Modified nucleoside compound, and use thereof in oligonucleotides

Abstract

Provided is a deuterated nucleoside phosphoramidite compound. The compound can be used for preparing an oligonucleotide drug, and can improve the stability, long-acting property, and efficacy of the oligonucleotide drug.

Description

A modified nucleoside compound and its application in oligonucleotides Technical Field

[0001] This invention relates to the field of biopharmaceuticals, specifically to a modified nucleoside compound and, more particularly, oligonucleotides prepared therefrom. Background Technology

[0002] Oligonucleotides are polymers of nucleotides that, as nucleic acid inhibitor molecules, can regulate the levels of target mRNAs within cells and have shown early promise in the treatment of genetic diseases, metabolic diseases, inflammatory diseases, cancer, and viral infections. Nucleic acid inhibitor molecules can regulate mRNA expression through a variety of mechanisms, including RNA interference (RNAi).

[0003] RNAi is a conserved pathway found in most eukaryotes, in which a double-stranded RNA molecule (dsRNA) inhibits the expression of a target mRNA with a complementary sequence to the dsRNA. In one typical RNAi pathway, the longer dsRNA is cleaved by a nuclease (Dicer) into a shorter RNA duplex called small interfering RNA (“siRNA”). siRNA has been shown to associate with the nuclease, trans-activating response RNA-binding protein (TRBP), and Argonaute 2 (“Ago2”) to form a complex, sometimes referred to as the RNA-induced silencing complex (“RISC”). Ago2 is a nuclease that uses the antisense strand (also known as the guide strand) of the siRNA to guide the sequence-specific cleavage of the target mRNA.

[0004] Various double-stranded RNAi inhibitor molecular structures have been developed over the years. For example, early work on RNAi inhibitor molecules focused on double-stranded nucleic acid molecules mimicking natural siRNA, where each strand has 19-25 nucleotides and includes at least one 3' overhang with 1 to 5 nucleotides (see, for example, U.S. Patent No. 8,372,968). Subsequently, longer double-stranded RNAi inhibitor molecules were developed, which were cleaved in vivo by endonucleases into active RNAi inhibitor molecules (see, for example, U.S. Patent No. 8,883,996). Subsequent work developed extended double-stranded nucleic acid inhibitor molecules in which at least one end of at least one strand extends beyond the double-stranded target region of the molecule, one of the strands comprising a thermodynamically stable tetracyclic structure (see, for example, U.S. Patent Nos. 8,513,207, 8,927,705, WO 2010 / 033225, and WO 2016 / 100401). These structures include single-strand extensions (on one or both sides of the molecule) and double-strand extensions.

[0005] In recent years, many modified nucleosides have been used for RNAi, such as common modifications like 2'-fluorine, 2'-methoxy, 2'-methoxyethyl, UNA, LNA, BNA, GNA, and 5'-(E)-vinyl phosphate. In some cases, chemical modifications are introduced into nucleic acid inhibitor molecules to obtain potentially desired properties after in vivo administration. These modifications include those designed to, for example, antagonize nucleases or other enzymes that interfere with the structure or activity of oligonucleotides, increase cellular uptake of oligonucleotides, or improve the pharmacokinetic properties of oligonucleotides. Introducing modified nucleosides into the corresponding oligonucleotide sequences improves the pharmacokinetic properties and bioavailability of oligonucleotide drugs in vivo. Further research is needed on the chemical modification of oligonucleotide drugs to obtain oligonucleotide drugs with superior properties in all aspects. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention aims to develop a deuterated nucleoside compound with more stable conformation and metabolic stability. When applied to oligonucleotide molecules (including but not limited to siRNA, antisense nucleic acids, saRNA, miRNA aptamers, and lncRNA), it can replace currently available modified nucleosides while improving the activity and metabolic stability of oligonucleotide molecules.

[0007] One objective of this invention is to provide a deuterated nucleoside phosphoramidide compound of Formula 1 and its stereoisomers:

[0008] in,

[0009] R 1 R 2 R 3 R 4 R 5 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN.

[0010] Z represents -O-, -S-, -Se-, -NR 6 or -CR 6 R 7 , where R 6 and R 7 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, or substituted or unsubstituted cycloalkyl.

[0011] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base;

[0012] P1 is a hydroxyl protecting group; preferably, P1 is DMTr or MMTr.

[0013] P2 is R 8 OR 8 , where R 8 It is CH2CH2CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, or substituted or unsubstituted C2-C6 alkynyl.

[0014] According to the present invention, the nucleoside compound represented by Formula 1 is as shown in Formula 2a or Formula 2b:

[0015] in,

[0016] R 1 R 2 R 3 R 4 R 5 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN.

[0017] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base;

[0018] P1 is a hydroxyl protecting group; preferably, P1 is DMTr or MMTr.

[0019] P2 is R 8 OR 8 R8 is CH2CH2CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, or substituted or unsubstituted C2-C6 alkynyl.

[0020] The compounds represented by formula 2a or 2b according to the present invention are shown as those represented by formula 3a or 3b:

[0021] in,

[0022] R 2 R 3 R 4Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN.

[0023] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base;

[0024] P1 is a hydroxyl protecting group; preferably, P1 is DMTr or MMTr.

[0025] P2 is R 8 OR 8 , where R 8 It is CH2CH2CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy.

[0026] The compounds represented by formula 3a or 3b according to the present invention are shown as those represented by formula 4a or 4b:

[0027] in,

[0028] R 3 R 4 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN.

[0029] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base;

[0030] P2 is R 8 OR 8 , where R 8 It is CH2CH2CN, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted C1-C3 alkoxy.

[0031] The compounds represented by formula 4a or 4b according to the present invention are shown as those represented by formula 5a or 5b:

[0032] in

[0033] R 3 R 4 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN.

[0034] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base.

[0035] The nucleoside compound represented by Formula 1 of the present invention is as shown in Formula 6a or Formula 6b:

[0036] in,

[0037] R 3 R 4 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN.

[0038] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base.

[0039] The nucleoside compound represented by Formula 1 of the present invention is as shown in Formula 7a or Formula 7b:

[0040] in,

[0041] R 3 R 4 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN.

[0042] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base.

[0043] The nucleoside compound represented by Formula 1 of the present invention is as shown in Formula 8a or Formula 8b:

[0044] in

[0045] R 3 R 4 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN.

[0046] R 9 Selected from C1-C6 alkyl groups;

[0047] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base.

[0048] The nucleoside compound represented by Formula 1 of the present invention is as shown in Formula 9a or Formula 9b:

[0049] in,

[0050] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base.

[0051] The nucleoside compound represented by Formula 1 of the present invention is as shown in Formula 10a or Formula 10b:

[0052] in

[0053] W represents hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN.

[0054] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base.

[0055] The nucleoside compound represented by Formula 1 of the present invention is as shown in Formula 11a or Formula 11b:

[0056] in,

[0057] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base.

[0058] In some embodiments of the present invention, when Base is a natural nucleobase, a modified nucleobase, a universal base, or an aryl group, it includes, but is not limited to, the following structures:

[0059] Furthermore, the Base is selected from the following structure:

[0060] The compounds described in this invention are specifically selected from:

[0061] The present invention also provides the use of the above-mentioned compounds as intermediates in the preparation of modified oligonucleotides.

[0062] The method for preparing modified oligonucleotides according to the present invention is as follows: the compound replaces the 2'-F modified nucleotide in the oligonucleotide.

[0063] Furthermore, the oligonucleotide is selected from siRNA, antisense nucleic acid, saRNA, miRNA, nucleic acid aptamer, and lncRNA.

[0064] Furthermore, the oligonucleotide comprises at least one 2'-F modified nucleotide, and the preparation method involves replacing at least one 2'-F modified nucleotide in the oligonucleotide with the compound to obtain the modified oligonucleotide.

[0065] Furthermore, the preparation was carried out under different Oligo synthetic conditions, and depending on the type of oxidant, the structure of the compound in the modified oligonucleotide was as follows: Y represents S, O, or BH2.

[0066] The present invention also provides a modified oligonucleotide comprising at least one structure shown in Formula II:

[0067] in,

[0068] R 1 R 2 R 3 R 4 R 5 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN.

[0069] Z represents -O-, -S-, -Se-, -NR 6 or -CR 6 R 7 , where R 6 and R 7 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, or substituted or unsubstituted cycloalkyl.

[0070] Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base;

[0071] P3 is R 8 OR 8 , where R 8 Y is OH, CH2CH2CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl; Y is S, O, BH2.

[0072] Furthermore, the oligonucleotide is selected from siRNA, antisense nucleic acid, saRNA, miRNA, nucleic acid aptamer, and lncRNA.

[0073] Furthermore, the siRNA comprises at least one, two, or three structures represented by Formula II.

[0074] Furthermore, the siRNA comprises a sense strand and an antisense strand, the sense strand or antisense strand having a length of 15-30 nucleotides, and the two single strands having at least more than 15 complementary bases.

[0075] Furthermore, the siRNA comprises a sense strand and an antisense strand, the sense strand having a length of 19 nucleotides and the antisense strand having a length of 21 nucleotides, and the two single strands having at least more than 15 complementary bases.

[0076] Further, the modified oligonucleotide consists of a sense strand sequence and an antisense strand sequence, and at least one of the following sites of the modified oligonucleotide contains the structure shown in Formula II: positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 starting from the 5' end of the sense strand sequence, and positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 starting from the 5' end of the antisense strand sequence.

[0077] Furthermore, at least one of the following sites in the modified oligonucleotide comprises the structure shown in Formula II: positions 7, 8, 9, 10, and 11 starting from the 5' end of the sense strand sequence, and positions 2 and 14 starting from the 5' end of the antisense strand sequence.

[0078] The modified oligonucleotides of the present invention are shown in Table 1, and the sense strand sequence and antisense strand sequence of the modified oligonucleotides are shown in Table 1.

[0079] The present invention also provides the use of the above-described compounds and the above-described modified conjugates in the preparation of medicaments for treating and / or preventing pathological conditions or diseases associated with INHBE gene overexpression.

[0080] Further, the pathological condition or disease is an immune-related disease, a disease related to dyslipidemia, or a disease related to dysglucose abnormalities; more preferably, the disease related to dyslipidemia is hyperlipidemia, hypertriglyceridemia, pancreatitis, familial chylomicronemia syndrome, diabetes, type 2 diabetes, non-alcoholic liver disease, cirrhosis, liver cancer, pancreatic cancer, heart disease, myocardial infarction, angina pectoris, or atherosclerosis.

[0081] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0082] The carbon, hydrogen, oxygen, sulfur, nitrogen, or F, Cl, Br, I mentioned in the groups and compounds described in this application include their isotopes, and the carbon, hydrogen, oxygen, sulfur, or nitrogen mentioned in the groups and compounds described in this application may optionally be further replaced by one or more of their corresponding isotopes, wherein the isotopes of carbon include 12 C 13 C and 14 C, the isotopes of hydrogen include protium (H), deuterium (D, also called heavy hydrogen), and tritium (T, also called superheavy hydrogen), and the isotopes of oxygen include 16 O、 17 O and 18 O, isotopes of sulfur include 32 S, 33 S, 34 S and 36 S, nitrogen isotopes include 14 N and 15 N, isotopes of fluorine include 17 F and 19 F, isotopes of chlorine include 35 Cl and 37 Cl, isotopes of bromine include 79 Br and 81 Br.

[0083] In this invention, "oligonucleotide" refers to a polymer of linked nucleosides, each nucleoside being independently modified or unmodified, comprising an oligonucleotide sequence of approximately 10-50 single-stranded or double-stranded nucleotide base pairs. In some embodiments, the oligonucleotide has a nucleobase sequence at least partially complementary to the core sequence of a target gene expressed in cells. In some embodiments, the oligonucleotide, upon delivery to a cell expressing the gene, regulates the expression of the corresponding target gene. Target gene expression can be regulated in vitro or in vivo. "Oligonucleotide" includes, but is not limited to: single-stranded antisense oligonucleotides, short interfering RNA (nucleic acid), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and dicer enzyme substrates.

[0084] In this invention, "nucleic acid drug" refers to an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can reduce or inhibit the translation of messenger RNA (mRNA) in a sequence-specific manner.

[0085] Nucleic acid molecules can exert their effects through RNA interference mechanisms (e.g., by interacting with the mRNA interference pathway mechanism (RNA-induced silencing complex RISC) in mammalian cells) or any other mechanism or pathway. While the term "nucleic acid drug" as used in this invention is considered to primarily exert its effects through RNA interference mechanisms, the nucleic acid drug is not limited to or restricted to any particular pathway or mechanism of action. Types of nucleic acid drug molecules include, but are not limited to: single-stranded antisense oligonucleotides, short interfering RNA (nucleic acid), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer enzyme substrates. The nucleic acid drug of this invention comprises an oligonucleotide chain having at least a partial complementarity to the mRNA serving as the target. In some embodiments, the nucleic acid drug of this invention is double-stranded and comprises an antisense strand and a sense strand at least partially complementary to the antisense strand.

[0086] The terms “silence,” “reduction,” “inhibition,” “downregulation,” or “knockdown” refer to a reduction or decrease in the expression level of a given gene when it is directly administered into cells, tissues, organs, or animals treated with the nucleic acid drug molecules described in this invention, compared to administration into cells, tissues, organs, or animals that have not been so treated.

[0087] The term "sequence" or "nucleotide sequence" refers to the order or sequence of nucleobases or nucleotides, expressed alphabetically using standard nucleotide nomenclature.

[0088] The term "base" is a nucleobase or modified nucleobase as defined in this invention. In some embodiments, the heterocyclic base moiety is a pyrimidine, a substituted pyrimidine, a purine, or a substituted purine, as well as a nitrogen-containing heterocycle. In some embodiments, the heterocyclic base moiety is an unconventional purine or a substituted purine. In some embodiments, the heterocyclic base moiety is an unconventional pyrimidine or a substituted pyrimidine. In some embodiments, the heterocyclic base moiety contains a non-natural five-membered nitrogen-containing heterocycle. In some embodiments, the heterocyclic base moiety may include one or more protecting groups.

[0089] In this invention, "nucleotide base" or "nucleobase" refers to heterocyclic pyrimidine or purine compounds, wherein "conventional nucleoside base" includes the bases of conventional nucleotides such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). Nucleobases can be modified or substituted with novel unconventional nucleobases. Nucleotide bases include naturally occurring nucleotide bases as well as non-naturally occurring nucleotide bases. It should be obvious to those skilled in the art that various nucleotide bases previously considered "non-naturally occurring" have later been discovered in nature. Therefore, "nucleotide base" includes not only known purine and pyrimidine heterocycles but also their heterocyclic analogs and tautomers. Exemplary examples of nucleotide bases include adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-diazaxanthine, 7-diazaguanine, N4,N4-bridged ethylidene cytosine, N6,N6-bridged ethylidene-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolpyridine, isocytosine, isoguanine, inosine, and the “non-naturally occurring” nucleotide bases described in U.S. Patent No. 5,432,272 to Benner et al. The term “nucleotide base” includes each and all of these examples and their analogues and tautomers. Particularly important nucleotide bases include adenine, guanine, thymine, cytosine, and uracil, which are considered to be naturally occurring nucleotide bases relevant to human therapeutic and diagnostic applications.

[0090] The term "complementarity," used to describe the relationship between a first nucleotide sequence (e.g., the sense strand of a nucleic acid drug or target mRNA) and a second nucleotide sequence (e.g., a single-stranded antisense oligonucleotide or a double-stranded antisense strand of a nucleic acid drug), refers to the ability of an oligonucleotide or oligonucleotide containing the first nucleotide sequence to hybridize with an oligonucleotide or oligonucleotide containing the second nucleotide sequence under certain conditions (under mammalian physiological conditions or similar in vitro conditions) to form base pairs and a double-stranded or double-helix structure. A complementary sequence contains either a Watson-Crick base pair or a non-Watson-Crick base pair and contains native or modified nucleotides or nucleotide analogs to a degree sufficient to satisfy the hybridization requirements described above. For example, for the purpose of determining identity or complementarity, monomers a and Af are complementary to U (or T) and are equivalent to A.

[0091] The term "sense strand" refers to the nucleotide sequence on an RNA molecule that carries the information of the amino acids that encode the protein. It is also called the coding strand, sense strand, or positive strand, while the other nucleotide sequence that is complementary to it is called the antisense strand.

[0092] The term "antisense strand" refers to a nucleotide sequence in the mRNA of the target gene that is substantially anticomplementary or substantially anticomplementary to the antisense strand.

[0093] The terms “optional” or “optionally” mean that the event or environment described below may, but does not have to, occur, and the description includes the possibility that the event or environment may or may not occur. For example, “optionally alkyl-substituted heterocyclic group” means that an alkyl group may, but does not have to, be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0094] The term "substitution" refers to one or more hydrogen atoms in a group, preferably up to five, and more preferably one to three hydrogen atoms, being independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, which can be substituted, whether experimentally or theoretically, by means of existing technology and experimental conditions. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0095] The minimum and maximum carbon atom content in hydrocarbon groups are indicated by a prefix, for example, the prefix C. a～ C b Alkyl groups indicate any alkyl group containing "a" to "b" carbon atoms. Therefore, for example, C 1～6 Alkyl groups are straight-chain or branched alkyl groups containing 1 to 6 carbon atoms.

[0096] Alkyl refers to a straight-chain or branched hydrocarbon group in an alkane molecule, such as methyl-CH3, ethyl-CH2CH3, or methylene-CH2-. The alkyl group can also be part of other groups, such as C1-C6 alkoxy groups or C1-C6 alkylamino groups. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, and n-hexyl groups. Alkyl groups can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxy, carboxyl, or carboxylic acid ester groups.

[0097] "Alkenyl" refers to a straight-chain or branched hydrocarbon group having at least two carbon atoms and at least one vinyl unsaturation site (>C=C<). For example, C a-b Alkenyl refers to an alkenyl group having a to b carbon atoms and is intended to include, for example, vinyl, propenyl, isopropenyl, 1,3-butadienyl, etc.

[0098] "Alynyl" refers to a straight-chain monovalent hydrocarbon group or a branched monovalent hydrocarbon group containing at least one triple bond. The term "alkynyl" is also intended to include those hydrocarbon groups having one triple bond and one double bond. For example, C 2-6 The term "alkynyl" is intended to include ethynyl, propynyl, etc.

[0099] "Alkoxy" refers to -O-(alkyl) or -O-(cycloalkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester group.

[0100] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0101] "Halogenated alkyl" refers to an alkyl group that is substituted with one or more halogens, wherein the alkyl group is as defined above.

[0102] "Ester group" refers to -C(O)O (alkyl) or -C(O)O (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0103] "Acyl" refers to a compound containing a -C(O)R group, where R is an alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl group.

[0104] A heteroatom is an atom in an organic compound that is other than carbon and hydrogen. It generally refers to an atom that replaces carbon in the molecular skeleton (especially in ring systems). Nitrogen, oxygen, sulfur, phosphorus, boron, chlorine, bromine, and iodine are common heteroatoms. If an organic compound contains a ring that includes heteroatoms, it is called a heterocyclic compound.

[0105] "Heteroaryl" refers to an aromatic ring group having a conjugated planar ring system and containing heteroatoms. It can be a 3- to 8-membered (e.g., 3, 4, 5, 6, 7, 8-membered) monocyclic, a 5- to 12-membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12-membered) bicyclic, or a 10- to 15-membered (e.g., 10, 11, 12, 13, 14, 15-membered) tricyclic system, and contains 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) heteroatoms selected from N, O, or S. Non-limiting examples of heteroaryl include triazolyl, pyridyl, furanyl, thiophene, pyranyl, pyrroloyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, piperidinylbenzimidazolyl, benzopyridyl, and pyrrolopyridyl. The heteroaryl group may optionally be further substituted by one or more substituents.

[0106] "Heterocyclic group" or "heterocycle" refers to a saturated or unsaturated aromatic heterocycle or a non-aromatic heterocycle. When it is an aromatic heterocycle, its definition is the same as the definition of "heteroaryl" above. When it is a non-aromatic heterocycle, it can be a 3- to 10-membered (e.g., 3, 4, 5, 6, 7, 8, 9, 10-membered) monocyclic, a 4- to 12-membered (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12-membered) bicyclic, or a 10- to 15-membered (e.g., 10, 11, 12, 13, 14, 15-membered) tricyclic system, and contains 1 to 4 (e.g., 1, 2, 3, 4) heteroatoms selected from N, O, or S, preferably a 3- to 8-membered heterocyclic group. Non-limiting examples of "heterocyclic group" or "heterocycle" include oxoheterobutyl, azaheterobutyl, thioheterobutyl, 1,3-dioxopentyl, 1,4-dioxopentyl, 1,3-dioxahexacycloyl, azaheptanyl, oxoheptanyl, thioheptanyl, triazolyl, pyridyl, piperidinyl, furanyl, thiophene, pyranyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, pyridazinyl, piperazinyl, homopiperazinyl, imidazolyl, piperidinyl, morpholinyl, thiomorpholinyl, thiaxylyl, 1,3-dithiaalkyl, dihydrofuranyl, dithiapentylyl Cycloyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydropyrroleyl, tetrahydroimidazoyl, tetrahydrothiazoyl, tetrahydropyranyl, benzimidazolyl, benzopyridyl, pyrrolopyridyl, benzodihydrofuranyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolyl, 2H-pyranyl, 4H-pyranyl, dioxacyclohexyl, 1,3-dioxapentyl, pyrazolinyl, dithiaalkyl, dithiamonyl, dihydrothiophenyl, pyrazolyl, imidazolinyl, imidazolinyl, 1,2,3,4-tetrahydroisoquinolinyl. The "heterocyclic group" or "heterocycle" may optionally be further substituted with one or more substituents.

[0107] In this invention, a "protecting group" refers to an unstable chemical moiety known in the art for preventing reactive groups (e.g., hydroxyl, amino, carboxyl, and thiol groups) from undergoing undesirable reactions during synthesis. Protecting groups are typically used selectively and / or orthogonally to protect other reactive sites in the reaction and are subsequently removed to release unprotected groups, making them available for further reactions. In some embodiments, "substituted" groups or substituents contain protecting groups.

[0108] Representative hydroxyl protecting groups used in this invention are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, all of which are incorporated herein by reference in their entirety. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used in this invention include dimethoxytriphenylmethyl (DMT), monomethoxytriphenylmethyl, 9-phenyloxanthracene-9-yl (Pixyl), and 9-(p-methoxyphenyl)oxanthracene-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used in this invention include Tr (triphenylmethyl), MMTr (4-methoxytriphenylmethyl), DMTr (4,4'-dimethoxytriphenylmethyl), and TMTr (4,4',4”-trimethoxytriphenylmethyl).

[0109] The compounds and compositions described in this invention may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state, depending on the environment in which the compound or composition is situated. Therefore, as used in this invention, the structures described herein take into account that certain functional groups, such as OH, SH, or NH, can be protonated or deprotonated. The disclosure of this invention is intended to cover the compounds and compositions described above, regardless of their protonation state based on environmental pH, as will be readily understood by those skilled in the art.

[0110] The terms "salt" and "available salt" refer to acidic and / or basic salts formed by the above-described compounds or their stereoisomers with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkyl ammonium salts. These salts can be obtained directly during the final separation and purification of the compounds. Alternatively, they can be obtained by mixing the above-described compounds, or their stereoisomers, with an appropriate (e.g., equimolar) amount of acid or base. These salts may be obtained by precipitating in solution and collecting by filtration, or by recovery after solvent evaporation, or by freeze-drying after reaction in an aqueous medium. Detailed Implementation

[0111] Unless otherwise specified, the instruments used in this invention are not conventional instruments, and the reagents used are all conventional reagents.

[0112] The structure of a compound is determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS).

[0113] NMR shifts (δ) are given in units of 10⁻⁶ (ppm). NMR measurements were performed using a Broker Avance III 400 NMR spectrometer with deuterated dimethyl sulfoxide (DMSO-d₆), deuterated chloroform (CDCl₃), and deuterated methanol (CD₃OD) as solvents, and tetramethylsilane (TMS) as the internal standard. MS measurements were performed using Agilent 6120B (ESI) and Agilent 6120B (APCI). Thin-layer chromatography (TLC) used Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The TLC plates used had a diameter of 0.15 mm–0.20 mm, and the purified products used had a diameter of 0.4 mm–0.5 mm.

[0114] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0115] Example Section

[0116] Example 1: Synthesis of compound FD2:

[0117] Step 1, Synthesis of Compound 2:

[0118] Pyridine (400 mL) was added to dried compound 1 (50 g, 0.205 mol, 1.0 eq.) and stirred at room temperature until completely dissolved. The reaction was cooled to 0-5 °C, and TIPSCl2 (71.0 g, 0.225 mol, 1.1 eq.) was slowly added dropwise over approximately 30 minutes. The reaction was brought back to room temperature and allowed to proceed overnight until the reactants of compound 1 were completely dissolved. The reaction was quenched by adding methanol (50 mL) and water (300 mL), and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent, yielding the crude compound. The crude compound was purified by column chromatography to obtain compound 2 (75.6 g, 0.156 mol, 76.1% yield).

[0119] Step 2, Synthesis of Compound 3:

[0120] To a dry compound 2 (75.6 g, 0.156 mol), 800 mL of ultradry dichloromethane was added and stirred at room temperature until completely dissolved. Pyridine (49.4 g, 0.624 mol, 4.0 eq.) and acetic anhydride (39.8 g, 0.39 mol, 2.5 eq.) were added to the reaction mixture, and the reaction was stirred at room temperature for 30 minutes. Chromium trioxide (39.0 g, 0.39 mol, 2.5 eq.) was then added to the reaction mixture, and the reaction was stirred at room temperature for 2–3 hours. TLC analysis confirmed that compound 2 had reacted completely. The reaction was quenched by adding an aqueous sodium thiosulfate solution. The mixture was filtered through diatomaceous earth, and the residue was washed twice with dichloromethane to obtain the filtrate. The filtrate was separated to obtain the organic phase. The aqueous phase was extracted once with dichloromethane, and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent, yielding the crude compound. The crude compound is used directly in the next reaction without purification.

[0121] Step 3, Synthesis of Compound 4:

[0122] To crude compound 3, 400 mL of ultra-dry tetrahydrofuran and 50 mL of deuterated ethanol were added and stirred until completely dissolved. The reaction mixture was then cooled to -5 to 0 °C and stirred for 30 minutes. At this temperature, NaBD4 (9.8 g, 0.234 mmol, 1.5 eq.) was slowly added in portions to the reaction mixture. After the addition was complete, the mixture was stirred until TLC analysis showed that compound 3 had completely reacted. After the reaction was complete, methanol and a saturated ammonium chloride aqueous solution were added to quench the reaction. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent, yielding crude compound 4. The crude compound was subjected to column chromatography to obtain compound 4 (41.0 g, 84.15 mmol, overall yield of 53.9%).

[0123] Step 4, Synthesis of Compound 5:

[0124] To compound 4 (41.0 g, 84.15 mmol), 300 mL of ultradry tetrahydrofuran was added and stirred until completely dissolved. Then, 84.2 mL of 1.0 mol / L TBAF tetrahydrofuran solution was added to the reaction mixture, and the reaction was carried out overnight at room temperature until compound 4 was completely reacted. The reaction mixture was concentrated at low temperature to remove the solvent, yielding crude compound 5. The crude compound was purified by C18 reverse-phase chromatography to give compound 5 (17.3 g, 70.60 mmol, 83.9% yield).

[0125] Step 5, Synthesis of Compound 6:

[0126] To compound 5 (17.3 g, 70.60 mmol), 150 mL of ultradry pyridine was added and stirred until completely dissolved. DMAP (12.9 g, 105.9 mmol, 1.5 eq.) was added to the reaction mixture, followed by slow, fractional addition of TrtCl (41.3 g, 84.72 mmol, 1.2 eq.). The reaction was stirred at room temperature until compound 5 was completely reacted. The reaction was quenched with methanol and water, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent, yielding crude compound 6. The crude compound was subjected to column chromatography to obtain compound 6 (30.6 g, 62.81 mmol, 89% yield). 1 H NMR(600MHz,DMSO-d6)δ11.37-11.29(m,1H),7.46-7.20(m,16H),6.02(s,1H),5.57-5.52(m ,2H),5.43-5.37(m,1H),3.94–3.86(m,2H),3.31-3.23(m,1H),2.23-3.17(m,1H),ESI-LCMS m / z 486.1[MH] - .

[0127] Step 6, Synthesis of Compound 7:

[0128] To compound 6 (30.6 g, 62.81 mmol), 200 mL of ultradry DMF was added and stirred until completely dissolved. Collidine (45.8 g, 0.377 mol, 6.0 eq.) and silver nitrate (19.2 g, 0.113 mol, 1.8 eq.) were added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes. TrtCl (43.8 g, 0.157 mol, 2.5 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature overnight until compound 6 was completely reacted. The reaction was quenched with methanol and water. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent, yielding the crude compound. The crude compound was purified by column chromatography to give compound 7 (36.2 g, 49.64 mmol, 79%). 1 H NMR(400MHz,DMSO-d6)δ7.51–7.18(m,30H),7.17-7.08(m,1H),6.10(s,1H),5.39-5.27(m,1H),4 .01–3.87(m,1H),3.66(s,1H),3.25-3.12(m,3H),3.11–3.01(m,1H),2.72–2.63(m,1H),ESI-LCMS m / z 728.1[MH]-

[0129] Step 7, Synthesis of Compound 8:

[0130] To compound 7 (36.2 g, 49.64 mmol), ultra-dry dichloromethane was added and stirred until completely dissolved. The reaction was cooled to -5 to 0 °C, and DAST (24.0 g, 0.149 mol, 3.0 eq.) was slowly added dropwise to the reaction. After the addition was complete, the reaction was continued at this temperature until compound 7 was completely reacted. At this temperature, a saturated aqueous solution of ammonium chloride was slowly added to the reaction to quench the reaction. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent to obtain the crude compound. The crude compound was purified by column chromatography to obtain compound 8 (16.5 g, 22.56 mmol, 45.4% yield). 1 H NMR (600MHz, DMSO-d6) δ11.40–11.28(m,1H),7.57–7.45(m,1H),7.34–7.18(m,30H),5.78–5.73(m, 1H),5.35-5.27(m,1H),4.27–4.18(m,1H),4.13-3.95(m,2H),3.21-3.12(m,1H),3.00–2.91(m,1H). 19 FNMR(600MHz,DMSO-d6)δ-193.72,ESI-LCMS m / z 730.1[MH] -

[0131] Step 8, Synthesis of Compound 9:

[0132] Compound 8 (16.5 g, 22.56 mmol) was added to ultradry dichloromethane (150 mL) and stirred until completely dissolved. TES (30 mL) was added to the reaction mixture and stirred for 5 minutes, followed by slow dropwise addition of TFA (12.9 g, 0.113 mol, 5.0 eq.). The reaction was allowed to proceed at room temperature for approximately 1 hour until compound 8 was completely reacted. The reaction was quenched by adding ammonia-methanol solution (50 mL). The mixture was concentrated at low temperature to remove the solvent, yielding the crude compound. The crude compound was purified by C18 reverse-phase extraction to obtain compound 9 (4.8 g, 19.42 mmol, 86.1% yield). 1H NMR (400MHz, DMSO-d6) δ11.50–11.32(m,1H),7.99–7.85(m,1H),6.00–5.85(m,1H),5.68– 5.62(m,1H),4.21–4.09(m,1H),3.94–3.84(m,1H),3.81–3.71(m,1H),3.65–3.54(m,1H). 19 FNMR(400MHz,DMSO-d6)δ-203.05,ESI-LCMS m / z 248.1[M+H] -

[0133] Step 9, Synthesis of Compound 10:

[0134] To compound 9 (4.8 g, 19.42 mmol), 50 mL of ultradry pyridine was added and stirred until completely dissolved. DMTrCl (7.89 g, 23.29 mmol, 1.2 eq.) was then added to the reaction mixture. After the addition was complete, the reaction was allowed to proceed overnight at room temperature until compound 9 was fully reacted. After the reaction was complete, methanol and water were added to quench the reaction mixture. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent, yielding the crude compound. The crude compound was purified by column chromatography to give compound 10 (9.6 g, 17.48 mmol, 90% yield). 1 H NMR (600MHz, DMSO-d6) δ11.48–11.37(m,1H),7.82–7.71(m,1H),7.43–7.20(m,9H),6.98–6.85(m,4H),5.94–5.82( m,1H),5.71–5.61(m,1H),5.35–5.24(m,1H),4.43–4.25(m,1H),4.07–3.96(m,2H),3.74(s,6H),3.32–3.24(m,2H). 19 F NMR(600MHz,DMSO-d6)δ-200.18,ESI-LCMS m / z 548.1[MH] -

[0135] Step 10: Synthesis of compound FD2;

[0136] To the dried compound 10 (1.5 g, 2.73 mmol), 15 mL of ultra-dry dichloromethane was added and stirred until completely dissolved. DCI (0.29 g, 2.457 mmol, 0.9 eq.) was added to the reaction mixture and stirred for 5 minutes. CEP(N(iPr)₂)₂ (0.91 g, 3.0 mmol, 1.1 eq.) was added to the reaction mixture in one go, and the entire reaction was carried out under nitrogen protection. The reaction was carried out at room temperature for approximately 40 minutes until compound 10 was completely reacted. After the reaction was complete, a saturated sodium bicarbonate aqueous solution was added to quench the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated at low temperature to remove the solvent, yielding the crude compound. The crude compound was purified by C18 reverse-phase extraction to obtain compound FD₂ (1.7 g, 2.27 mmol, 83.2% yield). 1 H NMR(400MHz,DMSO-d6)δ11.42(s,1H),7.88–7.76(m,1H),7.48–7.16(m,9H),6.97–6.80(m,4H),5.97–5.79(m,1H),5.41–5.26(m,1H),4.77–4.4 3(m,1H),4.19–4.04(m,1H),3.84–3.66(m,7H),3.64–3.34(m,4H),3.29 –3.19(m,1H),2.81–2.82(m,1H),2.65–2.56(m,1H),1.21–0.87(m,12H). 19 F NMR(400MHz,DMSO-d6)δ-197.09,-197.12,-197.91,-197.93. 31 P NMR (162MHz, DMSO-d6) δ149.67,149.61,149.31,149.26, ESI-LCMS m / z 750.6[MH] -

[0137] Example 2, Synthesis of compound FD3:

[0138] Step 1, Synthesis of Compound 11:

[0139] Compound 10 (4.5 g, 8.19 mmol) was dissolved in 45 mL of ultradry DMF by stirring until completely dissolved. Imidazole (2.1 g, 32.76 mmol, 4.0 eq.) was then added to the reaction mixture, and the mixture was stirred for 30 minutes. TBSCl (2.48 g, 16.38 mmol, 2.0 eq.) was slowly added in portions to the reaction mixture, and the reaction was carried out overnight at room temperature with stirring until the compound was completely reacted. The reaction was quenched with methanol and water, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent, yielding the crude compound. The crude compound was purified by column chromatography to give compound 11 (4.7 g, 7.09 mmol, 86.6% yield). ESI-LCMS m / z 662.2 [MH] - .

[0140] Step 2, Synthesis of Compound 12:

[0141] Compound 11 (4.7 g, 7.09 mmol) was dissolved in acetonitrile (50 mL) by stirring until completely dissolved. Triethylamine (1.44 g, 14.18 mmol, 2.0 eq.) and DMAP (1.73 g, 14.18 mmol, 2.0 q.) were added to the reaction mixture and stirred for 30 minutes. Then, TPSCl (4.29 g, 14.18 mmol, 2.0 eq.) was slowly added in portions to the reaction mixture, and the reaction was stirred overnight at room temperature until compound 11 was completely reacted. Ammonia (20 mL) was added to the reaction mixture and stirred at room temperature. The reaction was monitored by TLC until complete. Water was added to the reaction mixture, and the mixture was extracted twice with ethyl acetate. The organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent, yielding the crude compound. The crude compound was used directly in the next reaction without purification. ESI-LCMS m / z 661.2 [MH] - .

[0142] Step 3, Synthesis of Compound 13:

[0143] To the dried crude compound 12, 50 mL of ultradry pyridine was added and stirred until completely dissolved. The reaction mixture was cooled to 0–5 °C, and B2Cl (2.0 g, 14.18 mmol, 2.0 eq.) was slowly added dropwise. After the addition was complete, the reaction mixture was stirred at this temperature for approximately 1 hour until compound 12 reacted completely. After the reaction was complete, methanol and water were added at this temperature to quench the reaction. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent, yielding the crude compound. The crude compound was subjected to column chromatography to obtain compound 13 (4.2 g, 5.48 mmol, 77.3% yield). 1 H NMR (400MHz, DMSO-d6): δ11.40(s,1H),8.55(s,1H),8.07-8.05(m,2H),7.71( m,1H),7.69-7.60(m,2H),7.58-7.31(m,9H),7.26-7.21(m,1H),6.99-6.96(m ,4H),6.09-6.05(m,1H),4.73-4.62(m,1H),4.16-4.13(m,1H),3.81(s,6H),3 .65-3.63(m,1H),3.28-3.27(m,1H),0.80(s,9H),0.11-0.00(m,6H).ESI-LCMS m / z767.2[M+H] + .

[0144] Step 4, Synthesis of Compound 14:

[0145] To compound 13 (4.02 g, 5.48 mmol), 40 mL of ultradry tetrahydrofuran was added and stirred until completely dissolved. 5.5 mL of 1.00 mol / L TBAF tetrahydrofuran solution was added to the reaction mixture, and the reaction was allowed to proceed overnight at room temperature until compound 13 was completely reacted. Water was added to the reaction mixture and stirred until homogeneous. The mixture was extracted twice with ethyl acetate, and the organic phases were combined. The organic phase was washed with 0.1 mol / L hydrochloric acid, water, and saturated brine, then dried over anhydrous sodium sulfate and concentrated to remove the solvent, yielding the crude compound. The crude compound was purified by column chromatography to give compound 14 (3.2 g, 4.91 mmol, 89.6% yield). 1H NMR (400MHz, DMSO-d6): δ11.30(s,1H),8.36-8.34(m,1H),7.99-7.65(m,2H),7.54-7.52(m,1H),7.50-7.16(m,12H),6.93- 6.91(m,4H),6.00-5.99(m,1H),5.7(s,1H),4.78-4.58(m,1H),4.46-4.37(m,1H),3.75(s,6H),3.45–3.35(m,2H).ESI-LCMS m / z 651.3[MH] - .

[0146] Step 5, Synthesis of compound FD3:

[0147] To the dried compound 14 (1.5 g, 2.30 mmol), ultradry dichloromethane was added and stirred until completely dissolved. DCI (0.24 g, 2.07 mmol, 0.9 eq.) was added to the reaction mixture, and the mixture was stirred for 5 minutes. The reaction was protected by nitrogen purging. CEP(N(iPr2)2 compound (0.77 g, 2.53 mmol, 1.1 eq.) was added to the reaction mixture, and the mixture was stirred at room temperature for 30 minutes until compound 14 reacted completely. The reaction was quenched by adding saturated sodium bicarbonate aqueous solution. The mixture was extracted twice with dichloromethane, and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to remove the solvent, yielding the crude compound. The crude compound was purified by a C18 reversed-phase column to give compound FD3 (1.5 g, 1.76 mmol, 76.5% yield). 1 H NMR (400MHz, DMSO-d6): δ11.31(s,1H),8.40-8.36(m,1H),8.00-7.99(m,2H) ,7.65-7.61(m,1H),7.54-7.14(m,12H),6.92-6.88(m,4H),6.02-5.98(m,1H) ,4.78-4.58(m,1H),4.23-4.21(m,1H),3.80-3.69(m,7H),3.59-3.46(m,4H), 3.37-3.32(m,1H),2.78-2.75(m,1H),2.62-2.59(m,1H),1.14-1.09(m,12H). 31 P NMR (162MHz, DMSO-d6): δ150.23,150.19,149.43,149.38.ESI-LCMS m / z 853.2[M+H] + .

[0148] Example 3: Synthesis of compound FD4 and compound FD

[0149] Following the same synthetic method as compound FD2, compounds FD4 and FD were obtained.

[0150] Example 4, Synthesis of compound FD12:

[0151] Compound FD12 was synthesized according to the above route.

[0152] Example 5, Synthesis of compound FD13:

[0153] Compound FD13 was synthesized according to the above route.

[0154] Example 6: Synthesis of compound FD17:

[0155] Compound FD17 was synthesized according to the above route.

[0156] Example 7, Synthesis of compound FD18:

[0157] Compound FD18 was synthesized according to the above route.

[0158] Example 8: Synthesis of compound FD22-compound FD113

[0159] Following the above synthetic route, compounds FD22 and FD113 were obtained.

[0160] Example 9: Synthesis of Conjugates

[0161] Using the previously synthesized FD compound (FD2-compound FD113) as a raw material, oligonucleotide (siRNA) conjugates were synthesized.

[0162] The synthesis of siRNA is no different from the usual solid-phase synthesis of phosphorus amide. When synthesizing nucleotides modified at various positions of the SS and AS chains, the original nucleotides in the parent sequence are replaced by the synthesized FD phosphorus amide monomer.

[0163] The synthesis process is briefly described as follows: For the sense and antisense strands of the siRNA sequence of the present invention, as well as the sense and antisense strands of the modified double-stranded siRNA (i.e., the modified double-stranded siRNA), CPG is used as a solid-phase carrier; GalNAc-modified CPG is used as the starting cycle for the synthesis of the sense strand, and universal CPG is used as the starting cycle for the synthesis of the antisense strand.

[0164] Using a YB-192S synthesizer, a solid-phase synthesis method of phosphoramidite was employed. Starting with a solid support, nucleoside monomers were sequentially linked in the 3'-5' direction to achieve a synthesis scale of 0.2 μmol.

[0165] The phosphorus amide monomer is linked through a four-step chemical reaction cycle: deprotection, coupling, oxidation / sulfidation, and capping. The phosphorus amide monomer is prepared as a 0.05M acetonitrile solution, with 0.3M BTT in acetonitrile as the activator, a 3% trichloroacetic acid / dichloromethane solution as the deprotecting agent, a 0.05M iodine / pyridine / tetrahydrofuran / aqueous solution (v / v / v = 2 / 1 / 7) as the oxidizing agent, acetic anhydride / acetonitrile solution as capping agent A (v / v = 2 / 8), pyridine / N-methylimidazolium / tetrahydrofuran solution as capping agent B (v / v / v = 10 / 16 / 74) as the capping agent, and a 0.05M DDTT solution of pyridine / acetonitrile as the thiochemical agent (v / v = 4 / 6).

[0166] After solid-phase synthesis, the support was transferred to a 2 mL centrifuge tube, and 0.8 mL of concentrated ammonia was added. The mixture was then sealed and reacted at 55 °C for 16 h. After cooling to room temperature, the solution was transferred to a 2 mL centrifuge tube and concentrated to dryness. 0.2 mL of anhydrous DMSO was added to dissolve the solution, followed by 0.25 mL of triethylamine trihydrofluoride. The reaction was carried out at 65 °C for 2 h. After the reaction was complete, the mixture was cooled to room temperature, and the crude sequence was obtained by ethanol precipitation.

[0167] The crude product was purified by reversed-phase HPLC, and the collected fraction was lyophilized. Ethanol precipitation was performed by adding 0.3 mL of 1M sodium acetate solution and 0.9 mL of ethanol to replace the sequence with sodium salt. Then, desalting was carried out using a 3KD ultrafiltration tube to remove excess free salt.

[0168] The sense and antisense chains were prepared into an aqueous solution of a certain concentration. The sense and antisense chains were mixed at a molar ratio of 1:1.05, incubated at 95°C for 5 minutes, and then naturally cooled to room temperature. The product was then freeze-dried to obtain the target product.

[0169] The modification schemes used in Table 1 are as follows:

[0170] In this diagram, uppercase letters A, C, G, U, and I represent adenosine-3'-phosphate, cytidine-3'-phosphate, guanosine-3'-phosphate, uridine-3'-phosphate, and inosine-3'-phosphate, respectively; lowercase letters a, u, g, c, and t represent nucleotides modified with a 2'-methoxy group; lowercase letter f indicates that the nucleotide adjacent to the right is modified with a 2'-fluoride group; lowercase letter d indicates that the nucleotide adjacent to the right is a 2'-deoxyribonucleotide; the two nucleotides adjacent to each other with an asterisk (*) are linked by a thiophosphate group; FD1 to FD113 represent the FD-modified nucleoside compounds described above. By replacing the 2'-F-modified nucleotide in the siRNA, under the oligonucleotide synthesis conditions of this embodiment, the structures of FD1 to FD113... Become And replace the 2'-F modified nucleotide in the siRNA; taking the positive strand of conjugate 1 with the sequence shown in SEQ ID No. 1 as an example: the FD2 structure in the sequence is L96 indicates that the delivery vector GalNAc(L96) is conjugated at this location; the letter eVP indicates that the nucleotide adjacent to the right of the identifier eVP is a vinyl phosphate modified nucleotide.

[0171] GalNAc(L96) has the following structure:

[0172] Table 1: Conjugate Sequences

[0173] The structural characterization methods and results of the conjugates are detailed in Table 2:

[0174] Representative LC-MS test method: When the test sample is subjected to denaturing IP·RP-LC detection, the complementary double strands are untied into single strands (sense and antisense strands). Then, the parent ions of the sense and antisense strands are fragmented by tandem mass spectrometry. All detected fragment ions are analyzed and resolved using the software CONFIRM Sequence. The sequence of the test sample is consistent with the theoretical sequence, that is, the deviation between the actual molecular weight (MW) and the theoretical molecular weight (MW) is less than 0.05%. The results are shown in Table 2.

[0175] Table 2. Molecular weight (MW) of the conjugates

[0176] The effects of the present invention are illustrated below through experimental results:

[0177] Experimental Section

[0178] Experimental Example 1: In vitro activity assay (in vitro silencing activity of INHBE target mRNA and siRNA containing FD modification)

[0179] Activity screening steps:

[0180] Cell culture and transfection

[0181] Cell culture: Hep3B cells (ATCC) were cultured at 37°C and 5% CO2 in MEM complete medium (Gibco, with 10% FBS) until near confluence. The cells were then digested with trypsin and seeded into 96-well plates. 2.0 × 103 Hep3B cells and 1.0 mL of MEM complete medium (Gibco, with 10% FBS) were added to each well. The cells were cultured at 37°C and 5% CO2 for 16-24 h before transfection.

[0182] Cell transfection: Add 0.15 μL of lipofectamine RNAiMax (Invitrogen) to each well of opti-MEM, then add 5.0 μL of siRNA and mix. Add this mixture to a PCR tube and incubate at room temperature for 5 minutes. Finally, add this siRNA mixture to the cells and continue culturing for 24 hours before RNA extraction. Single-dose experiments were performed at concentrations of 10 nM and 0.1 nM or 0.1 nM and 0.01 nM siRNA duplexes. IC50 assays were performed at concentrations of 10 nM, 1.0 nM, 0.1 nM, 0.01 nM, 0.001 nM, 0.0001 nM, and 0.00001 nM siRNA duplexes.

[0183] RNA extraction

[0184] Using the Total RNA Isolation Kit (Omega, CAT: R6834-02): Collect cells, wash with 1% PBS, then add lysis buffer (containing 2% β-mercaptoethanol) to lyse the cells. Follow the instructions for the RNA isolation kit. Finally, add 30 μL of RNase-free water, let stand for 2 minutes, and then centrifuge at 14000g for 2 minutes to collect RNA.

[0185] cDNA synthesis

[0186] cDNA synthesis was performed using the TransGold gDNA Removal and cDNA Synthesis Kit (TransGold Biotechnology Co., Ltd., Beijing, China, Cat#AE311-03). 1 μg of total RNA was added to each sample, and cDNA synthesis was performed using a gradient thermal cycler (LongGene, A600) following the manufacturer's instructions.

[0187] Real-time quantitative PCR

[0188] Add the synthesized cDNA and the mixed stock solution (containing primers, qPCR premix and ultrapure water) to a 384-well plate (Bokcom Biosystems Cat#PC-0040-9U) to make the final real-time quantitative PCR system contain 0.25 μM each of upstream and downstream primers of the target gene (AGT) or internal reference gene (GADPH) and 1×SYBR Green premix (Applied Biosystems Cat#A25742).

[0189] The ΔΔCt measurement method was used in ABI QuantStudio. TM 6. Perform real-time fluorescence PCR in a real-time fluorescence PCR system. Perform 3-4 independent transfection tests for each double-stranded strain, with 3-4 assays per transfection.

[0190] The results of the in vitro activity assays of the siRNA conjugates are shown in Table 3. Pc, whose maternal sequence exhibits INHBE gene repression, was used as a positive control.

[0191] The Pc molecule, positive chain: c*u*gucafCafGfAfCuccacuucauL96

[0192] Antisense strand: a*fU*gadAggnUggagucfUgfUgacag*u*a

[0193] Table 3. Transfection activity of the conjugates in Hep3B cells

[0194] As is well known to those skilled in the art, positions 2 and 14 of the antisense strand of siRNA (starting from the 5' end of the antisense strand) and positions 7, 8, 9, 10, and 11 of the sense strand (starting from the 5' end of the sense strand) are highly sensitive to modification and require a high degree of steric hindrance from the modified nucleoside. Generally, nucleosides modified with 2'-F are used. This invention found that replacing the FD-modified nucleoside compounds at positions 2 and 14 (starting from the 5' end of the antisense strand) and positions 7, 8, 9, 10, and 11 (starting from the 5' end of the sense strand) of the conjugates in Table 1 with nucleotides modified with methoxy, MOE, LNA, etc., significantly reduced or even eliminated the transfection activity of the resulting conjugates. However, this invention found that replacing the 2'-F-modified nucleotides at the modification-sensitive positions 2 and 14 (starting from the 5' end of the antisense strand) and positions 7, 8, 9, 10, and 11 (starting from the 5' end of the sense strand) of the siRNA with FD-modified nucleoside compounds maintained or slightly improved the transfection activity of the resulting conjugates. The solution of this invention is not obvious.

[0195] Experimental Example 2: Cytotoxicity Test

[0196] Cell culture: Hep3B cells (ATCC) were cultured at 37°C and 5% CO2 in MEM complete medium (Gibco, with 10% FBS) until near confluence. Then, the cells were digested with trypsin and seeded into 24-well plates. 0.5 × 10⁵ Hep3B cells and 0.5 mL of MEM complete medium (Gibco, with 10% FBS) were added to each well. After culturing at 37°C and 5% CO2 for 16-24 h, transfection was performed.

[0197] Cell transfection: Add 0.75 μL of lipofectamine RNAiMax (Invitrogen) to each well of 24.25 μL opt-MEM, then add 25 μL of siRNA and mix. Add the mixture to a PCR tube and incubate at room temperature for 5 minutes. Finally, add this siRNA mixture to the cells and continue culturing for 48 or 96 hours before performing cytotoxicity testing. This experiment was performed at 10 nM and 1 nM siRNA double-stranded concentrations.

[0198] Cytotoxicity test:

[0199] Cytotoxicity was tested using a CCK-8 assay kit (Beyotime, CAT: #C0040). After 48 / 96 hours of cell transfection, the culture medium was removed, and 500 μL of the corresponding complete culture medium (containing 10% CCK-8) was added to each well. The cells were incubated at 37°C for 30-60 minutes in the dark. The OD values ​​of the samples were detected using a microplate reader (Tecan CAT: #spark 20M) with a wavelength of 450 nm and a reference wavelength of 620 nm.

[0200] Conclusion: The siRNA conjugates with the FD-modified nucleoside compounds described above did not exhibit cytotoxicity.

[0201] Experimental Example 3: siRNA Immunogenicity Test

[0202] hPBMC cells (Shanghai Saili Biotechnology Co., Ltd.) from three different donors were centrifuged and resuspended in RPMI-1640 cell culture medium (containing 10% FBS and 1% Penicillin-streptomycin solution). After mixing, the cells were incubated overnight at 37°C with 5% CO2. The test compound was diluted to 20× working solution. Prepare a mixture of Opti-Mem and Opti-Mem at a ratio of 1.5:23.5, vortex, and then use. Mix the prepared 20× working solution with... The mixture was prepared at a 1:1 ratio in 96-well V plates. The overnight culture medium was discarded, and hPBMC cells were resuspended in RPMI-1640 cell culture medium (containing 10% FBS and 1% Penicillin-streptomycin solution). After cell counting, the cells were diluted to the required transfection density and added to the 96-well plates. The final cell count was 2.0 × 10⁵ cells / well, with a total volume of 200 μL. The plates were then incubated for 24 hours in a 5% CO₂, 37°C incubator.

[0203] 24 h after hPBMC cell transfection, cell supernatant was collected, and the levels of IFN alpha, IL-6, and TNF alpha in the hPBMC supernatant were detected using the ProcartaPlex Mix & Match 3-plex Kit. The fold change of each factor in the test compounds was calculated: fold change for the test siRNA, nake siRNA, and polyIC = detected factor concentration / factor concentration in the transfection reagent well; fold change for GS9688 = detected factor concentration / factor concentration in the DMSO well.

[0204] Conclusion: siRNA conjugates containing FD-modified nucleoside compounds as described above do not induce immune activation.

[0205] Experiment Example 4: In vivo activity assay of ANGPTL3 / TTr target mRNA and siRNA containing FD-modified nucleosides

[0206] Evaluation of siRNA sequence activity in wild-type mice:

[0207] The activity of siRNA conjugates with FD-modified nucleosides in vivo was evaluated using wild-type C57BL / 6 mice.

[0208] C57BL / 6 mice aged 6-8 weeks were used, and the mice were placed in the facility for acclimatization for more than 3 days before administration. Each group of 3-5 mice was injected subcutaneously with a single dose of the compound (conjugates in Table 4). Blood was collected via the orbital sinus before administration and on days 7, 14, 21, 28, 35, 42, and 56 after administration. After the blood samples were left at room temperature for two hours, they were centrifuged at 5500 rpm for 10 minutes at 4°C to separate and collect serum, and the expression level of TTr protein in the animal serum was detected.

[0209] Table 4. siRNA conjugates (TTr target mRNAs) used for in vivo activity evaluation

[0210] Table 5. In vivo drug delivery of siRNA

[0211] Table 6. siRNA conjugates used for in vivo activity evaluation

[0212] Table 7. In vivo drug delivery of siRNA

[0213] Conclusion: The siRNA conjugates with the FD-modified nucleoside compounds described above did not affect the knockdown activity of mouse serum TTR or ANGPTL3 proteins, indicating that the FD-modified nucleoside compounds have good compatibility at key positions of siRNA (positions 2 and 14 of the antisense strand; positions 9, 10, and 11 of the sense strand).

[0214] Experimental Example 5: In vitro stability assay of oligonucleotide single strands containing FD-modified nucleosides

[0215] Stability evaluation of single-stranded oligonucleotides containing FD-modified nucleosides against 3' and 5' exonucleases:

[0216] The modified oligonucleotide single-chain acid was prepared into a solution with a final concentration of 0.1 mg / mL and placed in 50 mM Tris (pH 7.2), 10 mM MgCl2 or 50 mM sodium acetate (pH 6.5), 10 mM MgCl2, respectively. 150 mU / mL SVPDE or 500 mU / mL phosphodiesterase II was added, respectively. Samples were taken at different time points (0 h, 1 h, 2 h, 5 h, 10 h, 15 h, 20 h, 25 h, 48 h) and the content of the full-length oligonucleotide single chain was analyzed by electrophoresis or quantitatively detected by HPLC to evaluate the stability of the oligonucleotide single chain in 3' exonuclease and 5' exonuclease.

[0217] Table 8: Oligonucleotide single strands used to evaluate stability against 3' exonucleases

[0218] Table 9: Oligonucleotide single strands used to evaluate stability against 5' exonuclease

[0219] Conclusion: Oligonucleotide single chains with FD2 and FD3 modified nucleoside compounds as described above have higher stability than oligonucleotide single chains with fU and fC modifications.

Claims

1. A deuterated nucleoside phosphoramidide compound as shown in Formula 1 and its stereoisomers: in, R 1 R 2 R 3 R 4 R 5 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN. Z represents -O-, -S-, -Se-, -NR 6 or -CR 6 R 7 , where R 6 and R 7 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, or substituted or unsubstituted cycloalkyl. Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base; P1 is a hydroxyl protecting group; preferably, P1 is DMTr or MMTr. P2 is R 8 OR 8 , where R 8 It is CH2CH2CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, or substituted or unsubstituted C2-C6 alkynyl.

2. The compound according to claim 1, characterized in that: The compounds represented by Formula 1 are shown as those in Formula 2a or Formula 2b: in, R 1 R 2 R 3 R 4 R 5 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN. Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base; P1 is a hydroxyl protecting group; preferably, P1 is DMTr or MMTr. P2 is R 8 OR 8 R8 is CH2CH2CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, or substituted or unsubstituted C2-C6 alkynyl.

3. The compound according to claim 2, characterized in that: The compounds represented by formula 2a or 2b are shown as those represented by formula 3a or 3b: in, R 2 R 3 R 4 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN. Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base; P1 is a hydroxyl protecting group; preferably, P1 is DMTr or MMTr. P2 is R 8 OR 8 , where R 8 It is CH2CH2CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy.

4. The compound according to claim 3, characterized in that: The compounds represented by formula 3a or 3b are shown as those represented by formula 4a or 4b: in, R 3 R 4 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN. Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base; P2 is R 8 OR 8 , where R 8 It is CH2CH2CN, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted C1-C3 alkoxy.

5. The compound according to claim 4, characterized in that: The compounds represented by formula 4a or 4b are shown as those represented by formula 5a or 5b: in, R 3 R 4 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN. Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base.

6. The compound according to any one of claims 1-5, characterized in that: The Base is selected from the following structure:

7. The compound according to claim 6, characterized in that: The Base is selected from the following structure:

8. The compound according to any one of claims 1-7, characterized in that: The compounds are specifically selected from:

9. Use of the compound according to any one of claims 1-8 as an intermediate in the preparation of modified oligonucleotides.

10. The use according to claim 9, characterized in that: The oligonucleotides are selected from siRNA, antisense nucleic acid, saRNA, miRNA, nucleic acid aptamers, and lncRNA.

11. The use according to claim 9, characterized in that: The oligonucleotide contains at least one 2'-F modified nucleotide, and the preparation method is to replace at least one 2'-F modified nucleotide in the oligonucleotide with the compound to obtain the modified oligonucleotide.

12. A modified oligonucleotide comprising at least one structure of Formula II: in, R 1 R 2 R 3 R 4 R 5 Each of these elements is independent of the following: hydrogen, deuterium, halogen, cycloalkyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, and CN. Z represents -O-, -S-, -Se-, -NR 6 or -CR 6 R 7 , where R 6 and R 7 Each of them is independently hydrogen, halogen, acyl, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic, or substituted or unsubstituted cycloalkyl. Base can be a natural nucleobase, a modified nucleobase, a universal base, an aryl base, or a hydrogen base; P3 is R 8 OR 8 , where R 8 It can be OH, CH2CH2CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C2-C6 alkenyl, or substituted or unsubstituted C2-C6 alkynyl. Y represents S, O, and BH2.

13. The modified oligonucleotide according to claim 12, characterized in that: The oligonucleotides are selected from siRNA, antisense nucleic acid, saRNA, miRNA, nucleic acid aptamers, and lncRNA.

14. The modified oligonucleotide according to any one of claims 12-13, characterized in that: The oligonucleotide is selected from siRNA, which comprises a sense strand and an antisense strand, the sense strand or antisense strand having a length of 15-30 nucleotides, and the two single strands having at least more than 15 complementary bases.

15. The modified oligonucleotide according to any one of claims 12-14, characterized in that: The modified oligonucleotide consists of a sense strand sequence and an antisense strand sequence, and the modified oligonucleotide contains at least one of the following sites: positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 starting from the 5' end of the sense strand sequence, and positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 starting from the 5' end of the antisense strand sequence.

16. The modified oligonucleotide according to claim 15, characterized in that: The modified oligonucleotide contains at least one of the following sites: positions 7, 8, 9, 10, and 11 starting from the 5' end of the sense strand sequence, and positions 2 and 14 starting from the 5' end of the antisense strand sequence.

Images