Or A₁', shown in the structure of linker such as formula (iv).In certain embodiments, 1 n.

In certain embodiments, in conjugate group shown in formula (2) or formula (2 '), A_xIt is each independently selected from A₁、 A₂、A₃、A₁’、A₂' or A₃', shown in the structure of linker such as formula (i).In certain embodiments, n is 1 or 6.

In certain embodiments, in conjugate group shown in formula (2) or formula (2 '), Ax is each independently selected from A₁Or A₁', shown in the structure of linker such as formula (ii).In certain embodiments, n₁It is 1, and n₂It is 4.

In certain embodiments, in conjugate group shown in formula (3) or formula (3 '), Ax be each independently selected from for A₁、A₂、A₃、A₁’、A₂' or A₃', shown in the structure of linker such as formula (i).In certain embodiments, n is 1 or 6.

In certain embodiments, in conjugate group shown in formula (4), A_xFor A₁Or A₁', the structure of linker such as formula (v) shown in.In certain embodiments, 4 n.

In certain embodiments, formula (1)-formula (3), in conjugate group shown in formula (1 ')-formula (3 '), Q is selected from: gallbladder Sterol and its derivative, polyethylene glycol, fluorescence probe, biotin, polypeptide, vitamin, tissue-targeting molecule.

In the present invention, oligonucleotides can be single-stranded oligonucleotide or double chain oligonucleotide.Optionally, few core of the invention Thuja acid may include one or more nucleotide by modification.In certain embodiments, one or more of by repairing The nucleotide of decorations is each independently selected from: 2 '-methoxy ethyl modified nucleosides acid, 2 '-O- alkyl modified nucleotide (such as 2 '- O- methyl modified nucleotide), 2 '-O- allyl modified nucleosides acid, 2 '-C- allyl modified nucleosides acid, 2 '-fluorine modification nucleosides Acid, 2 '-deoxidation modified nucleoside acid, 2 '-hydroxyl modified nucleotide, lock nucleotide, hexitol nucleic acid (Hexitol nucleic Acid, HNA), solution lock nucleic acid (unlocked nucleic acid, UNA).In certain embodiments, the process is modified Nucleotide is selected from 2 '-O- alkyl modified nucleotide, 2 '-fluorine modification nucleotide.

In certain embodiments, the oligonucleotides has end modified object, it is preferable that the end modified object choosing From: cholesterol, polyethylene glycol, fluorescence probe, biotin, polypeptide, vitamin, tissue-targeting molecule and any combination thereof.

In certain embodiments, the phosphoric acid skeleton of the oligonucleotides is by modification, it is preferable that described to be modified to sulphur Generation modification.

In certain embodiments, the oligonucleotides is siRNA.In certain embodiments, the siRNA includes mutual Mend the positive-sense strand and antisense strand for forming double-strand.In certain embodiments, the siRNA includes such as SEQ ID NO:1, SEQ ID Sequence shown in NO:2, SEQ ID NO:3 or SEQ ID NO:4.

In the oligonucleotides of modification of the invention, conjugate group can connect the different location in oligonucleotides.

In certain embodiments,Each independently with 3 ' of any bar chain in the oligonucleotides It is connected among end, 5 ' ends or sequence.In certain embodiments,Pass through tricresyl phosphate ester bond and widow Nucleotide is connected.In certain embodiments, M and M_TBetween or M and M between be connected by tricresyl phosphate ester bond.

In certain embodiments, oligonucleotides is single-stranded oligonucleotide.In certain embodiments, 1 Y,It is connected with 3 ' ends of the oligonucleotides or 5 ' ends.In certain embodiments, 2 Y, 2Respectively it is connected with 3 ' ends of the oligonucleotides and 5 ' ends.

In certain embodiments, oligonucleotides is double chain oligonucleotide.In certain embodiments, 1 Y,It is connected with 3 ' ends of any bar chain in the oligonucleotides or 5 ' ends.In certain embodiments In, Y 2,2Respectively it is connected with 3 ' ends of the same chain in the oligonucleotides and 5 ' ends. In certain embodiments, 2 Y, 2Respectively with 3 ' end phases of two chains in the oligonucleotides Connection.In certain embodiments, 2 Y, 2It is respectively last with 5 ' of two chains in the oligonucleotides End is connected.In certain embodiments, 3 Y, 3In, there are two respectively and in the oligonucleotides 3 ' ends of same chain be connected with 5 ' ends, third is connected with 3 ' ends of another chain or 5 ' ends.At certain In a little embodiments, Y 4,4Respectively with the 3 ' ends and 5 ' ends of two chains in the oligonucleotides It is connected.

In the oligonucleotides of modification of the invention, M_TIt can have identical or different knot between M or between multiple M Structure.In certain embodiments, X is not 0, M_TWith at least one M A having the same_xAnd/or linker structure.In certain implementations In scheme, X, which is greater than 1, X M, has identical or different structure.In certain embodiments, Y is greater than 1, YWith identical or different structure.

In the oligonucleotides of modification of the invention, one or more may includeAnd it is possible to M Or the structure of MT, the number of M or numberIt is adjusted.

In certain embodiments, 1 Y, X 0, and the compound have one in following characteristics:

(1)M_TStructure such as formula (1) shown in, A_xFor A₁’、A₂' or A₃', shown in the structure of linker such as formula (i), it is preferable that N is 1 or 6；

(2)M_TStructure such as formula (1) shown in, A_xFor A₁', shown in the structure of linker such as formula (ii), it is preferable that n₁It is 1, And n₂It is 4；

(3)M_TStructure such as formula (1) shown in, A_xFor A₁', shown in the structure of linker such as formula (iii), it is preferable that n₁It is 1, n₂It is 3, and n₃It is 4；

(4)M_TStructure such as formula (1) shown in, A_xFor A₁', shown in the structure of linker such as formula (iv), it is preferable that n 1；

(5)M_TStructure such as formula (2) shown in, A_xFor A₁’、A₂' or A₃', shown in the structure of linker such as formula (i), it is preferable that N is 1 or 6；

(6)M_TStructure such as formula (2) shown in, A_xFor A₁', shown in the structure of linker such as formula (ii), it is preferable that n₁It is 1, And n₂It is 4；

(7)M_TStructure such as formula (3) shown in, A_xFor A₁’、A₂' or A₃', shown in the structure of linker such as formula (i), it is preferable that N is 1 or 6；

(8)M_TStructure such as formula (4) shown in, A_xFor A₁', shown in the structure of linker such as formula (v), preferably n is 1；

(9)M_TStructure such as formula (2) shown in, A_xFor A₁', shown in the structure of linker such as formula (iii)；Preferably, n₁It is 1, n₂It is 3, and n₃It is 4.

In certain embodiments, 1 Y, X 1,2 or 3, when X is 2 or 3, each M structure having the same, and The compound has one in following characteristics:

(1) shown in the structure of M such as formula (1 '), A_xFor A₁', shown in the structure of linker such as formula (i), it is preferable that n is 1 or 6； M_TStructure such as formula (1) shown in, A_xFor A₁', shown in the structure of linker such as formula (i), it is preferable that n is 1 or 6；

(2) shown in the structure of M such as formula (1 '), A_xFor A₁', shown in the structure of linker such as formula (ii), it is preferable that n₁It is 1, And n₂It is 4；M_TStructure such as formula (1) shown in, shown in the structure such as formula (ii) of Ax A1 ', linker, it is preferable that n₁It is 1, and n₂ It is 4；

(3) shown in the structure of M such as formula (1 '), A_xFor A₁', shown in the structure of linker such as formula (iii), it is preferable that n₁It is 1, n₂It is 3, and n₃It is 4；M_TStructure such as formula (1) shown in, shown in the structure such as formula (iii) of Ax A1 ', linker, it is preferable that n₁ It is 1, n₂It is 3, and n₃It is 4；

(4) shown in the structure of M such as formula (2 '), A_xFor A₁', shown in the structure of linker such as formula (i), it is preferable that n is 1 or 6； M_TStructure such as formula (2) shown in, A_xFor A₁', shown in the structure of linker such as formula (i), it is preferable that n is 1 or 6；

(5) shown in the structure of M such as formula (1 '), A_xFor A₁', shown in the structure of linker such as formula (ii), it is preferable that n₁It is 1, And n₂It is 4；M_TStructure such as formula (1) shown in, shown in the structure such as formula (ii) of Ax A1 ', linker, it is preferable that n₁It is 1, and n₂ It is 4；

(6) shown in the structure of M such as formula (1 '), A_xFor A₃', shown in the structure of linker such as formula (ii), it is preferable that n₁It is 1, And n₂It is 4；M_TStructure such as formula (1) shown in, shown in the structure such as formula (ii) of Ax A3 ', linker, it is preferable that n₁It is 1, and n₂ It is 4.

In certain embodiments, the structure of 1 Y, X 2, two M is identical, as shown in formula (1 '), A_xFor A₁', linker Structure such as formula (iv) shown in, it is preferable that n 1；Shown in the structure of MT such as formula (4), A_xFor A₁', the structure of linker such as formula (v) It is shown, it is preferable that n 4.

The illustrative compound of the present invention includes:

Wherein, n₁、n₂、n₃And n is each independently selected from the integer between 1-10.

On the other hand, this application provides the compound that can be used for modified oligonucleotide, ligand is had on the compound With can be with the chemical group of oligonucleotides chain reaction, and the linking arm that ligand is connect with the chemical group.

It therefore, is A this application involves general formula_x-linker-R₁、A_x-linker-R₂、A_x-linker-R₃And A_x-linker- R₄Compound, wherein A_xFor ligand, linker is linking arm,

R₁For

R₂ForWherein, m₁And m₂The integer being each independently selected between 1-10,

R₃For

R₄For

R₁、R₂、R₃In, Z is the protecting group of hydroxyl, it is preferable that Z is each independently 4,4- dimethoxy triphenylmenthane base (DMTr) or 4- methoxyl group triphenylchloromethane base (MMT).

In certain embodiments, A_xFor the ligand of people's asialoglycoprotein receptor (ASGPR).

In certain embodiments, A_xFor galactolipin, acetylamino galactosamine, the polysaccharide containing galactolipin, contain acetylamino The polysaccharide of galactolipin, galactose derivative (such as the ester of galactolipin, such as galactolipin acetic acid esters) or acetylamino galactosamine spread out Biology (such as the ester of acetylamino galactosamine, such as acetylamino galactosamine acetic acid esters).Optionally, A_xOn also each independently With modification group, such as carbonylic alkyl or ester group alkyl, the alkyl are preferably C_1-6Alkyl or C_6-12Alkyl.

In certain embodiments, A_xIt is selected from:

In certain embodiments, the structure of linker such as formula (i), formula (ii), formula (iii), formula (iv) or formula (v) institute Show:

Wherein, n is selected from the integer between 1-10, it is preferable that n is 1 or 6；

Wherein, n₁And n₂The integer being each independently selected between 1-10, it is preferable that n₁It is 1, it is preferable that n₂It is 4；

Wherein, n₁、n₂、n₃The integer being each independently selected between 1-10, it is preferable that n₁It is 1, it is preferable that n₂It is 3, it is excellent Selection of land, n₃It is 4；

Wherein, n is selected from the integer between 1-10, it is preferable that n 1；

Wherein, n is selected from the integer between 1-10, it is preferable that n 4.

It is A for general formula_x-linker-R₁Compound, in certain embodiments, the compound have following characteristics In one:

(1)A_xFor A₁、A₂Or A₃, shown in the structure of linker such as formula (i), it is preferable that n is 1 or 6；

(2)A_xFor A₁, shown in the structure of linker such as formula (ii), it is preferable that n₁It is 1, and n₂It is 4；

(3)A_xFor A₁, shown in the structure of linker such as formula (iii), it is preferable that n₁It is 1, n₂It is 3, and n₃It is 4；

(4)A_xFor A₁, shown in the structure of linker such as formula (iv), it is preferable that n 1.

In certain embodiments, general formula A_x-linker-R₁Compound be selected from:

Wherein, n is selected from the integer between 1-10,

Wherein, n₁And n₂The integer being each independently selected between 1-10,

Wherein, n₁、n₂、n₃The integer being each independently selected between 1-10.

It is A for general formula_x-linker-R₂Compound, in certain embodiments, the compound have following characteristics In one:

(2)A_xFor A₁, shown in the structure of linker such as formula (ii), it is preferable that n₁It is 1, and n₂It is 4.

In certain embodiments, general formula A_x-linker-R₂Compound be selected from:

Wherein, n, m₁、m₂The integer being each independently selected between 1-10,

Wherein, n₁、n₂、m₁、m₂The integer being each independently selected between 1-10,

Wherein, n₁、n₂、n₃、m₁、m₂The integer being each independently selected between 1-10.

For for A_x-linker-R₃Compound, in certain embodiments, A_xFor A₁、A₂Or A₃, the structure of linker As shown in formula (i), it is preferable that n is 1 or 6.

For for A_x-linker-R₄Compound, in certain embodiments, A_xFor A₁, the structure of linker such as formula (v) It is shown, it is preferable that n 4.

In one aspect, this application provides the methods that a kind of pair of oligonucleotides is modified, including will be one or more Compound (such as 2,3,4,5,6,7,8,9 or 10) is connected on oligonucleotides, it is one or It is A that multiple compounds, which are each independently selected from general formula as defined above,_x-linker-R₁、A_x-linker-R₂、A_x-linker-R₃ Or A_x-linker-R₄Compound.Preferably, in the method, by the compoundIt is anti-that chemistry occurs It should realize connection.In certain embodiments, the method is used for synthesis in solid state.

This application provides the methods that another kind modifies oligonucleotides, comprising the following steps:

Step (1): providing oligonucleotides, and the first compound be connected on oligonucleotides, obtains comprising conjugate M Oligonucleotides, it is A that first compound, which is selected from general formula as defined above,_x-linker-R₁、A_x-linker-R₂Or A_x- linker-R₃Compound,

Step (2): second compound is connected on the conjugate M of previous step formation, the second compound is selected from General formula as defined above is A_x-linker-R₁、A_x-linker-R₂、A_x-linker-R₃Or A_x-linker-R₄Compound.

Optionally, the method also includes step (3): it is one or many (such as 2-9 times) to repeat step (2).

Optionally, the method also includes step (4): it is one or many to repeat step (1), step (2) and step (3) (such as 2-9 times).

Preferably, in the step (1) and (2), by the first compound or second compoundOccur Connection is realized in chemical reaction.

It can be single-stranded oligonucleotide or Double stranded oligonucleotide for the oligonucleotides in any one method of modifying of the invention Acid.Optionally, the oligonucleotides may include one or more nucleotide by modification.In certain embodiments, institute It states one or more nucleotide by modification and is each independently selected from: 2 '-methoxy ethyl modified nucleoside acid, 2 '-O- alkyl Modified nucleoside acid (such as 2 '-O- methyl modified nucleotide), 2 '-O- allyl modified nucleoside acid, 2 '-C- allyl modified nucleosides Acid, 2 '-fluorine modification nucleotide, 2 '-deoxidation modified nucleoside acid, 2 '-hydroxyl modified nucleotide, lock nucleotide, HNA, UNA.At certain In a little embodiments, the nucleotide by modification is selected from 2 '-O- alkyl modified nucleotide, 2 '-fluorine modification nucleotide.

It is A present invention also provides general formula as defined above_x-linker-R₁、A_x-linker-R₂、A_x-linker-R₃Or A_x-linker-R₄Compound purposes, the compound is for modifying oligonucleotides.Present invention also provides one kind Kit, being selected from general formula as defined above it includes at least one is A_x-linker-R₁、A_x-linker-R₂、A_x-linker-R₃ Or A_x-linker-R₄Compound.In certain embodiments, the kit also includes for synthesizing and/or modifying few core Thuja acid reagent (such as: solid support, DNA monomer, RNA monomer, modification monomer, activator, oxidant, deprotecting regent, Buffer and any combination thereof).

In one aspect, this application provides a kind of pharmaceutical compositions, and it includes of the invention comprising oligonucleotides and sews The compound of conjunction object group and optional pharmaceutically acceptable carrier.In certain embodiments, described pharmaceutical composition For preventing and/or treating disease relevant to liver in subject.

Pharmaceutically acceptable any dosage form can be made in pharmaceutical composition of the invention.In certain embodiments, institute The dosage form stated be selected from powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, aerosol, Powder spray.In certain embodiments, the preparation can be such as oral, parenteral, straight with any suitable administration mode The modes such as intestines, transpulmonary or local administration are applied to the patient or subject for needing to prevent and/or treat.When for being administered orally When, the preparation can be oral preparation, such as oral solid formulation, such as tablet, capsule, pill, granule；Or, mouth Take liquid preparation, such as oral solution, oral suspensions, syrup.The oral preparation also may include suitable filler, Adhesive, disintegrating agent, lubricant etc..When being used for parenteral administration, the preparation can be injection, including injection, note It penetrates with aseptic powdery and concentrated solution for injection.For injection, the conventional method in existing pharmaceutical field can be used to be given birth to It produces.When preparing injection, additives can be added without in the preparation, suitable add can also be added according to the property of drug Agent.

In one aspect, this application provides the use of the compound comprising oligonucleotides and conjugate group of the invention On the way, the compound is for preventing and/or treating disease relevant to liver in subject.

In one aspect, this application provides a kind of sides of disease relevant to liver in prevention and/or treatment subject Method, including a effective amount of compound comprising oligonucleotides and conjugate group of the invention is bestowed subject in need.

In certain embodiments, the disease relevant to liver is selected from: hereditary angioedema, I type familial junket Propylhomoserin mass formed by blood stasis, Alagille syndrome, alpha-1-amtitrypsin deficiency, bile acid biosynthesis and metabolic deficiency, Biliary atresia, capsule Property fibrotic liver disease, idiopathic neonatal hepatitis, mitochondria hepatopathy, progressive familial intrahepatic cholestasis, primary hardening Property cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, hyperlipemia, Steatohepatitis, nonalcoholic fatty liver disease (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia and involve with The disease for the Hepatic glucose production that the similar exception of type-2 diabetes mellitus increases, hepatitis, liver property porphyrin.

In certain embodiments, the subject be mammal, such as bovid, equid, caprid, Porcine animals, canid, felid, rodent, primate；For example, described, subject is a human.

In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology The normally understood meaning of personnel institute.Also, laboratory operation step involved in herein is to be widely used in corresponding field Conventional steps.Meanwhile for a better understanding of the present invention, the definition and explanation of relational language is provided below.

As used in this article, term " oligonucleotides " refers to the chemical modification containing multiple connections or unmodified nucleotide Oligomeric compounds, have less than about 100 nucleotide length (such as 1-20 nucleotide or 1-50 nucleotide).? In certain embodiments, oligonucleotides may include non-nucleic acid conjugation group.In certain embodiments, oligonucleotides includes ribose Nucleic acid (RNA) or DNA (DNA).In certain embodiments, oligonucleotide is double-strand or single-stranded.? In certain embodiments, oligonucleotide siRNA, aptamer or antisense nucleic acid.

As used in this article, term " conjugate " or " conjugate group " mean the atom for being bound to oligonucleotides or original Subbase group.In some cases, conjugate group changes the one or more properties for the oligonucleotides that they are connected to, including But it is not limited to pharmacodynamics, pharmacokinetics, combination, absorption, cell distribution, cellular uptake, charge and/or removes property.

As used in this article, term " receptor " refers to the large biological molecule that glycoprotein or lipoprotein are constituted, and is present in thin In after birth, endochylema or nucleus, different receptors has special structure and configuration.As used in this article, term " ligand " is Refer to the substance that there is recognition capability to receptor and can be in combination.In certain embodiments, the ligand is to asialoglycoprotein Glycoprotein receptor (asialoglycoprotein receptor, ASGPR) has the ligand of affinity.In certain embodiments In, the ligand is carbohydrate, such as monosaccharide and polysaccharide, including but not limited to: galactolipin, GalNAc, Mannose, glucose, aminoglucose and fucose.

As used in this article, term " polysaccharide " refer to as multiple monosaccharide groups with glycosidic bond be connected made of polymer.This In invention, polysaccharide includes oligosaccharides and oligosaccharide.In general, " oligosaccharides " refer to as 2-10 monosaccharide groups with glycosidic bond be connected made of gather Close object, " oligosaccharide " refer to as 20 or less monosaccharide groups with glycosidic bond be connected made of polymer.

As used in this article, term " about " should be readily appreciated by one skilled in the art, and will be with the upper of place used in it Hereafter there is a degree of variation.If according to the context of term application, to those skilled in the art, meaning It is not clearly, then " about " meaning that deviation is no more than positive and negative the 10% of the certain number value or range.

As used in this article, term " prevention " refers to prevention or postpones the generation of disease.

As used in this article, term " treatment " refers to the progress cured or at least partly prevent disease, or alleviates disease Symptom.

As used in this article, term " effective quantity " is the amount for referring to effectively realize expected purpose.For example, prevention disease Effective quantity refers to, can effectively prevent, prevents or postpone the amount of the generation of disease.Such effective quantity is measured in art technology Within the limit of power of personnel.

Advantageous effect of the invention

Compared with the existing technology compared with, the invention has the following advantages:

With Alnylam pharmacy and ISIS Pharmaceuticals Inc.'s related invention (US20150119444A1, US20150119445A1, US20150126718A1 it) compares, the present invention has following significant difference:

1. chemical structure is different.Two company's design structures are that more feeler ASGPR ligands are once connect with oligonucleotide；This hair The structure of bright design is that list ASGPR ligand is repeatedly coupled with oligonucleotide with nucleic acid phosphoramidite solid-state chemical reaction method, realizes polygamy Body modification.The advantage of single multiple synthesis in solid state coupling of ASGPR ligand is that (1) shortens synthesis step, it is easy to accomplish amplification life It produces.Synthesis in solid state connects each ligand coupling reaction efficiency and is greater than 98%, connects 3 ligand efficiency reactions and is greater than 94%, solid phase Synthesis can be automatically performed by equipment, need not be purified in connection reaction.Compared to liquid phase synthesis by 3 ligand combinations be single molecule again It is attached, more efficient, speed is faster, it is easier to amplification production.(2) oligonucleotide modification range is expanded.According to report (lee et al.,Carbohydrates in Chemistry and Biology；4:549,2000) in three feeler structures GalNAc is respectively with centre distanceSame ASGPR receptor is non-equally more advantageous to combine. Because preparation method limits, GalNAc and center are equidistant (17 atoms) in three feeler structures of two companies application.And this Inventing each ASGPR ligand individually connect with oligonucleotide, can simplicity control centre Zhi Peiti space length, adjusting ligand Quantity forms hundreds of combinations, expands oligonucleotide modification type, is conducive to find the higher patent medicine molecule of activity.(3) it expands ASGPR ligand classes.On the basis of GalNAc, a series of new ASGPR ligand substrates are prepared for, by these new ligands Screening be beneficial to the exploitation of more oligonucleotide original new drugs.

2. ligand is covalently attached synthetic method difference with oligonucleotide.Alnylam drugmaker uses three feeler GalNAc Ligand is connect with solid support, and the solid support after modification is used for oligonucleotide synthesis in solid state, so that ligand is connected to widow Nucleic acid 3 ' is held.ISIS pharmacy has been attempted three feeler GalNAc ligands being connected to 5 ' ends with nucleic acid phosphoramidite solid-phase synthesis End, but because steric hindrance is big, the connection that fails (Efficient Synthesis and Biological Evaluation 5 '-GalNAc of of), therefore develop a kind of three feeler GalNAc ligand of liquid phase and terminal amino group modifies oligonucleotide liquid phase connection method, The method achieve the reaction time 3 hours, reaction efficiency was greater than 95%.The method of the present invention is independent with oligonucleotide company by each ligand It connects, molecule steric hindrance is small, and joint efficiency is high.

3. it is different with combination to modify oligonucleotide site.End site is usually with cholesterol, poly- in oligonucleotide drug molecule The modification such as ethylene glycol (PEG), improves pharmacokinetic property.The three feeler GalNAc of Alnylam and ISIS Pharmaceuticals Inc.'s design It is terminal modified that ligand is only used for oligonucleotide last-in-chain(LIC), occupies terminal modification site, reduces the type that can be applied to oligonucleotide modification. Novel compound of present invention can modify oligonucleotide any position in synthesis in solid state, other modifications are not influenced by end. The preparation that noval chemical compound mixes modification oligonucleotide with end cholesterol is described in the embodiment of the present invention.

Embodiment of the present invention is described in detail below in conjunction with drawings and examples, still, art technology Personnel will be understood that, following drawings and embodiment are merely to illustrate the present invention, rather than the restriction to the scope of the present invention.According to attached The following detailed description of figure and preferred embodiment, various purposes of the invention and advantageous aspect carry out those skilled in the art Saying will be apparent.

Detailed description of the invention

Fig. 1 shows in embodiment 35, the GalNAc binding curve of each sample.

Sequence information

The information of sequence of the present invention is provided in following table:

Sequence number (SEQ ID NO :)	Description
		1	Artificial sequence
2	Artificial sequence
		3	Artificial sequence
4	Artificial sequence

Sequence 1 (SEQ ID NO:1): 19nt

CAGCAAGUGUGACAGUCAU

Sequence 2 (SEQ ID NO:2): 25nt

AUGACUGUCACACUUGCUGGCCUGU

Sequence 3 (SEQ ID NO:3): 19nt

CAGGCCAGCAAGUGUGACA

Sequence 4 (SEQ ID NO:4): 21nt

UGUCACACUUGCTGGCCUGUC

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is It can be with conventional products that are commercially available.

1 compound R of embodiment '₁The synthesis of-H

Using silk amine alcohol as raw material, bibliography (Choi J Y, BorchRF.Highly efficient synthesis of enantiomerically enriched 2-hydroxymethylaziridines by enzymatic Desymmetrization. [J] .Organic letters, 2007,9 (2): 215-218) it is prepared for compound 1, and further Prepare compound R '₁- H obtains white solid, two step total recoverys 49%.¹HNMR (400MHz, DMSO-d6) δ: 7.41-7.37 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J= 8.2Hz,4H),5.16(s,2H),4.63-4.58(m,1H),4.05-3.97(m,1H),3.74(s,6H),3.04-2.99(m, 2H),2.95-2.90(m,2H).MS(ESI),m/z:416.3([M+Na]⁺).

2 compound R of embodiment '₂The synthesis of-H

Referring to the method for embodiment 1, prepare compound R '₂- H is white solid, yield 55%.¹HNMR(400MHz, DMSO-d6) δ: 7.42-7.37 (d, J=7.2Hz, 2H), 7.35-7.29 (t, J=6.9Hz, 2H), 7.28-7.19 (m, 5H), 6.92-6.86 (d, J=8.2Hz, 4H), 5.17 (s, 1H), 4.63-4.59 (m, 1H), 3.74 (s, 6H), 3.05-2.99 (m, 2H), 2.96-2.90 (m, 2H), 2.88-2.81 (m, 4H) .MS (ESI), m/z:430.3 ([M+Na]⁺).

3 compound R of embodiment '₃The synthesis of-H

Using L- hydroxyproline methyl ester hydrochloride as raw material, referring to the method for embodiment 1, prepare compound R '₃- H is obtained white Color solid, yield 45%.¹HNMR (400MHz, DMSO-d6) δ: 7.42-7.37 (d, J=7.2Hz, 2H), 7.35-7.29 (t, J =6.9Hz, 2H), 7.28-7.19 (m, 5H), 6.92-6.86 (d, J=8.2Hz, 4H), 5.17 (s, 1H), 4.63-4.59 (m, 1H),3.74(s,6H),3.05-2.99(m,3H),2.90-2.86(m,2H),2.77-2.71(m,1H),1.88-1.81(m, 2H).MS(ESI),m/z:442.5([M+Na]⁺).

4 compound A of embodiment₁-I₁Synthesis

(1) synthesis of compound 3

In 1L round-bottomed flask, δ-penta lactones (100g, 1mol), sodium hydroxide (40g, 1mol), deionized water is added 400mL, mixing, 70 DEG C are reacted 6 hours, and TLC monitors end of reaction, are spin-dried for reaction solution, are spin-dried for, are obtained white after 200mL toluene is added Color solid 140g.

(2) synthesis of compound 4

In 1L round-bottomed flask, be added compound 3 (140g, 1mol), anhydrous propanone 500mL, cylite (205.2g, 1.2mol), catalyst tetrabutylammonium bromide (16.2g, 0.05mol), is heated to reflux, TLC monitoring reaction, rear fully reacting for 24 hours, After reaction solution is cooled to room temperature, acetone is removed under reduced pressure, residue is dissolved in 500mL ethyl acetate, successively molten with saturation sodium bisulfate Liquid 200mL, saturated sodium bicarbonate solution 200mL and saturated salt solution 200mL washing, organic phase is dry with anhydrous sodium sulfate, dense Contracting, through the isolated transparent oily liquid 175g of silicagel column (petroleum ether: ethyl acetate V:V=1:1), yield 84%.

(3) synthesis of compound 5

In 1L round-bottomed flask, D- galactose hydrochloride (100g, 0.46mol), anhydrous pyridine 450mL is added, delays under ice bath It is slow that acetic anhydride 325mL, triethylamine (64.5mL, 0.46mol), DMAP (2g, 0.016mol) is added.Normal-temperature reaction is stayed overnight, and is precipitated A large amount of solids filter, and filter cake is eluted with 0.5N HCl solution 200mL, obtain white solid 162.5g, yield 90%.¹H NMR (400MHz, DMSO-d6) δ: 7.88 (d, J=9.2Hz, 1H), 5.63 (d, J=8.8Hz, 1H), 5.26 (d, J=3.1Hz, 1H), 5.05 (d, J=11.3,3.3Hz, 1H), 4.36 (m, 4H), 2.11 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H), 1.90 (s,3H),1.78(s,3H).

(4) synthesis of compound 6

In 250mL round-bottomed flask, compound 5 (10g, 25.7mmol), anhydrous methylene chloride 100mL is added, stirs 10 points Trimethylsilyl trifluoromethanesulfonate (7mL, 38.7mmol) is added after clock, overnight, reaction solution is slowly poured into bicarbonate for room temperature reaction Stirring 0.5 hour in the aqueous solution (200mL) of sodium (7g, 79.5mmol), organic phase separation, anhydrous sodium sulfate is dry, depressurizes dense Contracting, obtains pale yellow gum 7.78g, yield 92%.

(5) synthesis of compound 7

In 100mL round-bottomed flask, compound 6 (5g, 15.2mmol), compound 4 (3.8g, 18.25mmol) are dissolved in nothing Water 1, in 2- dichloroethanes 50mL, Trimethylsilyl trifluoromethanesulfonate (0.55mL, 3mmol) is added after ten minutes in stirring, room temperature Cross reaction overnight, reaction solution is extracted with dichloromethane, and organic phase is washed twice with saturated sodium bicarbonate solution 50mL, anhydrous slufuric acid Sodium is dry, is concentrated under reduced pressure, through silicagel column (petroleum ether: ethyl acetate V:V=3:2) isolated transparent oily liquid, 6.94g, Yield 85%.¹HNMR (400MHz, DMSO-d6) δ: 7.69 (d, J=9.3Hz, 1H), 7.33-7.16 (m, 5H), 5.28 (d, J =5.3Hz, 1H), 4.95 (s, 2H), 4.93 (q, J=4.2Hz, 1H), 4.40 (d, J=8.6Hz, 1H), 4.00-3.86 (m, 3H), 3.73-3.56 (m, 2H), 3.36-3.21 (m, 1H), 2.53 (t, J=8.2Hz, 2H), 2.11 (s, 3H), 1.89 (s, 3H),1.83(s,3H),1.65(s,3H),1.59–1.36(m,4H).MS(ESI),m/z:560.2([M+Na]⁺).

(6) compound A₁-I₁Synthesis

In 50mL round-bottomed flask, compound 7 (3.3g, 6.1mmmol), Pd/C (0.33g, 10%) be dissolved in 5mL methanol and In 20mL ethyl acetate, hydrogen balloon is accessed, normal-temperature reaction is overnight.The filtering of reaction solution diatomite, the elution of diatomite methanol, filtrate subtract Pressure concentration is spin-dried for, and obtains white solid 2.8g, yield 95.5%.¹HNMR (400MHz, DMSO-d6) δ: 11.98 (s, 1H), 7.79- 7.75 (d, J=8.9Hz, 1H), 5.20 (s, 1H), 5.0-4.95 (q, J=4.2Hz, 1H), 4.46-4.51 (d, J=7.2Hz, 1H), 4.15-4.07 (m, 3H), 3.89-3.79 (m, 1H), 3.80-3.69 (m, 1H), 3.46-3.36 (m, 1H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.59-1.42 (m, 4H) .MS(ESI),m/z:470.5([M+Na]⁺).

5 compound A of embodiment₁-I₂Synthesis

(1) synthesis of compound 8

In 100mL round-bottomed flask, compound 6 (5g, 15.2mmol), 10- undecylenic alcohol (3.1g, 18.24mmol) are dissolved in In 50mL anhydrous methylene chloride, Trimethylsilyl trifluoromethanesulfonate (0.55mL, 3.0mmol) is added after ten minutes in stirring, room temperature Overnight, reaction solution is extracted with dichloromethane, and organic phase is washed twice with saturated sodium bicarbonate solution 50mL, anhydrous sodium sulfate for reaction It is dry, it is concentrated under reduced pressure, through the isolated white solid 6.59g of silicagel column (petroleum ether: ethyl acetate V:V=3:2), yield 87%.¹HNMR (400MHz, DMSO-d6) δ: 7.82 (d, J=3.3Hz, 1H), 5.86-5.73 (m, 1H), 5.22 (s, 1H), 5.02-4.9 (m, 3H), 4.5-4.98 (s, J=3.5Hz, 1H), 4.08-3.99 (m, 3H), 3.9-3.88 (m, 1H), 3.73- 3.65(m,1H),3.48-3.38(m,1H),2.12(s,3H),2.05-2.01(m,2H),2.00(s,3H),1.88(s,3H), 1.66(s,3H),1.5-1.4(m,2H),1.39-1.3(m,2H),1.29-1.19(m,10H).MS(ESI),m/z:522.4([M +Na]⁺).

(2) compound A₁-I₂Synthesis

In 100mL round-bottomed flask, compound 8 (4g, 8.02mmol), methylene chloride 50mL, acetonitrile 50mL, deionization is added Water 70mL, is added portionwise NaIO₄(6.86g, 32.1mmol), normal-temperature reaction 48h, TLC monitor end of reaction.Reaction solution addition is gone Ionized water 100mL, methylene chloride extract three times (50mL × 3), merge organic phase, and anhydrous sodium sulfate is dry, and reduced pressure is spin-dried for, Obtain filbert gum-like product 4.1g, yield 99%.¹HNMR (400MHz, DMSO-d6) δ: 11.99 (s, 1H), 7.82 (d, J= 3.3Hz, 1H), 5.22 (s, 1H), 5.02-4.9 (m, 1H), 4.5-4.98 (s, J=3.5Hz, 1H), 4.08-3.99 (m, 3H), 3.9-3.88(m,1H),3.73-3.65(m,1H),3.48-3.38(m,1H),2.12(s,3H),2.05-2.01(m,2H), 2.00(s,3H),1.88(s,3H),1.66(s,3H),1.5-1.4(m,2H),1.39-1.3(m,2H),1.29-1.19(m, 10H).MS(ESI),m/z:540.26([M+Na]⁺).

6 compound A of embodiment₂-I₁Synthesis

(1) synthesis of compound 9

9 reference literature of compound [2] Hudson, C.S.；Johnson,J.J.Am.Chem.Soc.1915,37,1270– 1275 synthesis.

H NMR (400MHz, DMSO-d6) δ: 5.20 (s, 2H), 4.95 (q, J=4.2Hz, 2H), 4.51 (d, J= 7.2Hz, 1H), 4.46 (d, J=7.2Hz, 1H), 4.15-3.97 (m, 6H), 3.89-3.79 (m, 2H), 2.23 (s, 3H), 2.15 (s, 6H), 2.00 (s, 6H), 1.95 (s, 6H), 1.87 (s, 3H) .MS (ESI), m/z:701.6 ([M+Na]⁺).

(2) synthesis of compound 10

500mL round-bottomed flask, compound 9 (20g, 29.5mmol), compound 4 (9.2g, 44.3mmol) be dissolved in 200mL without In water methylene chloride, BF is added dropwise under ice bath₃-OEt₂(14.8mL), reaction keep ice bath reaction for 24 hours, and TLC monitors end of reaction.Silicon Diatomaceous earth filtering, filtrate are dissolved in 500mL ethyl acetate, are successively washed with saturated sodium bicarbonate solution 200mL and saturated salt solution 200mL It washs.Organic phase is dry with anhydrous magnesium sulfate, is concentrated under reduced pressure, separates white through silicagel column (petroleum ether: ethyl acetate V:V=3:2) Color solid 17.06g, yield 70%.MS(ESI),m/z:849.26([M+Na]⁺).

(3) compound A₂-I₁Synthesis

In 100mL round-bottomed flask, compound 10 (10g, 12.1mmol), Pd/C (1g, 10%), be dissolved in 10mL methanol and In 50mL ethyl acetate, nitrogen displaced air accesses hydrogen balloon, and normal-temperature reaction is overnight.The filtering of reaction solution diatomite, diatomite first Alcohol elution, filtrate decompression are spin-dried for, and obtain white solid 8.5g, yield 95.5%.H NMR (400MHz, DMSO-d6) δ: 11.98 (s, 1H), 5.20 (s, 2H), 4.95 (q, J=4.2Hz, 2H), 4.51 (d, J=7.2Hz, 1H), 4.46 (d, J=7.2Hz, 1H), 4.15–3.97(m,6H),3.89–3.79(m,2H),3.80–3.69(m,1H),3.46–3.36(m,1H),2.22-2.14(t,J =7.2Hz, 2H), 2.15 (s, 6H), 2.00 (s, 6H), 1.95 (s, 6H), 1.87 (s, 3H), 1.59-1.42 (m, 4H) .MS (ESI),m/z:759.26([M+Na]⁺).

7 compound A of embodiment₂-I₂Synthesis

(1) synthesis of compound 11

In 500mL round-bottomed flask, compound 9 (20g, 29.5mmol), 10- undecylenic alcohol (6g, 35.4mmol) are dissolved in In the anhydrous DCM of 200mL, BF is added dropwise under ice bath₃-OEt₂(14.8mL) keeps ice bath reaction for 24 hours, and TLC monitors end of reaction.Diatom Soil filtering, filtrate are dissolved in 500mL ethyl acetate, are successively washed with saturated sodium bicarbonate solution 200mL and saturated salt solution 200mL It washs.Organic phase is dry with anhydrous magnesium sulfate, is concentrated under reduced pressure, separates white through silicagel column (petroleum ether: ethyl acetate V:V=3:2) Color solid 19.5g, yield 83.9%, MS (ESI), m/z:811.25 ([M+Na]⁺).

(2) compound A₂-I₂Synthesis

It is raw material with compound 11, referring to A₁-I₂Synthesis.Yield 87%.¹HNMR (400MHz, DMSO-d6) δ: 11.82 (s, 1H), 5.86-5.73 (m, 1H), 5.22 (s, 1H), 5.2-4.9 (m, 6H), 4.5-4.98 (s, J=3.5Hz, 2H), 4.08- 3.99(m,3H),3.9-3.88(m,2H),3.73-3.65(m,2H),3.48-3.38(m,1H),2.12(s,6H),2.05- 2.01(m,2H),2.00(s,6H),1.88(s,6H),1.66(s,3H),1.5-1.4(m,2H),1.39-1.3(m,2H), 1.29-1.19(m,8H).MS(ESI),m/z:829.7([M+Na]⁺).

8 compound A of embodiment₃-I₁Synthesis

(1) synthesis of compound 12

The synthesis of reference compound 5.White solid, yield 91%.¹H NMR (400MHz, DMSO-d6) δ: 5.63 (d, J =8.8Hz, 1H), 5.26 (d, J=3.1Hz, 1H), 5.05 (d, J=11.3,3.3Hz, 1H), 4.36 (m, 4H), 2.11 (s, 3H),2.03(s,3H),1.98(s,3H),1.90(s,3H),1.78(s,3H).MS(ESI),m/z:391.21([M+1]⁺).

(2) synthesis of compound 13

It is raw material, the synthesis of reference compound 10 with compound 12.Transparent oily liquid, yield 86%.

¹HNMR (400MHz, DMSO-d6) δ: 7.33-7.16 (m, 5H), 5.28 (d, J=5.3Hz, 1H), 4.95 (s, 2H), 4.93 (q, J=4.2Hz, 1H), 4.40 (d, J=8.6Hz, 1H), 4.00-3.86 (m, 3H), 3.73-3.56 (m, 1H), 3.36-3.21 (m, 2H), 2.53 (t, J=8.2Hz, 2H), 2.11 (s, 3H), 1.89 (s, 3H), 1.83 (s, 3H), 1.65 (s, 3H),1.59–1.36(m,4H).MS(ESI),m/z:561.2([M+Na]⁺).

(3) compound A₃-I₁Synthesis

It is raw material, reference compound A with compound 13₁-I₁Synthesis.White solid, yield 93.5%.¹HNMR (400MHz, DMSO-d6) δ: 11.98 (s, 1H), 5.20 (s, 1H), 4.95 (q, J=4.2Hz, 1H), 4.51 (d, J=7.2Hz, 1H), 4.15-3.97 (m, 3H), 3.89-3.79 (m, 1H), 3.80-3.69 (m, 1H), 3.46-3.36 (m, 1H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.59-1.42 (m, 4H) .MS(ESI),m/z:471.5([M+Na]⁺).

9 compound A of embodiment₃-I₂Synthesis

(1) synthesis of compound 14

It is raw material, the synthesis of reference compound 11 with compound 12.White solid, yield 88%.¹HNMR(400MHz, DMSO-d6) δ: 5.86-5.73 (m, 1H), 5.22 (s, 1H), 5.2-4.9 (m, 3H), 4.5-4.98 (s, J=3.5Hz, 1H), 4.08-3.99(m,3H),3.9-3.88(m,1H),3.73-3.65(m,1H),3.48-3.38(m,1H),2.12(s,3H), 2.05-2.01(m,2H),2.00(s,3H),1.88(s,3H),1.66(s,3H),1.5-1.4(m,2H),1.39-1.3(m, 2H),1.29-1.19(m,10H).MS(ESI),m/z:523.5([M+Na]⁺).

(2) compound A₃-I₂Synthesis

It is raw material, reference compound A with compound 14₁-I₂Synthesis.Filbert gum-like product, yield 97%.¹HNMR (400MHz, DMSO-d6) δ: 11.99 (s, 1H), 5.22 (s, 1H), 5.02-4.9 (m, 1H), 4.5-4.98 (s, J=3.5Hz, 1H),4.08-3.99(m,3H),3.9-3.88(m,1H),3.73-3.65(m,1H),3.48-3.38(m,1H),2.12(s, 3H),2.05-2.01(m,2H),2.00(s,3H),1.88(s,3H),1.66(s,3H),1.5-1.4(m,2H),1.39-1.3 (m,2H),1.29-1.19(m,10H).MS(ESI),m/z:541.3([M+Na]⁺).

10 compound A of embodiment₁-IV₁Synthesis

(1) synthesis of compound 15

It is raw material, the synthesis of reference compound 8 with compound 6.MS(ESI),m/z:484.2([M+1]⁺).

(2) compound A₁-IV₁Synthesis

It is raw material, reference compound A with compound 15₁-I₂Synthesis.¹HNMR (400MHz, DMSO-d6) δ: 11.88 (s, 1H), 7.77-7.73 (d, J=8.9Hz, 1H), 5.21 (s, 1H), 5.0-4.96 (q, J=4.2Hz, 1H), 4.45-4.51 (d, J =7.2Hz, 1H), 4.12-4.07 (m, 3H), 3.88-3.78 (m, 1H), 3.72-3.68 (m, 2H), 3.62-3.58 (m, 2H), 3.56-3.46 (m, 4H), 2.15 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H) .MS (ESI), m/z:502.6 ([M+1]⁺).

11 compound A of embodiment₁-I₁-R₁Synthesis

(1) synthesis of compound 16

In 250mL round-bottomed flask, compound A is added₁-I₁(10g, 22.35mmol), 1- ethyl-(3- dimethylamino third Base) phosphinylidyne diimmonium salt hydrochlorate (EDC.HCL) (5.14g, 26.82mmol), n-hydroxysuccinimide (2.83g, 24.59mmol), methylene chloride 100mL.Compound R is added after stirring at normal temperature reaction 0.5h₁- H (8.79g, 22.35mmol), TLC monitoring is reacted, fully reacting after 4h.Reaction solution is successively washed with saturated sodium bicarbonate solution 50mL and saturated salt solution 50mL It washs, organic phase anhydrous sodium sulfate is dry, concentration, through silicagel column (methylene chloride: methanol V:V=20:1) isolated white solid 15.8g, yield 86%.MS(ESI),m/z:845.2([M+Na]⁺).

(2) compound A₁-I₁-R₁Synthesis

It in 250mL bottle with two necks, is added compound 16 (5g, 6.08mmol), nitrogen protection, anhydrous acetonitrile 100mL, double is added (diisopropylaminoethyl) (2- cyanoethoxy) phosphine (3.66g, 12.16mmol), is slowly added dropwise the second of ethlythiotetrazole under stirring Nitrile solution (2.5M) (1.22mL, 3.04mmol) reacts 0.5h, and TLC monitoring is reacted, fully reacting after 0.5h.Reduced pressure removes Acetonitrile is removed, methylene chloride 100mL dissolution, saturated salt solution 100mL washing is added.Organic phase is dry with anhydrous sodium sulfate, concentration, Through the isolated white solid 5.16g of silicagel column (petroleum ether: ethyl acetate V:V=1:3), yield 83%.¹H NMR (400MHz, DMSO-d6) δ: 7.84-7.79 (d, J=8.9Hz, 1H), 7.65-7.60 (d, J=8.9Hz, 1H), 7.41-7.37 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J= 8.2Hz, 4H), 5.20 (s, 1H), 5.0-4.95 (q, J=4.2Hz, 1H), 4.51-4.46 (d, J=7.2Hz, 1H), 4.15- 4.06(m,3H),4.05-3.96(m,1H),3.84-3.80(m,2H),3.89-3.79(m,1H),3.74(s,6H),3.71- 3.69 (m, 1H), 3.46-3.36 (m, 1H), 3.04-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.88-2.84 (m, 2H), 2.59-2.54 (m, 2H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.77 (s, 12H), 1.59-1.42 (m, 4H) .MS (ESI), m/z:1045.5 ([M+Na]⁺).

12 compound A of embodiment₁-I₁-R₂Synthesis

(1) synthesis of compound 17

With compound A₁-I₁For raw material, the synthesis of reference compound 16.White solid, yield 82.5%.MS(ESI),m/ z:859.2([M+Na]⁺).

(2) compound A₁-I₁-R₂Synthesis

It is raw material, reference compound A with compound 17₁-I₁-R₁Synthesis.White solid, yield 84.2%.¹H NMR (400MHz, DMSO-d6) δ: 7.83-7.79 (d, J=8.8Hz, 1H), 7.42-7.37 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J=8.2Hz, 4H), 5.20 (s, 1H), 5.0-4.95 (q, J=4.2Hz, 1H), 4.51-4.46 (d, J=7.2Hz, 1H), 4.15-3.97 (m, 3H), 4.05-3.96 (m, 1H), 3.84-3.80 (m, 2H), 3.89-3.79 (m, 1H), 3.74 (s, 6H), 3.71-3.69 (m, 1H), 3.46-3.36 (m, 1H), 3.04-2.99 (m, 2H), 2.98-2.95 (m, 2H), 2.89-2.93 (m, 4H), 2.88-2.84 (m, 2H), 2.60-2.55 (m, 2H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.77 (s,12H),1.59–1.42(m,4H).MS(ESI),m/z:1059.6([M+Na]⁺).

13 compound A of embodiment₁-I_1-R₃Synthesis

(1) synthesis of compound 18

With compound A₁-I₁For raw material, the synthesis of reference compound 16.White solid, yield 86%.MS(ESI),m/z: 871.2([M+Na]⁺).

(2) compound A₁-I₁-R₃Synthesis

It is raw material, reference compound A with compound 18₁-I₁-R₁Synthesis.White solid, yield 84.2%.

¹HNMR (400MHz, DMSO-d6) δ: 7.73-7.70 (d, J=7.9Hz, 1H), 7.42-7.37 (d, J=7.2Hz, 2H), 7.35-7.29 (t, J=6.9Hz, 2H), 7.28-7.19 (m, 5H), 6.92-6.86 (d, J=8.2Hz, 4H), 5.20 (s, 1H), 5.0-4.95 (q, J=4.2Hz, 1H), 4.51-4.46 (d, J=7.2Hz, 1H), 4.15-3.97 (m, 3H), 3.89- 3.79(m,3H),3.74(s,6H),3.70–3.67(m,1H),3.46–3.36(m,1H),3.05–2.99(m,3H),2.90– 2.86 (m, 3H), 2.77-2.71 (m, 1H), 2.60-2.55 (m, 2H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.88-1.81 (m, 2H), 1.77 (s, 12H), 1.59-1.42 (m, 4H).MS(ESI),m/z:1071.4([M+Na]⁺).

14 compound A of embodiment₁-I₂-R₁Synthesis

(1) synthesis of compound 19

With compound A₁-I₂For raw material, the synthesis of reference compound 16.White solid, yield 85.6%.MS(ESI),m/ z:915.5([M+Na]⁺).

(2) compound A₁-I₂-R₁Synthesis

It is raw material, reference compound A with compound 19₁-I₁-R₁Synthesis.White solid, yield 82.1%.¹HNMR (400MHz, DMSO-d6) δ: 7.82-7.78 (d, J=7.3Hz, 1H), 7.69-7.63 (d, J=7.3Hz, 1H), 7.41-7.37 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J= 8.2Hz, 4H), 5.22 (s, 1H), 5.02-4.9 (m, 1H), 4.5-4.98 (s, J=3.5Hz, 1H), 4.08-3.99 (m, 3H), 4.05–3.97(m,1H),3.9-3.88(m,1H),3.84–3.80(m,2H),3.74(s,6H),3.73-3.65(m,1H), 3.48-3.38 (m, 1H), 3.04-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.88-2.84 (m, 2H), 2.61-2.55 (m, 2H), 2.12 (s, 3H), 2.05-2.01 (m, 2H), 2.00 (s, 3H), 1.88 (s, 3H), 1.77 (s, 12H), 1.66 (s, 3H), 1.5-1.4(m,2H),1.39-1.3(m,2H),1.29-1.19(m,10H).MS(ESI),m/z:1115.2([M+Na]⁺).

15 compound A of embodiment₁-I₂-R₂Synthesis

(1) synthesis of compound 20

With compound A₁-I₂For raw material, the synthesis of reference compound 16.White solid, yield 84.2%.MS(ESI),m/ z:929.3([M+Na]⁺).

(2) compound A₁-I₂-R₂Synthesis

It is raw material, reference compound A with compound 20₁-I₁-R₁Synthesis.White solid, yield 81.1%.¹HNMR (400MHz, DMSO-d6) δ: 7.83-7.77 (d, J=7.3Hz, 1H), 7.41-7.37 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J=8.2Hz, 4H), 5.22 (s, 1H), 5.02-4.9 (m, 1H), 4.5-4.98 (s, J=3.5Hz, 1H), 4.08-3.99 (m, 3H), 3.9-3.88 (m, 1H), 3.84-3.80 (m, 2H),3.74(s,6H),3.73-3.65(m,1H),3.48-3.38(m,1H),3.15–3.11(m,4H),3.04–2.99(m, 2H), 2.95-2.90 (m, 2H), 2.88-2.84 (m, 2H), 2.61-2.55 (m, 2H), 2.12 (s, 3H), 2.05-2.01 (m, 2H), 2.00 (s, 3H), 1.88 (s, 3H), 1.77 (s, 12H), 1.66 (s, 3H), 1.5-1.4 (m, 2H), 1.39-1.3 (m, 2H),1.29-1.19(m,10H).MS(ESI),m/z:1129.4([M+Na]⁺).

16 compound A of embodiment₁-I₂-R₃Synthesis

(1) synthesis of compound 21

With compound A₁-I₂For raw material, the synthesis of reference compound 16.White solid, yield 80.5%.MS(ESI),m/ z:941.1([M+Na]⁺).

(2) compound A₁-I₂-R₃Synthesis

It is raw material, reference compound A with compound 21₁-I₁-R₁Synthesis.White solid, yield 81.1%

¹HNMR (400MHz, DMSO-d6) δ: 7.82 (d, J=3.3Hz, 1H), 7.42-7.37 (d, J=7.2Hz, 2H), 7.35-7.29 (t, J=6.9Hz, 2H), 7.28-7.19 (m, 5H), 6.92-6.86 (d, J=8.2Hz, 4H), 5.22 (s, 1H), 5.02-4.9 (m, 1H), 4.5-4.98 (s, J=3.5Hz, 1H), 4.08-3.99 (m, 3H), 3.9-3.88 (m, 1H), 3.84- 3.80(m,2H),3.76(s,6H),3.73-3.65(m,1H),3.48-3.38(m,1H),3.05–2.99(m,3H),2.90– 2.84(m,4H),2.77–2.71(m,1H),2.62–2.56(m,2H),2.12(s,3H),2.05-2.01(m,2H),2.00(s, 3H), 1.88 (s, 3H), 1.87-1.81 (m, 2H), 1.77 (s, 12H), 1.66 (s, 3H), 1.5-1.4 (m, 2H), 1.39-1.3 (m,2H),1.29-1.19(m,10H).MS(ESI),m/z:1141.2([M+Na]⁺).

17 compound A of embodiment₂-I₁-R₁Synthesis

(1) synthesis of compound 22

With compound A₂-I₁For raw material, the synthesis of reference compound 16.White solid, yield 80.4%.MS(ESI),m/ z:1134.7([M+Na]⁺).

(2) compound A₂-I₁-R₁Synthesis

It is raw material, reference compound A with compound 22₁-I₁-R₁Synthesis.White solid, yield 81.3%.H NMR (400MHz, DMSO-d6) δ: 7.61-7.57 (d, J=7.2Hz, 1H), 7.41-7.37 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J=8.2Hz, 4H), 5.20 (s, 2H), 4.95 (q, J =4.2Hz, 2H), 4.51 (d, J=7.2Hz, 1H), 4.46 (d, J=7.2Hz, 1H), 4.15-3.97 (m, 6H), 4.05-3.97 (m,1H),3.89–3.79(m,4H),3.75(s,6H),3.80–3.69(m,1H),3.46–3.36(m,1H),3.04–2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.88-2.84 (m, 2H), 2.61-2.55 (m, 2H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 6H), 2.00 (s, 6H), 1.95 (s, 6H), 1.87 (s, 3H), 1.77 (s, 12H), 1.59-1.42 (m, 4H) .MS(ESI),m/z:1334.2([M+Na]⁺).

18 compound A of embodiment₂-I₁-R₂Synthesis

(1) synthesis of compound 23

With compound A₂-Ι₁For raw material, the synthesis of reference compound 16.White solid, yield 86.1%.MS(ESI),m/ z:1148.3([M+Na]⁺).

(2) compound A₂-I₁-R₂Synthesis

It is raw material, reference compound A with compound 23₁-I₁-R₁Synthesis.White solid, yield 82.3%.H NMR (400MHz, DMSO-d6) δ: 7.40-7.36 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J=8.2Hz, 4H), 5.20 (s, 2H), 4.95 (q, J=4.2Hz, 2H), 4.51 (d, J= 7.2Hz, 1H), 4.46 (d, J=7.2Hz, 1H), 4.15-3.97 (m, 6H), 3.89-3.79 (m, 4H), 3.75 (s, 6H), 3.80–3.69(m,1H),3.46–3.41(m,4H),3.46–3.36(m,1H),3.04–2.99(m,2H),2.95–2.90(m, 2H), 2.88-2.84 (m, 2H), 2.61-2.55 (m, 2H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 6H), 2.00 (s, 6H), 1.95 (s, 6H), 1.87 (s, 3H), 1.77 (s, 12H), 1.59-1.42 (m, 4H) .MS (ESI), m/z:1348.2 ([M+Na]⁺).

19 compound A of embodiment₂-I₁-R₃Synthesis

(1) synthesis of compound 24

With compound A₂-I₁For raw material, the synthesis of reference compound 16.White solid, yield 82.9%.MS(ESI),m/ z:1160.5([M+Na]⁺).

(2) compound A₂-I₁-R₃Synthesis

It is raw material, reference compound A with compound 24₁-I₁-R₁Synthesis.White solid, yield 82.3%.H NMR (400MHz, DMSO-d6) δ: 7.44-7.39 (d, J=7.2Hz, 2H), 7.36-7.30 (t, J=6.9Hz, 2H), 7.28-7.19 (m, 5H), 6.92-6.86 (d, J=8.2Hz, 4H), 5.20 (s, 2H), 4.95 (q, J=4.2Hz, 2H), 4.51 (d, J= 7.2Hz, 1H), 4.46 (d, J=7.2Hz, 1H), 4.15-3.97 (m, 6H), 3.89-3.82 (m, 4H), 3.80-3.76 (m, 1H), 3.74 (s, 6H), 3.46-3.36 (m, 1H), 3.05-2.99 (m, 3H), 2.90-2.86 (m, 2H), 2.77-2.71 (m, 1H), 2.61-2.56 (m, 2H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 6H), 2.00 (s, 6H), 1.95 (s, 6H), 1.87 (s, 3H), 1.86-1.81 (m, 2H), 1.77 (s, 12H), 1.59-1.42 (m, 4H) .MS (ESI), m/z: 1360.26([M+Na]⁺).

20 compound A of embodiment₂-I₂-R₁Synthesis

(1) synthesis of compound 25

With compound A₂-I₂For raw material, the synthesis of reference compound 16.White solid, yield 86.3%.MS(ESI),m/ z:1204.6([M+Na]⁺).

(2) compound A₂-I₂-R₁Synthesis

It is raw material, reference compound A with compound 25₁-I₁-R₁Synthesis.White solid, yield 80.3%.¹HNMR (400MHz, DMSO-d6) δ: 7.71-7.66 (d, J=7.2Hz, 1H), 7.41-7.37 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J=8.2Hz, 4H), 5.86-5.73 (m, 1H), 5.22 (s, 1H), 5.2-4.9 (m, 6H), 4.5-4.98 (s, J=3.5Hz, 2H), 4.08-3.99 (m, 4H), 3.9-3.88 (m, 2H), 3.84-3.80 (m, 2H), 3.75 (s, 6H), 3.73-3.65 (m, 2H), 3.48-3.38 (m, 1H), 3.04-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.88-2.84 (m, 2H), 2.59-2.55 (m, 2H), 2.12 (s, 6H), 2.05-2.01 (m, 2H), 2.00 (s, 6H), 1.88 (s, 6H), 1.77 (s, 12H), 1.66 (s, 3H), 1.5-1.4 (m, 2H), 1.39-1.3 (m, 2H), 1.29-1.19(m,8H).MS(ESI),m/z:1404.7([M+Na]⁺).

21 compound A of embodiment₂-I₂-R₂Synthesis

(1) synthesis of compound 26

With compound A₂-I₂For raw material, the synthesis of reference compound 16.White solid, yield 87.3%.MS(ESI),m/ z:1218.4([M+Na]⁺).

(2) compound A₂-I₂-R₂Synthesis

It is raw material, reference compound A with compound 26₁-I₁-R₁Synthesis.White solid, yield 84.3%.¹HNMR (400MHz, DMSO-d6) δ: 7.41-7.37 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J=8.2Hz, 4H), 5.86-5.73 (m, 1H), 5.22 (s, 1H), 5.2-4.9 (m, 6H), 4.5- 4.98 (s, J=3.5Hz, 2H), 3.9-3.88 (m, 2H), 3.84-3.80 (m, 2H), 3.75 (s, 6H), 3.73-3.65 (m, 2H), 3.48-3.38 (m, 1H), 3.3.16-3.12 (m, 4H), 3.04-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.88- 2.84 (m, 2H), 2.59-2.55 (m, 2H), 2.12 (s, 6H), 2.05-2.01 (m, 2H), 2.00 (s, 6H), 1.88 (s, 6H), 1.77 (s, 12H), 1.66 (s, 3H), 1.5-1.4 (m, 2H), 1.39-1.3 (m, 2H), 1.29-1.19 (m, 8H) .MS (ESI), m/z:1418.7([M+Na]⁺).

22 compound A of embodiment₂-I₂-R₃Synthesis

(1) synthesis of compound 27

With compound A₂-I₂For raw material, the synthesis of reference compound 16.White solid, yield 88.0%.MS(ESI),m/ z:1230.2([M+Na]⁺).

(2) compound A₂-I₂-R₃Synthesis

It is raw material, reference compound A with compound 27₁-I₁-R₁Synthesis.White solid, yield 87.0%.¹HNMR (400MHz, DMSO-d6) δ: 7.42-7.37 (d, J=7.2Hz, 2H), 7.35-7.29 (t, J=6.9Hz, 2H), 7.28-7.19 (m, 5H), 6.92-6.86 (d, J=8.2Hz, 4H), 5.86-5.73 (m, 1H), 5.22 (s, 1H), 5.2-4.9 (m, 6H), 4.5- 4.98 (s, J=3.5Hz, 2H), 4.08-3.99 (m, 3H), 3.9-3.88 (m, 2H), 3.84-3.80 (m, 2H), 3.74 (s, 6H), 3.73-3.65 (m, 2H), 3.48-3.38 (m, 1H), 3.05-2.99 (m, 3H), 2.90-2.86 (m, 4H), 2.77-2.71 (m, 1H), 2.60-2.56 (m, 2H), 2.12 (s, 6H), 2.05-2.01 (m, 2H), 2.00 (s, 6H), 1.88 (s, 6H), 1.86- 1.81 (m, 2H), 1.77 (s, 12H), 1.66 (s, 3H), 1.5-1.4 (m, 2H), 1.39-1.3 (m, 2H), 1.29-1.19 (m, 8H).MS(ESI),m/z:1430.2([M+Na]⁺).

23 compound A of embodiment₃-I₁-R₁Synthesis

(1) synthesis of compound 28

With compound A₃-I₁For raw material, the synthesis of reference compound 16.White solid, yield 88.2%.MS(ESI),m/ z:846.3([M+Na]⁺).

(2) compound A₃-I₁-R₁Synthesis

It is raw material, reference compound A with compound 28₁-I₁-R₁Synthesis.White solid, yield 84.2%.¹HNMR (400MHz, DMSO-d6) δ: 7.61-7.56 (d, J=7.2Hz, 1H), 7.41-7.37 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J=8.2Hz, 4H), 5.20 (s, 1H), 4.95 (q, J =4.2Hz, 1H), 4.51 (d, J=7.2Hz, 1H), 4.15-3.97 (m, 4H), 3.89-3.79 (m, 3H), 3.75 (s, 6H), 3.73-3.69 (m, 1H), 3.46-3.36 (m, 1H), 3.04-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.88-2.84 (m, 2H), 2.59-2.54 (m, 2H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.77 (s, 12H), 1.59-1.42 (m, 4H) .MS (ESI), m/z:1046.5 ([M+Na]⁺).

24 compound A of embodiment₃-I₁-R₂Synthesis

(1) synthesis of compound 29

With compound A₃-I₁For raw material, the synthesis of reference compound 16.White solid, yield 88.9%.MS(ESI),m/ z:860.3([M+Na]⁺).

(2) compound A₃-I₁-R₂Synthesis

It is raw material, reference compound A with compound 29₁-I₁-R₁Synthesis.White solid, yield 84.7%.¹HNMR (400MHz, DMSO-d6) δ: 7.41-7.37 (d, J=7.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J=8.2Hz, 4H), 5.20 (s, 1H), 4.95 (q, J=4.2Hz, 1H), 4.51 (d, J= 7.2Hz,1H),3.89-3.79(m,3H),3.75(s,6H),3.73-3.69(m,1H),3.46-3.36(m,1H),3.04- 2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.88-2.84 (m, 2H), 2.82-2.78 (m, 4H), 2.59-2.54 (m, 2H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.77 (s, 12H), 1.59-1.42 (m, 4H) .MS (ESI), m/z:1060.5 ([M+Na]⁺).

25 compound A of embodiment₃-I₁-R₃Synthesis

(1) synthesis of compound 30

With compound A₃-I₁For raw material, the synthesis of reference compound 16.White solid, yield 84.3%.MS(ESI),m/ z:872.7([M+Na]⁺).

(2) compound A₃-I₁-R₃Synthesis

It is raw material, reference compound A with compound 30₁-I₁-R₁Synthesis.White solid, yield 86.2%.¹HNMR (400MHz, DMSO-d6) δ: 7.46-7.39 (d, J=7.2Hz, 2H), 7.37-7.31 (t, J=6.9Hz, 2H), 7.28-7.19 (m, 5H), 6.92-6.86 (d, J=8.2Hz, 4H), 5.20 (s, 1H), 4.95 (q, J=4.2Hz, 1H), 4.51 (d, J= 7.2Hz,1H),4.15-3.97(m,3H),3.89-3.79(m,3H),3.76(s,6H),3.72-3.68(m,1H),3.46- 3.36 (m, 1H), 3.05-2.99 (m, 3H), 2.90-2.86 (m, 2H), 2.88-2.84 (m, 2H), 2.77-2.71 (m, 1H), 2.59-2.54 (m, 2H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.86-1.81 (m, 2H), 1.77 (s, 12H), 1.59-1.42 (m, 4H) .MS (ESI), m/z:1072.8 ([M+ Na]⁺).

26 compound A of embodiment₃-I₂-R₁Synthesis

(1) synthesis of compound 31

With compound A₃-I₂For raw material, the synthesis of reference compound 16.White solid, yield 86.2%.MS(ESI),m/ z:916.4([M+Na]⁺).

(2) compound A₃-I₂-R₁Synthesis

It is raw material, reference compound A with compound 31₁-I₁-R₁Synthesis.White solid, yield 85.1%.¹HNMR (400MHz, DMSO-d6) δ: 7.75-7.71 (d, J=7.2Hz, 1H), 7.42-7.39 (d, J=7.2Hz, 2H), 7.32-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J=8.2Hz, 4H), 5.22 (s, 1H), 5.02-4.9 (m, 1H), 4.5-4.98 (s, J=3.5Hz, 1H), 4.05-3.99 (m, 4H), 3.9-3.88 (m, 1H), 3.84-3.80 (m, 2H), 3.76 (s, 6H), 3.73-3.65 (m, 1H), 3.48-3.38 (m, 1H), 3.04-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.87-2.84 (m, 2H), 2.58-2.54 (m, 2H), 2.12 (s, 3H), 2.05-2.01 (m, 2H), 2.00 (s, 3H), 1.88 (s, 3H), 1.77 (s, 12H), 1.66 (s, 3H), 1.5-1.4 (m, 2H), 1.39-1.3 (m, 2H), 1.29-1.19 (m, 10H).MS(ESI),m/z:1116.6([M+Na]⁺).

27 compound A of embodiment₃-I₂-R₂Synthesis

(1) synthesis of compound 32

With compound A₃-I₂For raw material, the synthesis of reference compound 16.White solid, yield 82.9%.MS(ESI),m/ z:930.7([M+Na]⁺).

(2) compound A₃-I₂-R₂Synthesis

It is raw material, reference compound A with compound 32₁-I₁-R₁Synthesis.White solid, yield 84.3%.¹HNMR (400MHz, DMSO-d6) δ: 7.44-7.39 (d, J=7.2Hz, 2H), 7.32-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J=8.2Hz, 4H), 5.22 (s, 1H), 5.02-4.9 (m, 1H), 4.5-4.98 (s, J= 3.5Hz, 1H), 3.9-3.88 (m, 1H), 3.84-3.80 (m, 2H), 3.76 (s, 6H), 3.73-3.65 (m, 1H), 3.48-3.38 (m, 1H), 3.04-2.99 (m, 2H), 2.97-2.94 (m, 2H), 2.93-2.88 (m, 4H), 2.87-2.84 (m, 2H), 2.58- 2.54 (m, 2H), 2.12 (s, 3H), 2.05-2.01 (m, 2H), 2.00 (s, 3H), 1.88 (s, 3H), 1.77 (s, 12H), 1.66 (s, 3H), 1.5-1.4 (m, 2H), 1.39-1.3 (m, 2H), 1.29-1.19 (m, 10H) .MS (ESI), m/z:1130.6 ([M+ Na]⁺).

28 compound A of embodiment₃-I₂-R₃Synthesis

(1) synthesis of compound 33

With compound A₃-I₂For raw material, the synthesis of reference compound 16.White solid, yield 83.8%.MS(ESI),m/ z:942.4([M+Na]⁺).

(2) compound A₃-I₂-R₃Synthesis

It is raw material, reference compound A with compound 33₁-I₁-R₁Synthesis.White solid, yield 85.2%.¹HNMR (400MHz, DMSO-d6) δ: 7.46-7.41 (d, J=7.2Hz, 2H), 7.37-7.31 (t, J=6.9Hz, 2H), 7.28-7.19 (m, 5H), 6.92-6.86 (d, J=8.2Hz, 4H), 5.22 (s, 1H), 5.02-4.9 (m, 1H), 4.5-4.98 (s, J= 3.5Hz, 1H), 4.08-3.99 (m, 3H), 3.9-3.88 (m, 1H), 3.84-3.80 (m, 2H), 3.75 (s, 6H), 3.73-3.65 (m, 1H), 3.48-3.38 (m, 1H), 3.05-3.00 (m, 3H), 2.91-2.86 (m, 2H), 2.88-2.84 (m, 2H), 2.77- 2.71 (m, 1H), 2.59-2.54 (m, 2H), 2.12 (s, 3H), 2.05-2.01 (m, 2H), 2.00 (s, 3H), 1.88 (s, 3H), 1.86-1.81 (m, 2H), 1.77 (s, 12H), 1.66 (s, 3H), 1.5-1.4 (m, 2H), 1.39-1.3 (m, 2H), 1.29-1.19 (m,10H).MS(ESI),m/z:1142.5([M+Na]⁺).

29 compound A of embodiment₁-II₁-R₁Synthesis

(1) synthesis of compound 34

In 250mL round-bottomed flask, compound N-benzyloxycarbonyl group -- 6-aminocaprolc acid (10g, 37.69mmol), 1- second is added Base-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC.HCL) (8.67g, 45.23mmol), N- hydroxysuccinimidyl acyl Imines (4.67g, 41.46mmol), methylene chloride 100mL.Compound R is added after stirring at normal temperature reaction 0.5h '₁- H (14.8g, 37.69mmol), TLC monitoring is reacted, fully reacting after 4h.Reaction solution successively uses saturated sodium bicarbonate solution 50mL and saturation food Salt water 50mL washing, organic phase anhydrous sodium sulfate is dry, and concentration is separated through silicagel column (ethyl acetate: petroleum ether V:V=4:1) Obtain white solid 19.8g, yield 82.1%.MS(ESI),m/z:663.1([M+Na]⁺).

(2) synthesis of compound 35

100mL round-bottomed flask, compound 34 (5g, 7.8mmmol), Pd/C (0.5g, 10%) are dissolved in 10mL methanol and 40mL In ethyl acetate, hydrogen balloon is accessed, TLC monitoring is reacted, fully reacting after 6h.The filtering of reaction solution diatomite, the leaching of diatomite methanol It washes, filtrate decompression concentration is spin-dried for, and obtains white solid 3.8g, yield 96.1%.¹HNMR (400MHz, DMSO-d6) δ: 7.64- 7.61 (d, J=7.2Hz, 1H), 7.43-7.37 (d, J=8.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27- 7.19 (m, 5H), 6.92-6.87 (d, J=8.2Hz, 4H), 5.12 (m, 2H), 4.63-4.58 (m, 1H), 4.05-3.97 (m, 1H), 3.73 (s, 6H), 3.5-3.42 (m, 2H), 3.04-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.12-2.06 (m, 2H), 1.52-1.45 (m, 2H), 1.42-1.35 (m, 2H), 1.28-1.20 (m, 2H) .MS (ESI), m/z:529.3 ([M+Na ]⁺).

(3) synthesis of compound 36

In 250mL round-bottomed flask, compound A is added₁-I₁(10g, 22.37mmol), 1- ethyl-(3- dimethylamino third Base) phosphinylidyne diimmonium salt hydrochlorate (EDC.HCL) (5.15g, 26.85mmol), n-hydroxysuccinimide (2.83g, 24.61mmol), methylene chloride 100mL.Compound 35 (11.32g, 22.37mmol) is added after reacting 0.5h in stirring at normal temperature, TLC Monitoring is reacted, fully reacting after 6h.Reaction solution is successively washed with saturated sodium bicarbonate solution 50mL and saturated salt solution 50mL, is had Machine is mutually dry with anhydrous sodium sulfate, concentration, through silicagel column (methylene chloride: methanol V:V=20:1) isolated white solid 17.3g, yield 82.7%.MS(ESI),m/z:958([M+Na]⁺).

(4) compound A₁-II₁-R₁Synthesis

It is raw material, reference compound A with compound 36₁-I₁-R₁Synthesis.White solid, yield 84.7%.¹HNMR (400MHz, DMSO-d6) δ: 7.84-7.80 (d, J=7.2Hz, 1H), 7.72-7.66 (m, 1H), 7.64-7.61 (d, J= 7.2Hz, 1H), 7.43-7.37 (d, J=8.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.92-6.87 (d, J=8.2Hz, 4H), 5.20 (s, 1H), 5.0-4.95 (q, J=4.2Hz, 1H), 4.51-4.46 (d, J= 7.2Hz, 1H), 4.15-4.11 (m, 3H), 4.05-3.97 (m, 1H), 3.89-3.79 (m, 3H), 3.76 (s, 6H), 3.74- 3.69 (m, 1H), 3.54-3.49 (m, 2H), 3.46-3.36 (m, 1H), 3.04-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.88-2.84 (m, 2H), 2.59-2.54 (m, 2H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 3H), 2.12-2.06 (m, 2H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.77 (s, 12H), 1.59-1.42 (m, 6H), 1.41-1.35 (m, 2H), 1.28-1.20 (m, 2H) .MS (ESI), m/z:1158.5 ([M+Na]⁺).

30 compound A of embodiment₁-II₁-R₂Synthesis

(1) synthesis of compound 37

With compound R '₂- H is raw material, the synthesis of reference compound 34.White solid, yield 84.3%.MS(ESI),m/ z:677.5([M+Na]⁺).

(2) synthesis of compound 38

It is raw material, the synthesis of reference compound 35 with compound 37.White solid, yield 88.2%.1HNMR(400MHz, DMSO-d6) δ: 7.44-7.38 (d, J=8.2Hz, 2H), 7.34-7.29 (t, J=6.9Hz, 2H), 7.28-7.20 (m, 5H), 6.92-6.87 (d, J=8.2Hz, 4H), 5.12 (m, 2H), 4.63-4.58 (m, 1H), 3.73 (s, 6H), 3.5-3.42 (m, 2H), 3.04-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.88-2.82 (m, 4H), 2.12-2.06 (m, 2H), 1.52-1.45 (m, 2H), 1.42-1.35 (m, 2H), 1.28-1.20 (m, 2H) .MS (ESI), m/z:543.3 ([M+Na]⁺).

(3) synthesis of compound 39

It is raw material, the synthesis of reference compound 36 with compound 38.White solid, yield 80.7%.MS(ESI),m/z: 972.6([M+Na]⁺).

(4) compound A₁-II₁-R₂Synthesis

It is raw material, reference compound A with compound 39₁-I₁-R₁Synthesis.White solid, yield 84.1%.¹HNMR (400MHz, DMSO-d6) δ: 7.83-7.79 (d, J=7.2Hz, 1H), 7.72-7.66 (m, 1H), 7.42-7.36 (d, J= 8.2Hz, 2H), 7.33-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.92-6.87 (d, J=8.2Hz, 4H), 5.20 (s, 1H), 5.0-4.95 (q, J=4.2Hz, 1H), 4.51-4.46 (d, J=7.2Hz, 1H), 4.15-4.10 (m, 3H), 3.89-3.79 (m, 3H), 3.76 (s, 6H), 3.74-3.69 (m, 1H), 3.54-3.49 (m, 2H), 3.46-3.36 (m, 1H), 3.04-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.88-2.84 (m, 6H), 2.59-2.54 (m, 2H), 2.22-2.14 (t, J =7.2Hz, 2H), 2.15 (s, 3H), 2.12-2.06 (m, 2H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.77 (s, 12H), 1.59-1.42 (m, 6H) 1.41-1.35 (m, 2H), 1.28-1.20 (m, 2H) .MS (ESI), m/z:1172.7 ([M+ Na]⁺).

31 compound A of embodiment₁-III₁-R₁Synthesis

(1) synthesis of compound 40

It is raw material, the synthesis of reference compound 34 with compound 35.White solid, yield 82.6%.MS(ESI),m/z: 776.7([M+Na]⁺).

(2) synthesis of compound 41

It is raw material, the synthesis of reference compound 35 with compound 40.White solid, yield 96.7%.¹HNMR(400MHz, DMSO-d6) δ: 7.71-7.67 (m, 1H), 7.65-7.62 (d, J=7.2Hz, 1H), 7.45-7.39 (d, J=8.2Hz, 2H), 7.34-7.29 (t, J=6.9Hz, 2H), 7.26-7.19 (m, 5H), 6.93-6.88 (d, J=8.2Hz, 4H), 5.14 (m, 2H), 4.64-4.58 (m, 1H), 4.05-3.98 (m, 1H), 3.72 (s, 6H), 3.5-3.43 (m, 4H), 3.05-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.12-2.06 (m, 4H), 1.52-1.45 (m, 4H), 1.41-1.35 (m, 4H), 1.29-1.20 (m, 4H).MS(ESI),m/z:642.3([M+Na]⁺).

(3) synthesis of compound 42

It is raw material, the synthesis of reference compound 36 with compound 41.White solid, yield 86.3%.MS(ESI),m/z: 1071.4([M+Na]⁺).

(4) compound A₁-III₁-R₁Synthesis

It is raw material, reference compound A with compound 42₁-I₁-R₁Synthesis.White solid, yield 84.1%.¹HNMR (400MHz, DMSO-d6) δ: 7.85-7.81 (d, J=7.2Hz, 1H), 7.78-7.74 (m, 1H), 7.72-7.66 (m, 1H), 7.65-7.61 (d, J=7.2Hz, 1H), 7.42-7.38 (d, J=8.2Hz, 2H), 7.34-7.29 (t, J=6.9Hz, 2H), 7.27-7.18 (m, 5H), 6.93-6.87 (d, J=8.2Hz, 4H), 5.22 (s, 1H), 5.0-4.96 (q, J=4.2Hz, 1H), 4.51-4.46 (d, J=7.2Hz, 1H), 4.15-4.11 (m, 3H), 4.05-3.97 (m, 1H), 3.89-3.79 (m, 3H), 3.76 (s, 6H), 3.74-3.69 (m, 1H), 3.54-3.49 (m, 4H), 3.46-3.36 (m, 1H), 3.04-2.99 (m, 2H), 2.95- 2.90 (m, 2H), 2.88-2.84 (m, 2H), 2.59-2.54 (m, 2H), 2.22-2.14 (t, J=7.2Hz, 2H), 2.15 (s, 3H), 2.12-2.06 (m, 4H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.77 (s, 12H), 1.59-1.42 (m, 8H), 1.41-1.35 (m, 4H), 1.28-1.20 (m, 4H) .MS (ESI), m/z:1271.2 ([M+Na]⁺).

32 compound A of embodiment₁-IV₁-R₁Synthesis

(1) synthesis of compound 43

With compound A₁-III₁For raw material, the synthesis of reference compound 16.White solid, yield 82.8%.MS(ESI), m/z:877.4([M+Na]⁺).

(2) compound A₁-III₁-R₁Synthesis

It is raw material, reference compound A with compound 40₁-I₁-R₁Synthesis.Yield 82.9%.¹HNMR(400MHz, DMSO-d6) δ: 7.76-7.72 (d, J=8.9Hz, 1H), 7.70-7.66 (d, J=8.0Hz, 1H), 7.40-7.36 (d, J= 7.2Hz, 2H), 7.32-7.28 (t, J=6.9Hz, 2H), 7.27-7.19 (m, 5H), 6.91-6.86 (d, J=8.2Hz, 4H, 5.21 (s, 1H), 5.0-4.96 (q, J=4.2Hz, 1H), 4.45-4.51 (d, J=7.2Hz, 1H), 4.12-4.07 (m, 3H), 4.05-3.97 (m, 1H), 3.88-3.78 (m, 3H), 3.74 (s, 6H), 3.72-3.68 (m, 2H), 3.62-3.58 (m, 2H), 3.56-3.46 (m, 4H), 3.04-2.99 (m, 2H), 2.95-2.90 (m, 2H), 2.89-2.85 (m, 2H), 2.58-2.53 (m, 2H), 2.15 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.88 (s, 3H), 1.76 (s, 12H) .MS (ESI), m/z:502.6 ([M+1]⁺).¹HNMR (400MHz, DMSO-d6) δ: MS (ESI), m/z:1077.3 ([M+Na]⁺).

33 compound A of embodiment₁-V₁-R₄Synthesis

(1) synthesis of compound 44

250mL round-bottomed flask, compound 6 (5g, 15.2mmol), 1,6- hexylene glycol (9g, 76mmol) are dissolved in anhydrous 1,2- In dichloroethanes 100mL, Trimethylsilyl trifluoromethanesulfonate (0.55mL, 3mmol) is added after stirring 30 minutes, room temperature crosses reaction Overnight, the extraction of reaction solution methylene chloride, organic phase are washed twice with saturated sodium bicarbonate solution 80mL, and anhydrous sodium sulfate is dry, It is concentrated under reduced pressure, through the isolated transparent oily liquid 5.86g of silicagel column (petroleum ether: ethyl acetate V:V=3:2), yield 86.2%.MS(ESI),m/z:470.2([M+Na]⁺).

(2) compound A₁-V₁-R₄Synthesis

It is raw material, reference compound A with compound 41₁-I₁-R₁Synthesis.Yield 84.1%.¹HNMR(400MHz, DMSO-d6) δ: 7.80-7.75 (d, J=8.9Hz, 1H), 5.21 (s, 1H), 5.02-4.95 (q, J=4.2Hz, 1H), 4.50- 4.76 (d, J=7.2Hz, 1H), 4.12-4.07 (m, 3H), 3.88-3.79 (m, 3H), 3.80-3.69 (m, 2H), 3.46-3.36 (m, 2H), 2.88-2.84 (m, 2H), 2.59-2.54 (m, 2H), 2.23-2.28 (m, 2H), 2.22-2.14 (m, 2H), 2.15 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.87 (s, 3H), 1.79 (s, 12H), 1.58-1.42 (m, 4H) .MS (ESI), m/z:870.5([M+Na]⁺).

The single-stranded oligonucleotide of the preparation modification of embodiment 34

In the present embodiment, for modification oligonucleotide according to 1 μm of ol specification synthesis of theoretical yield, process is as follows:

(1) the general solid support CPG or 3 ' of 1 μm of ol specification-cholesterol modification CPG (purchasing in Chemgenes) is weighed, Monomer, DNA monomer, 2 '-methoxy monomers, the 2 '-fluorine monomers of 2 '-O-TBDMS protection RNA phosphoramidite (are purchased in Sigma Aldrich) being dissolved in anhydrous acetonitrile makes its concentration reach 0.2M.For the oligonucleotide of phosphoric acid backbone thio-modification Using 0.2M PADS solution as thio reagents.It prepares 5- ethylmercapto group -1H-TETRAZOLE and (purchases in Chemgenes) acetonitrile solution conduct Activator (0.25M) prepares pyridine/aqueous solution of 0.02M iodine as oxidant and 3% trichloroacetic acid dichloromethane solution As deprotecting regent, it is placed in the corresponding reagent designated position of 394 model DNA/RNA automatic synthesizer of ABI.

(2) synthesis program is set, inputs specified oligonucleotides base sequence and starts the cycle over oligomerization after inspection is errorless Nucleotide synthesis, every step Coupling time 6 minutes, gala carbohydrate ligands correspond to monomer (A_x-linker-R_xCompound) Coupling time 10- 20 minutes.After automatic cycle, oligonucleotides synthesis in solid state is completed.

(3) with drying nitrogen dry up CPG, be transferred in 5mL EP pipe, be added ammonium hydroxide/ethanol solution (3/1) 2mL, 55 DEG C Heating 16~18 hours.It is centrifuged 10min under the revolving speed of 10000rpm and takes supernatant, obtains white after draining concentrated ammonia liquor/ethyl alcohol Colloidal solid.Solid is dissolved in 200 μ L 1M TBAF THF solutions, and room temperature is shaken 20 hours.It is slow that 0.5mL 1M Tris-HCl is added Fliud flushing (pH 7.4), room temperature are shaken 15 minutes, and being placed in centrifugation and draining machine to be evacuated to volume is original volume 1/2, remove THF.Solution is used 0.5mL chloroform extracts 2 times, and 1mL 0.1M TEAA sample solution is added, and mixed solution is poured into solid-phase extraction column, is removed in solution Excess salt.

(4) gained oligonucleotides acid concentration measures content by micro ultraviolet specrophotometer (KO5500).In Oligo In HTCS LC-MS system (Novatia) system, Mass Spectrometer Method analysis is completed.Returned after being scanned with level-one with Promass software One changes calculating nucleic acid molecular weight.

The single-stranded oligonucleotide of the preparation modification of embodiment 35

In the present embodiment, modified oligonucleotide is synthesized according to the specification of 1 μm of ol of theoretical yield, and process is as follows:

(1) the general solid support CPG or 3 ' of 1 μm of ol specification-cholesterol modification CPG (purchasing in Chemgenes) is weighed, DNA monomer, 2 '-methoxy monomers, 2 '-fluorine monomers (purchasing in Sigma Aldrich) are dissolved in anhydrous acetonitrile makes it Concentration reaches 0.2M.For phosphoric acid backbone thio-modification oligonucleotide using 0.2M PADS solution as thio reagents.It prepares 5- ethylmercapto group -1H-TETRAZOLE (purchases in Chemgenes) acetonitrile solution as activator (0.25M), prepare the pyridine of 0.02M iodine/ Aqueous solution, as deprotecting regent, is placed in 394 model of ABI as oxidant and 3% trichloroacetic acid dichloromethane solution The corresponding reagent designated position of DNA/RNA automatic synthesizer.

(2) synthesis program is set, inputs specified oligonucleotides base sequence and starts the cycle over oligomerization after inspection is errorless Nucleotide synthesis, every step Coupling time 6 minutes, gala carbohydrate ligands (A_x-linker-R_xCompound) correspond to monomer Coupling time 6- 10 minutes.After automatic cycle, oligonucleotides synthesis in solid state is completed.

(3) CPG is dried up with drying nitrogen, be transferred in 5mL EP pipe, ammonia spirit 2mL, 55 DEG C of heating 16~18 are added Hour.It is centrifuged 10min under the revolving speed of 10000rpm and takes supernatant, obtains white or yellow colloidal after draining concentrated ammonia liquor/ethyl alcohol Solid.1mL 0.1M TEAA sample solution is added, mixed solution is poured into solid-phase extraction column, removes excess salt in solution.

The double chain oligonucleotide of the preparation modification of embodiment 36

Process is as follows: after the preparation for completing the single-stranded oligonucleotide of modification, mixing according to UV absorption content 1:1, heating It after three minutes, is cooled to room temperature to 95 DEG C, forms double-strand.

In embodiment 34-36, it is lower than 50% modified oligonucleotide, for crude product purity with DNAPAc PA-100 ion The purifying of exchange column linear gradient, mobile phase A: 20mM NaOH；Mobile phase B: 20mM NaOH+2M NaCl mixed liquor.

The sequence of illustrative modified oligonucleotide and corresponding molecular weight detection the results are shown in Table 1.

Abbreviation explanation: N=RNA；DN=DNA；MN=2'OMe modification；FN=2'F modification.

Table 1

The cell targeted detection of 37 modified oligonucleotide of embodiment.

The modified oligonucleotide of zoopery, through 0.22 μm of film filtration treatment before injection.

1, mouse primary hepatocytes separate

Anesthetized mice cuts off skin and muscle layer, and perfusion conduit is inserted into portal vein by exposure liver, and inferior caval vein is cut off Osculum prepares liver perfusion.40 DEG C of preheating perfusion Solution I (Hank ' s, 0.5mM EGTA, pH 8) and Perfusion Solution II (Low-glucose DMEM, 100U/mL Type IV, pH 7.4), 37 DEG C of perfusion Solution I along portal catheterization be perfused liver, flow velocity 7mL/min, be perfused 5min, liver it is graying white until.Then with 37 Liver is perfused in DEG C perfusion Solution II, and 7min is perfused in flow velocity 7mL/min.After the completion of perfusion, removes liver and be placed in Solution III (10%FBS low-glucose DMEM, 4 DEG C) terminates digestion, and tweezers scratch liver coating, gently shake Discharge liver cell.Liver cell is filtered with 70 μm of cellular filters, abandons supernatant after 50g centrifugation 2min.With Solution IV (40% Percoll low-glucose DMEM, 4 DEG C) cell is resuspended, supernatant is abandoned after 100g centrifugation 2min.2%FBS low- is added Cell is resuspended in glucose DMEM, spare.Trypan Blue identification of cell vigor.

2, GalNAc binding curve and Kd value are measured

The mouse primary hepatocytes of fresh separated are taped against in 96 orifice plates, 2 × 10⁴A/hole, 100 holes μ L/.Every hole difference Add GalNAc-siRNA.Every GalNAc-siRNA be arranged final concentration of 0.9nM, 2.7nM, 8.3nM, 25nM, 50nM or 100nM.50g is centrifuged 2min after 4 DEG C of incubation 2h, abandons supernatant.Cell is resuspended in 10 μ g/mL PI, and 50g is centrifuged after dyeing 10min 2min.Cell is washed with the PBS of pre-cooling, abandons supernatant after 50g centrifugation 2min.Cell is resuspended in PBS.It is flat that flow cytometer measures living cells Equal fluorescence intensity MFI (Mean Fluorescence Intensity), 5 software of GraphPad Prism carry out nonlinear fitting And dissociation constant K_dValue calculates.As a result as shown in table 2, table 3 and Fig. 1.Statistics indicate that GalNAc-siRNA energy special target liver is thin Born of the same parents；The Kd value of its GalNAc ligand and cell receptor is between 7.6-53.4nM, with the prior art (PCT/US2014/046425) Preferred gala carbohydrate ligands, which are compared, has comparable high-affinity (Ki value 5.2-51.3nM), different conjugate structures What GalNAc-siRNA showed has certain difference with receptor binding capacity, and A3, A4 structure show relatively stronger receptor Affinity (Kd value is smaller, and affinity is bigger).

Each experimental group K of table 2._dIt is worth (nM) and B_maxValue

Each experimental group K of table 3._dIt is worth (nM) and B_maxValue

Liver targeting is tested in 38 body of embodiment

Test (ties up tonneau China purchased from Beijing to test using the SPF grade Balb/c-nu mouse 13 of male, 6~7 weeks week old Company of Animals Ltd.), 4 groups are randomly divided into, blank control group, P8G8 control group (not conjugated ligand), P8G8-A3 group, P8G8-B3 Group.Groups of animals number is respectively 2,3,4,4, and tail vein injection administration, dosage is about that (experimental design is shown in Table 10mg/kg 4).15min, 30min, 1h, 2h, 4h, 6h carry out living imaging to all animals before medicine, after administration, including white light and X-ray at Picture.After medicine after 6 hours euthanasia, takes out brain, salivary gland, heart, spleen, lungs, liver, kidney and enteron aisle and carry out isolated organ Imaging.

4. Liver targeting experimental design of table

In vitro imaging analysis (table 5- table 6) the results show that 6 hours after administration, P8G8-A3 group, P8G8-B3 group liver it is glimmering Luminous intensity is above negative control group.The result shows that P8G8-A3, P8G8-B3 have certain targeting to liver.

Isolated organ fluorescence intensity level statistical result (× 10 after 5. background correction of table⁸ps/mm²)

6. fluorescence intensity ratio result of table

	Salivary gland	Liver	Kidney	Enteron aisle
					P8G8-A3/P8G8	1.05	3.50	1.12	1.19
P8G8-B3/P8G8	1.10	2.88	0.92	1.30

Although a specific embodiment of the invention has obtained detailed description, those skilled in the art will appreciate that root According to all introductions having disclosed, details can be carry out various modifications and be changed, and these change in guarantor of the invention Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.

Sequence table

<110>Guangzhou Ribo Bio Co., Ltd.

<120>compound of the oligonucleotides modified and the oligonucleotides that can be used for synthetic modification

<130> IDC170149

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 19

<212> RNA

<213>artificial sequence

<220>

<223>oligonucleotides synthesized

<400> 1

cagcaagugu gacagucau 19

<210> 2

<211> 25

<212> RNA

<213>artificial sequence

<220>

<223>oligonucleotides synthesized

<400> 2

augacuguca cacuugcugg ccugu 25

<210> 3

<211> 19

<212> RNA

<213>artificial sequence

<220>

<223>oligonucleotides synthesized

<400> 3

caggccagca agugugaca 19

<210> 4

<211> 21

<212> RNA

<213>artificial sequence

<220>

<223>oligonucleotides synthesized

<400> 4

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Claims

1. including the compound of oligonucleotides and conjugate group, general formula isWherein, PN is few core Thuja acid, Y are selected from the integer between 1-10, and X is selected from the integer between 0-10, M_TSelected from such as formula (1), formula (2), formula (3) and formula (4) Shown in conjugate group, when X is not 0, X M is each independently selected to be sewed as shown in formula (1 '), formula (2 ') and formula (3 ') Object group is closed,

Wherein, A_xFor ligand, linker is linking arm, and Q is hydroxyl or modifier.

2. the compound of claim 1, in conjugate group shown in formula (1)-formula (4), formula (1 ')-formula (3 '), A_xIt is respectively independent Ground is the ligand of people's asialoglycoprotein receptor (ASGPR)；

Preferably, formula (1)-formula (4), in conjugate group shown in formula (1 ')-formula (3 '), A_xBe each independently selected from galactolipin, Acetylamino galactosamine, the polysaccharide containing galactolipin, the polysaccharide containing acetylamino galactosamine, galactose derivative (such as galactolipin Ester, such as galactolipin acetic acid esters) or acetylamino galactosamine derivative (such as the ester of acetylamino galactosamine, such as acetyl Amine-galactose acetic acid esters)；

Optionally, A_xOn also each independently have modification group, such as carbonylic alkyl or ester group alkyl, the alkyl be preferably C_1-6Alkyl or C_6-12Alkyl；

Preferably, A_xIt is selected from:

3. the compound of claims 1 or 2, in conjugate group shown in formula (1)-formula (4), formula (1 ')-formula (3 '), linker Structure each independently as shown in formula (i), formula (ii), formula (iii), formula (iv) or formula (v):

Wherein, n₁、n₂、n₃The integer being each independently selected between 1-10, it is preferable that n₁It is 1, it is preferable that n₂It is 3, it is preferable that n₃It is 4；

4. the compound of any one of claim 1-3, in conjugate group shown in formula (1) or formula (1 '),

A_xIt is each independently selected from A₁、A₂、A₃、A₁’、A₂' or A₃', shown in the structure of linker such as formula (i)；Or

A_xIt is each independently selected from A₁Or A₁', shown in the structure of linker such as formula (ii)；Or

A_xIt is each independently selected from A₁Or A₁', shown in the structure of linker such as formula (iii), or

A_xIt is each independently selected from A₁Or A₁', shown in the structure of linker such as formula (iv).

5. the compound of any one of claim 1-3, in conjugate group shown in formula (2) or formula (2 '),

A_xIt is each independently selected from A₁Or A₁', shown in the structure of linker such as formula (ii).

6. the compound of any one of claim 1-3, in conjugate group shown in formula (3) or formula (3 '), A_xIt selects each independently From for A₁、A₂、A₃、A₁’、A₂' or A₃', shown in the structure of linker such as formula (i).

7. the compound of any one of claim 1-3, in conjugate group shown in formula (4), A_xFor A₁Or A₁', the knot of linker Shown in structure such as formula (v).

8. the compound of any one of claim 1-7, in conjugate group shown in formula (1)-formula (3), formula (1 ')-formula (3 '), Q It is selected from: cholesterol and its derivative, polyethylene glycol, fluorescence probe, biotin, polypeptide, vitamin, tissue-targeting molecule.

9. the compound of any one of claim 1-8, wherein the oligonucleotides is single-stranded oligonucleotide or Double stranded oligonucleotide Acid；

Preferably, the oligonucleotides includes one or more nucleotide by modification；

Preferably, one or more of nucleotide by modification are each independently selected from: 2 '-methoxy ethyl modified nucleosides Acid, 2 '-O- alkyl modified nucleotide (such as 2 '-O- methyl modified nucleotide), 2 '-O- allyl modified nucleoside acid, 2 '-C- alkene Propyl modified nucleoside acid, 2 '-fluorine modification nucleotide, 2 '-deoxidation modified nucleoside acid, 2 '-hydroxyl modified nucleotide, lock nucleotide, Solve lock nucleic acid, hexitol nucleic acid；

Preferably, the nucleotide by modification is selected from 2 '-O- alkyl modified nucleotide, 2 '-fluorine modification nucleotide；

Preferably, the oligonucleotides has end modified object, it is preferable that the end modified object is selected from: cholesterol, poly- second two Alcohol, fluorescence probe, biotin, polypeptide, vitamin, tissue-targeting molecule and any combination thereof；

Preferably, the phosphoric acid skeleton of the oligonucleotides is by modification, it is preferable that described to be modified to thio-modification；

Preferably, the oligonucleotides is siRNA；

Preferably, the siRNA includes the positive-sense strand and antisense strand for being complementarily shaped to double-strand；

Preferably, the siRNA includes such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 institute The sequence shown.

10. the compound of any one of claim 1-9, whereinEach independently and in the oligonucleotides It is connected among 3 ' ends of any bar chain, 5 ' ends or sequence；

Preferably,It is connected by tricresyl phosphate ester bond with oligonucleotides；

Preferably, M and M_TBetween or M and M between be connected by tricresyl phosphate ester bond.

11. the compound of any one of claim 1-10, wherein the oligonucleotides is single-stranded oligonucleotide；

Preferably, 1 Y,It is connected with 3 ' ends of the oligonucleotides or 5 ' ends；

Preferably, 2 Y, 2Respectively it is connected with 3 ' ends of the oligonucleotides and 5 ' ends.

12. the compound of any one of claim 1-10, wherein the oligonucleotides is double chain oligonucleotide；

Preferably, 1 Y,It is connected with 3 ' ends of any bar chain in the oligonucleotides or 5 ' ends It connects；

Preferably, 2 Y, 2It is respectively last with 3 ' ends of the same chain in the oligonucleotides and 5 ' End is connected；

Preferably, 2 Y, 2Respectively it is connected with 3 ' ends of two chains in the oligonucleotides；

Preferably, 2 Y, 2Respectively it is connected with 5 ' ends of two chains in the oligonucleotides；

Preferably, 3 Y, 3In, there are two respectively last with 3 ' of the same chain in the oligonucleotides End is connected with 5 ' ends, and third is a to be connected with 3 ' ends of another chain or 5 ' ends；

Preferably, 4 Y, 4Respectively with 3 ' ends of two chains in the oligonucleotides and 5 ' end phases Connection.

13. the compound of any one of claim 1-12, wherein X is not 0, M_TWith at least one M A having the same_xAnd/or Linker structure；

Preferably, X, which is greater than 1, X M, has identical or different structure；

Preferably, Y is greater than 1, YWith identical or different structure.

14. the compound of any one of claim 1-13, wherein Y 1, X 0, and the compound has in following characteristics One:

(1)M_TStructure such as formula (1) shown in, A_xFor A₁’、A₂' or A₃', shown in the structure of linker such as formula (i)；

(2)M_TStructure such as formula (1) shown in, A_xFor A₁', shown in the structure of linker such as formula (ii)；

(3)M_TStructure such as formula (1) shown in, A_xFor A₁', shown in the structure of linker such as formula (iii)；

(4)M_TStructure such as formula (1) shown in, A_xFor A₁', shown in the structure of linker such as formula (iv)；

(5)M_TStructure such as formula (2) shown in, A_xFor A₁’、A₂' or A₃', shown in the structure of linker such as formula (i)；

(6)M_TStructure such as formula (2) shown in, A_xFor A₁', shown in the structure of linker such as formula (ii)；

(7)M_TStructure such as formula (3) shown in, A_xFor A₁’、A₂' or A₃', shown in the structure of linker such as formula (i)；

(8)M_TStructure such as formula (4) shown in, A_xFor A₁', shown in the structure of linker such as formula (v)；

(9)M_TStructure such as formula (2) shown in, A_xFor A₁', shown in the structure of linker such as formula (iii)；

Preferably, in the linker as shown in formula (i), n is 1 or 6；

Preferably, in the linker as shown in formula (ii), n₁It is 1, and n₂It is 4；

Preferably, in the linker as shown in formula (iii), n₁It is 1, n₂It is 3, and n₃It is 4；

Preferably, in the linker as shown in formula (iv), n 1.

15. the compound of any one of claim 1-13, wherein Y 1, X 1,2 or 3, when X is 2 or 3, each M has phase Same structure, and the compound has one in following characteristics:

(1) shown in the structure of M such as formula (1 '), A_xFor A₁', shown in the structure of linker such as formula (i)；M_TStructure such as formula (1) institute Show, A_xFor A₁', shown in the structure of linker such as formula (i)；

(2) shown in the structure of M such as formula (1 '), A_xFor A₁', shown in the structure of linker such as formula (ii)；M_TStructure such as formula (1) institute Show, A_xFor A₁', shown in the structure of linker such as formula (ii)；

(3) shown in the structure of M such as formula (1 '), A_xFor A₁', shown in the structure of linker such as formula (iii)；M_TStructure such as formula (1) institute Show, A_xFor A₁', shown in the structure of linker such as formula (iii)；

(4) shown in the structure of M such as formula (2 '), A_xFor A₁', shown in the structure of linker such as formula (i)；M_TStructure such as formula (2) institute Show, A_xFor A₁', shown in the structure of linker such as formula (i)；

(5) shown in the structure of M such as formula (1 '), A_xFor A₁', shown in the structure of linker such as formula (ii)；M_TStructure such as formula (1) institute Show, A_xFor A₁', shown in the structure of linker such as formula (ii)；

(6) shown in the structure of M such as formula (1 '), A_xFor A₃', shown in the structure of linker such as formula (ii)；M_TStructure such as formula (1) institute Show, A_xFor A₃', shown in the structure of linker such as formula (ii)；

Preferably, in the linker as shown in formula (i), n is 1 or 6；

Preferably, in the linker as shown in formula (iii), n₁It is 1, n₂It is 3, and n₃It is 4.

16. the compound of any one of claim 1-13, wherein the structure of Y 1, X 2, two M are identical, such as formula (1 ') institute Show, A_xFor A₁', shown in the structure of linker such as formula (iv)；Shown in the structure of MT such as formula (4), A_xFor A₁', the structure of linker is such as Shown in formula (v)；

Preferably, in the linker as shown in formula (iv), n 1.

17. a kind of pharmaceutical composition, it includes the compounds of any one of claim 1-16, and optionally pharmaceutically acceptable Carrier；

Preferably, described pharmaceutical composition is for preventing and/or treating disease relevant to liver in subject；

Preferably, the disease relevant to liver is selected from: hereditary angioedema, I type familial tyrosinemia, Alagille syndrome, alpha-1-amtitrypsin deficiency, bile acid biosynthesis and metabolic deficiency, Biliary atresia, cystic fibrosis Hepatopathy, idiopathic neonatal hepatitis, mitochondria hepatopathy, progressive familial intrahepatic cholestasis, primary sclerotic cholangitis, Transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, hyperlipemia, fatty liver Inflammation, nonalcoholic fatty liver disease (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia and involve and II type glycosuria Disease, the hepatitis, liver property porphyrin for the Hepatic glucose production that the similar exception of disease increases.