5'-spirophosphonate modified nucleotide

Abstract

The present invention relates to an oligonucleotide containing a nucleotide monomer having a structure as represented by formula (I), and further relates to an RNAi agent containing the nucleotide monomer. In addition, the present invention further relates to a composition containing the oligonucleotide or RNAi agent, a kit, and the related use.

Description

5'-spirophosphonate modified nucleotides

[0001] This international patent application claims priority to Chinese patent application number 202410874991.X, filed July 1, 2024, the entire contents of which are incorporated herein by reference for all purposes. TECHNICAL FIELD

[0002] The present invention is in the field of medicine, and specifically relates to spirophosphonate modified nucleotide monomers, as well as oligonucleotides, RNAi agents comprising the same, and related uses. BACKGROUND

[0003] Oligonucleotides are polymeric sequences of nucleotides (RNA, DNA, and analogs thereof). Nucleic acid inhibitor molecules are oligonucleotides that modulate RNA levels within cells and have shown early promise in the treatment of cancer, viral infections, and genetic disorders. Nucleic acid inhibitor molecules can modulate RNA expression via a diverse set of mechanisms, including RNA interference (RNAi).

[0004] RNAi is a conserved pathway found in most eukaryotes in which a double-stranded RNA molecule (dsRNA) inhibits the expression of a target gene with a sequence complementary to the dsRNA. In a typical RNAi pathway, longer dsRNAs are cleaved by Dicer into shorter RNA duplexes known as small interfering RNAs ("siRNAs"). The siRNAs have been shown to associate with Dicer, trans-activation 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 an endonuclease that uses the antisense strand of the siRNA (also known as the guide strand) to direct sequence-specific cleavage of a target mRNA.

[0005] A variety of double-stranded RNAi inhibitor molecule structures have been developed over the years. For example, early work on RNAi inhibitor molecules focused on double-stranded nucleic acid molecules that mimic natural siRNAs, with each strand having a size of 19-25 nucleotides, with at least one 3' overhang of 1 to 5 nucleotides (see, e.g., U.S. Patent No. 8,372,968). Subsequently, longer double-stranded RNAi inhibitor molecules were developed that are processed in vivo by Dicer into active RNAi inhibitor molecules (see, e.g., 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 targeting region of the molecule, including structures in which one of the strands includes a thermodynamically stable tetraloop structure (see, e.g., U.S. Patent No. 8,513,207, U.S. Patent No. 8,927,705, WO 2010 / 033225, and WO 2016 / 100401). Those structures include single-stranded extension structures (on one or both sides of the molecule) and double-stranded extension structures.

[0006] Single-stranded nucleic acid inhibitor molecules ("ssRNAi") are also known in the art. For example, recent work has demonstrated the activity of ssRNAi inhibitor molecules (see, e.g., Matsui et al., 2016, 24(5):946-55). Also, antisense molecules have been used for decades to reduce the expression of particular target genes. Pelechano and Steinmetz, Nature Review Genetics, 2013, 14:880-93. A number of variations on the common theme of these structures have been developed for a range of targets. Other single-stranded nucleic acid inhibitor molecules include, for example, microRNAs, ribozymes, microRNA antagomirs, and aptamers, all of which are known in the art.

[0007] In certain cases, chemical modifications have been introduced into nucleic acid inhibitor molecules to introduce properties that can be desirable under particular conditions, such as those experienced following in vivo administration. These modifications include modifications designed, for example, to stabilize against nuclease or other enzymes that degrade or interfere with the structure or activity of the oligonucleotide, to increase cellular uptake of the oligonucleotide, or to improve the pharmacokinetic properties of the oligonucleotide.

[0008] For example, synthetic oligonucleotides are generally terminated with a 5'-hydroxyl or 3'-hydroxyl group. It is possible to replace the terminal hydroxyl group with a phosphate group, which can be used, for example, to attach a linker, adaptor, or label or to directly link the oligonucleotide to another nucleic acid. In addition, it has been reported that a 5'-terminal phosphate group enhances the interaction between certain nucleic acid inhibitor molecules and Ago2. However, oligonucleotides with 5'-phosphate groups are generally susceptible to degradation via phosphatases or other enzymes, which can limit their bioavailability in vivo.

[0009] Accordingly, it is desirable to develop modifications to the 5'-terminal nucleotide of an oligonucleotide, such as a nucleic acid inhibitor molecule, that provide the functional effects of a phosphate group, but are more stable to environmental conditions to which the oligonucleotide will be exposed upon administration to a subject. These phosphate analogs will be more resistant to phosphatases and other enzymes, while minimizing negative effects on the function of the oligonucleotide (e.g., minimizing any reduction in knockdown of a genetic target when used in an RNAi inhibitor molecule). SUMMARY

[0010] The present invention provides oligonucleotides comprising 5'-spirocyclic phosphonate modified nucleotides and RNAi agents useful for inhibiting the expression of a target gene in vivo, for example, to achieve a therapeutic purpose. The modification patterns described herein can be applied universally to a variety of oligonucleotides or RNAi agents having different sequences and targets.

[0011] In particular, in one aspect, the present invention provides an oligonucleotide, or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof, comprising a nucleotide monomer of the structure shown in Formula (I):

[0012] wherein,

[0013] represents a single or double bond;

[0014] Ring A is selected from C 3-10 cycloalkylene, 3-10 membered heterocyclylene, C 6-10 arylene, or 5-10 membered heteroarylene;

[0015] Ring B is selected from C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl;

[0016] each of Ring A and Ring B is optionally substituted with 1, 2, 3, 4, or 5 R*;

[0017] R* is selected from H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1- 6haloalkoxy;

[0018] R1is selected from O, S or NR a ;

[0019] R2and R3are independently selected from OR b or SR c ;

[0020] R a is selected from H, C 1-6 alkyl or C 1-6 haloalkyl;

[0021] R b and R c are independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 3-7 cycloalkyl or 3-7 membered heterocyclyl;

[0022] X is selected from -0-, -S-, -NR d -, -CR e R f -, -CR e R f -CR e R f - or -CH=CH-;

[0023] R d , R e and R f are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0024] Y1, Y2, Y3and Y4are independently selected from H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-7 cycloalkyl or 3-7 membered heterocyclyl;

[0025] Z is selected from H, halogen, OR j , NR k R l , C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or C1-18 haloalkyl, preferably selected from H, halogen, OR j , NR k R l , C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl or C 1-10 haloalkyl, said Z is optionally further substituted with 1, 2 or 3 R#;

[0026] R j , R k and R l are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0027] R# is selected from H, halogen, OH, NH2, CN, C 1-6 alkoxy or C 1-6 haloalkoxy;

[0028] Q is selected from -0-, -S-, -NR g - or -CR h R i -;

[0029] R g , R h and R i are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0030] Base is selected from H, a modified or unmodified base;

[0031] said compound of formula (I) is optionally further substituted with 1, 2, 3, 4 or 5 substituents selected from:

[0032] H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy.

[0033] In another aspect, the present application provides an RNAi agent, characterized in that the RNAi agent comprises one or more nucleotide monomers as described herein;

[0034] Preferably, the ends of the RNAi agent are modified with one or more nucleotide monomers as described herein.

[0035] In another aspect, the present application provides a compound of Formula (I’), or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof, or a mixture thereof:

[0036] wherein,

[0037] represents a single or double bond;

[0038] Ring A is selected from C 3-10 cycloalkylene, 3-10 membered heterocyclylene, C 6-10 arylene, or 5-10 membered heteroarylene;

[0039] Ring B is selected from C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl;

[0040] each of Ring A and Ring B is optionally substituted with 1, 2, 3, 4, or 5 R*;

[0041] R* is selected from H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, or C 1- 6haloalkoxy;

[0042] R1is selected from O, S, or NR a ;

[0043] R2and R3are selected from OR b , OP2, SR c , or SP2;

[0044] R a is selected from H, C 1-6 alkyl, or C 1-6 haloalkyl;

[0045] R b and R c are independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 3-7 cycloalkyl, or 3-7 membered heterocyclyl;

[0046] P1is selected from a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0047] P2is selected from a protecting group, preferably a hydroxyl protecting group;

[0048] X is selected from -O-, -S-, -NRd -CR e R f -CR e R f -CR e R f - or -CH=CH-;

[0049] R d , R e and R f are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0050] Y1, Y2, Y3and Y4are independently selected from H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-7 cycloalkyl or 3-7 membered heterocyclyl;

[0051] Z is selected from H, halogen, OR j , NR k R l , C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or C 1-18 haloalkyl, preferably from H, halogen, OR j , NR k R l , C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl or C 1-10 haloalkyl, said Z being optionally substituted with 1, 2 or 3 R#;

[0052] R j , R k and R l are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0053] R#is selected from H, halogen, OH, NH2, CN, C 1-6 alkoxy or C 1-6 haloalkoxy;

[0054] Q is selected from -0-, -S-, -NR g - or -CR h R i-;

[0055] R g , R h and R i are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0056] Base' is selected from H, a modified or unmodified base or a leaving group;

[0057] said compound of formula (I') is optionally further substituted by 1, 2, 3, 4 or 5 substituents selected from:

[0058] H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy.

[0059] In another aspect, the present application provides a composition comprising one or more oligonucleotides described herein, or an RNAi agent described herein.

[0060] In another aspect, the present application provides a kit comprising one or more oligonucleotides described herein, or an RNAi agent described herein.

[0061] In another aspect, the present application provides a method of inhibiting the expression of a target nucleic acid, the method comprising administering to a subject a therapeutic amount of an oligonucleotide described herein, an RNAi agent described herein, or a composition described herein.

[0062] In another aspect, the present application provides a method of treating a disease, the method comprising administering to a subject in need of treatment for the disease a therapeutic amount of an oligonucleotide described herein, an RNAi agent described herein, or a composition described herein.

[0063] In a particular embodiment, the disease is selected from a cancer, an autoimmune disease, an inflammatory disease, a metabolic disease, a genetic disease or a rare disease. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 is an IC50 curve of different siRNAs on ApoB mRNA expression in Hep3b cells. 50 Figure 2 is the pharmacodynamic results of different siRNAs in C57BL / 6 mice.

[0065] Figure 2 is the pharmacodynamic results of different siRNAs in C57BL / 6 mice.

[0066] Figure 3 is the result of drug efficacy of different siRNAs in TG mice. DETAILED DESCRIPTION

[0067] The above features and advantages of the present application, and additional features and advantages, will be more clearly understood from the following detailed description when read in conjunction with the accompanying drawings.

[0068] The embodiments described herein with reference to the drawings are explanatory, illustrative, and for general understanding of the present application. The embodiments should not be construed to limit the scope of the present application. The same or similar elements and elements having the same or similar functions are denoted by the same reference numerals throughout the specification.

[0069] In the present application, unless otherwise specified, the scientific and technical terms used herein have meanings commonly understood by a person skilled in the art. Also, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory operation procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, for better understanding of the present application, the definitions and explanations of the related terms are provided below.

[0070] Definitions

[0071] Chemical Definitions

[0072] The definitions of specific functional groups and chemical terms are described in more detail below.

[0073] When a numerical range is listed, each value and sub-range within the range is intended to be included. For example, "C 1-6 "alkyl" includes C1, C2, C3, C4, C5, C6, C 1-6 , C 1-5 , C 1-4 , C 1-3 , C 1-2 , C 2-6 , C 2-5 , C 2-4 , C 2-3 , C 3-6 , C 3-5 , C 3-4 , C 4-6 , C 4-5 and C 5-6 alkyl.

[0074] "C 1-18 alkyl" means a straight-chain or branched-chain saturated hydrocarbon radical having from 1 to 18 carbon atoms. In some embodiments, C 1-12 alkyl and C 1- 10 alkyl are preferred. In some embodiments, C 1-8 alkyl and C1-6 Alkyl is preferred. In some embodiments, C 1-4 Alkyl and C 1-2 Alkyl is preferred. C 1-6 Examples of alkyl groups include: methyl (Ci), ethyl (C2), n-propyl (C3), i-propyl (C3), n-butyl (C4), t-butyl (C4), sec-butyl (C4), i-butyl (C4), n-pentyl (C5), 3-pentyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), t-amyl (C5), and n-hexyl (C6). The term "C 1-6 Alkyl" also includes heteroalkyl groups, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced with a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkyl groups can be optionally substituted with one or more substituents, e.g., with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Conventional alkyl abbreviations include: Me (-CH3), Et (-CH2CH3), iPr (-CH(CH3)2), nPr (-CH2CH2CH3), n-Bu (-CH2CH2CH2CH3), or i-Bu (-CH2CH(CH3)2).

[0075] "C 2-18 Alkenyl" refers to a straight or branched chain hydrocarbon group having from 2 to 18 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 2-12 Alkenyl and C 2-10 Alkenyl is preferred. In some embodiments, C 2-8 Alkenyl and C 2-6 Alkenyl is preferred. In some embodiments, C 2-4 Alkenyl is preferred. C 2-6 Examples of alkenyl groups include: ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. The term "C 2-6 Alkenyl" also includes heteroalkenyl groups, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced with a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkenyl groups can be optionally substituted with one or more substituents, e.g., with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0076] "C 2-18 Alkynyl" refers to a straight or branched chain hydrocarbon group having from 2 to 18 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 2-12 Alkynyl and C 2-10Alkynyl groups are preferred. In some embodiments, C 2-8 Alkynyl and C 2-6 Alkynyl groups are preferred. In some embodiments, C 2-4 Alkynyl groups are preferred. C 2-6 Examples of alkynyl groups include, but are not limited to: ethynyl (C2), 1- propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), pentynyl (C5), hexynyl (C6), and the like. The term "C 2-6 Alkynyl" also includes heteroalkynyl groups, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Alkynyl groups can be optionally substituted by one or more substituents, e.g., by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0077] "Halo" or "halogen" means fluoro (F), chloro (CI), bromo (Br), and iodo (I).

[0078] Thus, "C 1-18 Haloalkyl" means a "C 1-18 Alkyl" group as defined above, substituted by one or more halo groups. In some embodiments, C 1- 12 Haloalkyl groups are preferred. In some embodiments, C 1-10 Haloalkyl and C 1-8 Haloalkyl groups are preferred. In some embodiments, C 1-6 Haloalkyl and C 1-4 Haloalkyl groups are particularly preferred, more preferably C 1-2 Haloalkyl. Exemplary haloalkyl groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CC13, -CH2C1, -CHC12, 2,2,2-trifluoro- 1,1-dimethyl-ethyl, and the like. Haloalkyl groups can be substituted at any available attachment point, e.g., by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0079] "C 1-6 Alkoxy" means an -O-R group, wherein R is defined as "C 1-6 Alkyl" above.

[0080] "C 3-10 Cycloalkyl" means a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms and zero heteroatoms. In some embodiments, C 3-7 Cycloalkyl and C 3-6 Cycloalkyl groups are particularly preferred, more preferably C 5-6Cycloalkyl. Cycloalkyl also includes ring systems in which the above cycloalkyl ring is fused with one or more aryl or heteroaryl rings, where the point of attachment is on the cycloalkyl ring, and in such cases the number of carbons refers to the number of carbons in the cycloalkyl ring. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), and the like. Cycloalkyl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0081] "3-10 membered heterocyclyl" refers to a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 5 ring heteroatoms, wherein each heteroatom is independently selected from the group consisting of nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valence permits. In some embodiments, 5-10 membered heterocyclyl groups are preferred, which are 3- to 10-membered non-aromatic ring systems having ring carbon atoms and 1 to 5 ring heteroatoms; in some embodiments, 3-7 membered heterocyclyl groups are preferred, which are 3- to 7-membered non-aromatic ring systems having ring carbon atoms and 1 to 4 ring heteroatoms; 3-6 membered heterocyclyl groups are preferred, which are 3- to 6-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; 4-8 membered heterocyclyl groups are preferred, which are 4- to 8-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; 5-6 membered heterocyclyl groups are more preferred, which are 5- to 6-membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes ring systems in which the above-mentioned heterocyclyl ring is fused with one or more cycloalkyl rings, wherein the point of attachment is on the cycloalkyl ring, or with one or more aryl or heteroaryl rings, wherein the point of attachment is on the heterocyclyl ring; and in such cases, the number of ring members continues to refer to the number of ring members in the heterocyclyl ring system. Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to: azirdinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to: azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to: dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to: piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, but are not limited to: hexahydrotriazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to: azepanyl, oxepanyl, and thiepanyl. Exemplary 5-membered heterocyclyl groups fused with a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclyl groups) include, but are not limited to: indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinonyl, and the like.Exemplary 6-membered heterocyclyl groups fused to a C6aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl groups) include, but are not limited to: tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Heterocyclyl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0082] "C 6-10 Aryl" refers to a group having a single or multiple rings (for example, bicyclic) 4n+2 aromatic ring system (for example, having 6 or 10 π electrons shared in a cyclic array) with six to ten ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6aryl"; for example, phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C10aryl"; for example, naphthyl, for example, 1-naphthyl and 2-naphthyl). Aryl also includes ring systems in which the above aryl ring is fused to one or more cycloalkyl or heterocyclyl rings, with the point of attachment at the aryl ring, in which case the number of carbon atoms designates the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. 10 Aryl" refers to a group having a single or multiple rings (for example, bicyclic) 4n+2 aromatic ring system (for example, having 6 or 10 π electrons shared in a cyclic array) with six to ten ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6aryl"; for example, phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C10aryl"; for example, naphthyl, for example, 1-naphthyl and 2-naphthyl). Aryl also includes ring systems in which the above aryl ring is fused to one or more cycloalkyl or heterocyclyl rings, with the point of attachment at the aryl ring, in which case the number of carbon atoms designates the number of carbon atoms in the aryl ring system. Aryl groups can be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0083] "5-10 membered heteroaryl" refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 p electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valence permits. Heteroaryl bicyclic systems can include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems wherein an above-described heteroaryl ring is fused with one or more cycloalkyl or heterocyclyl rings, and the point of attachment is on the heteroaryl ring, in which case the number of carbon atoms indicates the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-6 membered heteroaryl groups are particularly preferred, which are 5-6 membered monocyclic or bicyclic 4n+2 aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms. Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl), and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Heteroaryl groups can be optionally substituted with one or more substituents, e.g., with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0084] "Carbonyl," whether used alone or as part of a larger moiety, e.g., "aminocarbonyl," "O- carbonyl," or "N-carbonyl," refers to -C(O)-.

[0085] "Oxo" means =0.

[0086] "Thio" means =S.

[0087] The divalent radical formed by removing two hydrogens from the above-defined alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups is collectively referred to as an "alkylene radical." The ring- forming radicals of cycloalkyl, heterocyclyl, aryl, and heteroaryl are collectively referred to as "ring radicals."

[0088] The alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups defined herein are optionally substituted.

[0089] Exemplary substituents on a carbon atom include, but are not limited to: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa , -ON(R bb )2, -N(R bb )2, -N(R bb )3 + X - , -N(OR cc )R bb , -SH, -SR aa , -SSR cc , -C(=O)R aa , -CO2H, -CHO, -C(OR cc )2, -CO2R aa , -OC(=O)R aa , -OCO2R aa , -C(=O)N(R bb )2, -OC(=O)N(R bb )2, -NR bb C(=O)R aa , -NR bb CO2R aa , -NR bb C(=O)N(R bb )2, -C(=NR bb )R aa , -C(=NR bb )OR aa , -OC(=NR bb )R aa , -OC(=NR bb )OR aa , -C(=NR bb )N(R bb )2, -OC(=NR bb )N(R bb )2, -NR bb C(=NR bb )N(R bb )2, -C(=O)NR bb SO2R aa , -NRbb SO2R aa , -SO2N(R bb )2, -SO2R aa , -SO2OR aa , -OSO2R aa , -S(=O)R aa , -OS(=O)R aa , -Si(R aa )3, -OSi(R aa )3, -C(=S)N(R bb )2, -C(=O)SR aa , -C(=S)SR aa , -SC(=S)SR aa , -SC(=O)SR aa , -OC(=O)SR aa , -SC(=O)OR aa , -SC(=O)R aa , -P(=O)2R aa , -OP(=O)2R aa , -P(=O)(R aa )2, -OP(=O)(R aa )2, -OP(=O)(OR cc )2, -P(=O)2N(R bb )2, -OP(=O)2N(R bb )2, -P(=O)(NR bb )2, -OP(=O)(NR bb )2, -NR bb P(=O)(OR cc )2, -NR bb P(=O)(NR bb )2, -P(R cc )2, -P(R cc )3, -OP(R cc )2, -OP(R cc )3, -B(R aa )2, -B(OR cc )2, -BR aa (OR cc ), alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0090] or two geminal hydrogens on a carbon atom are replaced with a group =O, =S, =NN(R bb)2, =NNR bb C(=O)R aa , =NNR bb C(=O)OR aa , =NNR bb S(=O)2R aa , =NR bb or =NOR cc substituted;

[0091] R aa each independently is selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R aa groups are joined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0092] R bb each independently is selected from: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R bb groups are joined to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R dd groups;

[0093] R cceach R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R groups; cc each R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R groups; dd each R is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R groups;

[0094] R dd each R is independently selected from: halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee , -ON(R ff )2, -N(R ff )2, -N(R ff )3 + X - , -N(OR ee )R ff , -SH, -SR ee , -SSR ee , -C(=O)R ee , -CO2H, -CO2R ee , -OC(=O)R ee , -OCO2R ee , -C(=O)N(R ff )2, -OC(=O)N(R ff )2, -NR ff C(=O)R ee , -NR ff CO2R ee , -NR ff C(=O)N(R ff )2, -C(=NR ff )OR ee , -OC(=NR ff )R ee , -OC(=NR ff )OR ee , -C(=NR ff )N(R ff )2, -OC(=NR ff )N(R ff )2, -NR ff C(=NR ff )N(R ff )2, -NR ff SO2R ee , -SO2N(R ff )2, -SO2R ee , -SO2OR ee , -OSO2R ee-S(=O)R ee -Si(R ee )3, -OSi(R ee )3, -C(=S)N(R ff )2, -C(=O)SR ee , -C(=S)SR ee , -SC(=S)SR ee , -P(=O)2R ee , -P(=O)(R ee )2, -OP(=O)(R ee )2, -OP(=O)(OR ee )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups, or two geminal R dd substituents can be combined to form =O or =S;

[0095] each R ee is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;

[0096] each R ff is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R ff groups combine to form a heterocyclyl or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R gg groups;

[0097] each R gg is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 alkyl, -ON(C 1-6 alkyl)2, -N(C 1-6 alkyl)2, -N(C 1-6 alkyl)3 + X - , -NH(C 1-6 alkyl)2 + X - , -NH2(C 1-6 alkyl) + X - , -NH3+ X - , -N(OC 1-6 alkyl)(C 1-6 alkyl), -N(OH)(C 1-6 alkyl), -NH(OH), -SH, -SC 1-6 alkyl, -SS(C 1-6 alkyl), -C(=O)(C 1-6 alkyl), -CO2H, -CO2(C 1- 6alkyl), -OC(=O)(C 1-6 alkyl), -OCO2(C 1-6 alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C 1-6 alkyl), -N(C 1-6 alkyl)C(=O)(C 1-6 alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C 1-6 alkyl)2, -NHC(=O)NH(C 1-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C 1-6 alkyl), -OC(=NH)(C 1-6 alkyl), -OC(=NH)OC 1-6 alkyl, -C(=NH)N(C 1-6 alkyl)2, -C(=NH)NH(C 1-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 alkyl)2, -OC(NH)NH(C 1- 6alkyl), -OC(NH)NH2, -NHC(NH)N(C 1-6 alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 alkyl), -SO2N(C 1-6 alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2C 1-6 alkyl, -SO2OC 1-6 alkyl, -OSO2C 1-6 alkyl, -SOC 1-6 alkyl, -Si(C 1-6 alkyl)3, -OSi(C 1-6 alkyl)3, -C(=S)N(C 1-6 alkyl)2, C(=S)NH(C 1-6alkyl), -C(=S)SC 1-6 alkyl, -C(=S)SC 1- 6alkyl, -SC(=S)SC 1-6 alkyl, -P(=O)2(C 1-6 alkyl), -P(=O)(C 1-6 alkyl)2, -OP(=O)(C 1-6 alkyl)2, -OP(=O)(OC 1-6 alkyl)2, C 1-6 alkyl, C 1-6 haloalkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C7cycloalkyl, C6-C 10 aryl, C3-C7heterocyclyl, C5-C 10 heteroaryl; or two geminal R gg substituents can combine to form =O or =S; wherein X - is a counterion.

[0098] Exemplary substituents on a nitrogen atom include, but are not limited to: hydrogen, -OH, -OR aa , -N(R cc )2, -CN, -C(=O)R aa , -C(=O)N(R cc )2, -CO2R aa , -SO2R aa , -C(=NR bb )R aa , -C(=NR cc )OR aa , -C(=NR cc )N(R cc )2, -SO2N(R cc )2, -SO2R cc , -SO2OR cc , -SOR aa , -C(=S)N(R cc )2, -C(=O)SR cc , -C(=S)SR cc , -P(=O)2R aa , -P(=O)(R aa )2, -P(=O)2N(R cc )2, -P(=O)(NR cc )2, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or two R ccThe groups combine to form a heterocyclic or heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution, wherein R aa R bb R cc and R dd As stated above.

[0099] Other definitions

[0100] The term "siRNA" in this article refers to a class of double-stranded RNA molecules that can mediate the silencing of a complementary target RNA (e.g., mRNA, such as transcripts of genes encoding proteins). siRNA is typically double-stranded, consisting of an antisense strand complementary to the target RNA and a sense strand complementary to that antisense strand. For convenience, such mRNA is also referred to herein as the mRNA to be silenced. Such genes are also referred to as target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. Additionally, RNAs other than mRNA (e.g., tRNA) and viral RNA can also be targeted.

[0101] The term "antisense strand" refers to a strand of siRNA that contains regions that are completely, sufficiently, or substantially complementary to the target sequence. The term "sense strand" refers to a strand of siRNA that includes regions that are completely, sufficiently, or substantially complementary to the regions defined herein as antisense strands.

[0102] The term "complementary region" refers to a region on the antisense strand that is completely, fully, or substantially complementary to the target mRNA sequence. In cases where the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the molecule or at the ends. Typically, the most tolerant mismatches are located in the end regions, for example, within 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends. The portion of the antisense strand most sensitive to mismatches is called the "seed region." For example, in a siRNA containing a 19-nt strand, the 19th position (from 5' to 3') can tolerate some mismatches.

[0103] The term "complementary" refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions may include 400 mM NaCl, 40 mM PIPES at pH 6.4, and 1 mM EDTA at 50 or 70°C for 12–16 hours. In terms of meeting the above requirements regarding their hybridization ability, a "complementary" sequence may also include base pairs formed entirely from non-Watson-Crick base pairs and / or from non-natural and modified nucleotides. Such non-Watson-Crick base pairs include, but are not limited to, G:U swing base pairings or Hoogstein base pairs.

[0104] A polynucleotide that is "at least partially complementary," "fully complementary," or "substantially complementary" to messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest. For example, a polynucleotide is at least partially complementary to a PCSK9 mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding PCSK9. The terms "complementary," "fully complementary," "substantially complementary," and "substantially complementary" herein can be used with respect to base pairing between the sense strand and the antisense strand of an siRNA, or between the antisense strand of an siRNA agent and a target sequence.

[0105] "Substantially complementary" means that the degree of complementarity need only be such that the molecule maintains its overall double-stranded character. In other words, while perfect complementarity is generally desired, in some cases, particularly in the antisense strand, one or more, e.g., 6, 5, 4, 3, 2, or 1, mismatches (relative to the target mRNA) can be included, but the sense strand and the antisense strand can still maintain the overall double-stranded character of the molecule.

[0106] "shRNA" refers to short hairpin RNA. shRNA includes two short inverted repeats. shRNA cloned into shRNA expression vectors includes two short inverted repeats separated by a loop sequence, which forms a hairpin structure, controlled by a pol III promoter. Subsequently, 5-6 T's are added as a transcription terminator for RNA polymerase III.

[0107] N-acetylgalactosamine (GalNAc) is a high-affinity targeting ligand for the asialogycoproprotein receptor (ASGPR). ASGPR is an ideal receptor for active targeting, which is specifically highly expressed on the surface of hepatocytes, and after binding with GalNAc, it enters the cell to form an endosome through endocytosis, thereby bringing a sufficient amount of nucleic acid drugs into the cell, realizing the liver-targeted delivery of nucleic acid drugs. N-acetylgalactosamine (GalNAc) can specifically bind to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes.

[0108] An "RNAi agent" refers to an agent containing an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of reducing or inhibiting the translation of an mRNA transcript of a target messenger RNA (mRNA) in a sequence-specific manner. As used herein, an RNAi agent can function through an RNA interference mechanism (e.g., by inducing RNA interference through interaction with the RNA interference pathway machinery of a mammalian cell (RNA-induced silencing complex or RISC)) or any other arbitrary mechanism or pathway. While it is believed that the term RNAi agent as used herein functions primarily through an RNA interference mechanism, the RNAi agent is not limited or restricted to any particular mechanism or pathway of action. RNAi agents include, but are not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates. The RNAi agents described herein are composed of oligonucleotides having strands that are at least partially complementary to an mRNA that is a target. In some embodiments, the RNAi agents described herein are double-stranded and composed of an antisense strand and a sense strand that is at least partially complementary to the antisense strand. The RNAi agents can be composed of modified nucleotides and / or one or more non-phosphodiester linkages. In some embodiments, the RNAi agents described herein are single-stranded.

[0109] The terms "silence," "reduce," "inhibit," "down-regulate," or "knockdown" when referring to the expression of a given gene mean that the expression of the gene, as determined by the level of RNA transcribed from the gene or the level of a polypeptide, protein, or protein subunit translated from an mRNA within a cell, cell mass, tissue, organ, or subject, is reduced when the cell, cell mass, tissue, organ, or subject is treated with an oligomeric compound such as an RNAi agent described herein, as compared to a second cell, cell mass, tissue, organ, or subject that is not so treated.

[0110] A "nucleoside" is a compound composed of a purine or pyrimidine base, and either ribose or deoxyribose, while a "nucleotide" is a compound composed of a purine or pyrimidine base, ribose or deoxyribose, and phosphate.

[0111] An "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) of, for example, less than 100, 200, 300, or 400 nucleotides in length, which can be single-stranded or double-stranded.

[0112] A "base" is the basic building block of synthetic nucleosides, nucleotides, and nucleic acids, which contains nitrogen among its constituent elements, also referred to as a "nitrogenous base," "nucleotide base," or "nucleobase." Herein, unless otherwise specified, the capital letters A, U, T, G, and C represent the base composition of a nucleotide, which are adenine, uracil, thymine, guanine, and cytosine, respectively. The base can be further modified, thus including but not limited to universal bases, hydrophobic bases, mixed bases, size-expanded bases, and fluorinated bases.

[0113] A "modification" of a nucleotide as described herein includes, but is not limited to, a methoxy modification, a fluoro modification, a phosphorothioate linkage, or a conventional protecting group protection, etc. For example, the fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group is replaced with fluorine to form a nucleotide, and the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced with a methoxy group.

[0114] A "modified nucleotide" herein includes, but is not limited to, a 2'-O-methyl-modified nucleotide, a 2'-fluoro-modified nucleotide, a 2'-deoxy-modified nucleotide, an inosine ribonucleotide, an abasic nucleotide, an inverted abasic deoxyribonucleotide, a nucleotide comprising a phosphorothioate group, a vinylphosphonate-modified nucleotide, a locked nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, and a terminal nucleotide, deoxyribonucleotide, or conventional protecting group protection, etc., linked to a cholesteryl derivative or dodecanoyl dodecylamide group. For example, the 2'-fluoro-modified nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group is replaced with fluorine to form a nucleotide. The 2'-deoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced with a methoxy group.

[0115] A "reactive phosphorus group" refers to a phosphorus-containing group contained in a nucleotide unit or a nucleotide analog unit, which can react with a hydroxyl group or an amine group contained in another molecule, especially another nucleotide unit or another nucleotide analog, through a nucleophilic attack reaction. Typically, such a reaction results in an ester-type internucleoside bond that links the first nucleotide unit or the first nucleotide analog unit to the second nucleotide unit or the second nucleotide analog unit. The reactive phosphorus group can be selected from a phosphoramidite, an H-phosphonate, an alkyl-phosphonate, a phosphate, or a phosphate mimic, including but not limited to: a natural phosphate, a phosphorothioate, a phosphorodithioate, a boranophosphate, a boranophosphorothioate, a phosphonate, a halogen-substituted phosphonate and phosphate, a phosphoramidate, a phosphodiester, a phosphotriester, a phosphorodithioate, a phosphorotriithioate, a diphosphate, and a triphosphate, preferably -P(OCH2CH2CN)(N(iPr)2).

[0116] A "protecting group" also referred to as a "protecting group," is any atom or group of atoms that is added to a molecule to prevent an existing group in the molecule from undergoing an undesired chemical reaction. The "protecting group" can be a labile chemical moiety known in the art that is used to protect a reactive group, such as a hydroxyl, amino, and thiol group, from undergoing undesired or untimely reactions during chemical synthesis. The protecting group is typically used selectively and / or orthogonally to protect a site during reactions at other reactive sites, and can then be removed to leave the unprotected group intact or available for further reactions.

[0117] A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkylcarbonyl and alkoxycarbonyl (e.g., tert-butoxycarbonyl (BOC), acetyl, or isobutyryl); arylalkylcarbonyl and arylalkoxycarbonyl (e.g., benzyloxycarbonyl); substituted methyl ether (e.g., methoxymethyl ether); substituted ethyl ether; substituted benzyl ether; tetrahydropyranyl ether; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy]methyl, or tert-butyldiphenylsilyl); esters, such as benzoate, pivaloyloxymethyl carbonates (e.g., methoxymethyl carbonate); sulfonates (e.g., tosylate or mesylate); acyclic ketals (e.g., dimethyl ethylidene ketal); cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); acyclic acetals; cyclic acetals (e.g., those described herein); acyclic hemiacetals; cyclic hemiacetals; cyclic dithio ketals (e.g., 1,3-dithiane or 1,3-dithiolane); orthoesters (e.g., those described herein); and triarylmethyl groups (e.g., trityl; monomethoxytrityl (MMTr); 4,4'-dimethoxytrityl (DMTr); 4,4',4"-trimethoxytrityl (TMTr); and those described herein). Preferred protecting groups are selected from acetyl (Ac), benzoyl (BzI), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethyl acetal (BOM), beta-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), triphenylmethyl (Trt), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), dimethoxytrityl, [bis-(4-methoxyphenyl)phenylmethyl] (DMT), trimethylsilyl ether (TMS), t-butyldimethylsilyl ether (TBDMS), tri-isopropylsilyloxymethyl ether (TOM), tri-isopropylsilyl ether (TIPS), methyl ether, ethoxyethyl ether (EE), N,N-dimethylformamidinium, and 2-cyanoethyl (CE).

[0118] A "hydroxyl protecting group" means a group that can avoid the hydroxyl group from undergoing a chemical reaction, and can be removed under certain conditions to restore the hydroxyl group. It mainly includes silyl-type protecting groups, acyl-type protecting groups, or ether-type protecting groups, and preferably the following:

[0119] Trimethylsilyl (TMS), Triethylsilyl (TES), Dimethylisopropylsilyl (DMIPS), Diethylisopropylsilyl (DEIPS), Tert-butyldimethylsilyl (TBDMS), Tert-butyldiphenylsilyl (TBDPS), Triisopropylsilyl (TIPS), Acetyl (Ac), Chloroacetyl, Dichloroacetyl, Trichloroacetyl, Trifluoroacetyl (TFA), Benzoyl, p-Methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), Allyloxycarbonyl (Alloc), 2,2,2-Trichloroethoxycarbonyl (Troc), Benzyloxycarbonyl (Cbz), Tert-Butyloxycarbonyl (Boc) Benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), bis-p-methoxytriphenylmethyl (DMTr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzylmethyl (PMBM), -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl, preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytriphenylmethyl, more preferably -C(O)CH2CH2C(O)OH.

[0120] A "leaving group" is an atom or functional group that detaches from a larger molecule during a chemical reaction. The leaving groups described herein include those commonly found in the art, including but not limited to halogen atoms (such as -X), ester groups (-OCOR), p-toluenesulfonate groups (-OTs), nitro groups (-ONO2), and hydroxyl groups (-OH).

[0121] As used herein, the term "pharmaceutically acceptable salt" refers to carboxylates and amino acid addition salts of the compounds of the present invention that are suitable for contact with patient tissues within the limits of reliable medical judgment, without producing undue toxicity, irritation, allergic reactions, etc., and are effective for their intended use in proportion to a reasonable benefit / risk ratio, including (where possible) zwitterionic forms of the compounds of the present invention.

[0122] This invention includes tautomers, which are functional group isomers resulting from the rapid movement of an atom in a molecule to two positions. A compound exists in different tautomer forms, and a compound is not limited to any particular tautomer, but is intended to encompass all tautomer forms.

[0123] The compounds of the present application can include one or more asymmetric centers and can thus occur as individual enantiomers, diastereomers, or as mixtures of stereoisomers, including racemates. The isolation of the isomers is achieved by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or the preferred isomers can be prepared by asymmetric synthesis.

[0124] The present application also includes isotopically-labelled compounds (isotopic variants), which are identical to those recited in Formula (I) but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulphur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Cl. Compounds of the present application, prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the present application. Certain isotopically-labelled compounds of the present application, for example those into which radioactive isotopes such as 3 H and 14 C) are useful in drug and / or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon-14, i.e., 14 C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., 2 H, can afford certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. Isotopically labelled compounds of Formula (I) of the present application and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes and / or in the examples and

[0125] A "subject" for administration includes, but is not limited to, humans (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)) and / or non-human animals, e.g., mammals, e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, swine, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," and "subject" are used interchangeably herein.

[0126] "Disease," "disorder," and "condition" are used interchangeably herein.

[0127] Generally, an "effective amount" of a compound refers to the quantity sufficient to elicit the desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of a compound of the application can vary depending on such factors as the biological target, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, health, and symptoms of the subject. An effective amount includes both therapeutic and prophylactic effective amounts.

[0128] "Combination" and related terms refer to the administration of a compound of the application and another therapeutic agent simultaneously or sequentially. For example, a compound of the application can be administered simultaneously or sequentially in separate unit dosage forms or concurrently in a single unit dosage form with another therapeutic agent.

[0129] Oligonucleotide or RNAi agent comprising nucleotide monomers of the structure of Formula (I)

[0130] In one embodiment, the present application relates to an oligonucleotide, or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof, comprising nucleotide monomers of the structure shown in Formula (I):

[0131] wherein,

[0132] represents a single or double bond;

[0133] Ring A is selected from C 3-10 cycloalkylene, 3-10 membered heterocyclylene, C 6-10 arylene, or 5-10 membered heteroarylene;

[0134] Ring B is selected from C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl;

[0135] each of Ring A and Ring B is optionally substituted with 1, 2, 3, 4, or 5 R*;

[0136] R* is selected from H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, or C 1- 6haloalkoxy;

[0137] R1is selected from O, S, or NR a ;

[0138] R2and R3are independently selected from OR b or SR c ;

[0139] R a is selected from H, C 1-6 alkyl, or C 1-6 haloalkyl;

[0140] R b and R c are independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 3-7 cycloalkyl, or 3-7 membered heterocyclyl;

[0141] X is selected from -O-, -S-, -NR d -, -CR e R f -, -CR e R f -CR e R f -, or -CH=CH-;

[0142] R d , R e , and R f are independently selected from H, halogen, C 1-6 alkyl, or C 1-6 haloalkyl;

[0143] Y1, Y2, Y3, and Y4are independently selected from H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-7 cycloalkyl, or 3-7 membered heterocyclyl;

[0144] Z is selected from H, halogen, OR j , NR k Rl , C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or C 1-18 haloalkyl, preferably selected from H, halogen, OR j , NR k R l , C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl or C 1-10 haloalkyl, said Z is optionally further substituted with 1, 2 or 3 R#;

[0145] R j , R k and R l are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0146] R# is selected from H, halogen, OH, NH2, CN, C 1-6 alkoxy or C 1-6 haloalkoxy;

[0147] Q is selected from -0-, -S-, -NR g - or -CR h R i -;

[0148] R g , R h and R i are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0149] Base is selected from H, a modified or unmodified base;

[0150] said compound of formula (I) is optionally further substituted with 1, 2, 3, 4 or 5 substituents selected from:

[0151] H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy.

[0152] In one embodiment, represents a single bond; in another embodiment, represents a double bond.

[0153] Ring A and Ring B

[0154] In one embodiment, Ring A is C 3-10 cycloalkylene; in another embodiment, Ring A is C 3-7 cycloalkylene; in another embodiment, Ring A is C 3-5 cycloalkylene, e.g., cyclopropylene, cyclobutylene, or cyclopentylene; in another embodiment, Ring A is 3-10 membered heterocyclylene; in another embodiment, Ring A is 3-7 membered heterocyclylene; in another embodiment, Ring A is 3-5 membered heterocyclylene; in another embodiment, Ring A is C 6-10 arylene; in another embodiment, Ring A is 5-10 membered heteroarylene.

[0155] In one embodiment, Ring A is unsubstituted; in another embodiment, Ring A is optionally substituted with 1, 2, 3, 4, or 5 R*.

[0156] In one embodiment, Ring B is C 3-10 cycloalkylene; in another embodiment, Ring B is C 3-7 cycloalkylene; in another embodiment, Ring B is C 3-5 cycloalkylene, e.g., cyclopropylene, cyclobutylene, or cyclopentylene; in another embodiment, Ring B is 3-10 membered heterocyclylene; in another embodiment, Ring B is 3-7 membered heterocyclylene; in another embodiment, Ring B is 3-5 membered heterocyclylene; in another embodiment, Ring B is C 6-10 arylene; in another embodiment, Ring B is 5-10 membered heteroarylene.

[0157] In one embodiment, Ring B is unsubstituted; in another embodiment, Ring B is optionally substituted with 1, 2, 3, 4, or 5 R*.

[0158] In one specific embodiment, Ring A and Ring B together form In another specific embodiment, Ring A and Ring B together form In another specific embodiment, Ring A and Ring B together form

[0159] In one more specific embodiment, Ring A and Ring B together form In another more specific embodiment, Ring A and Ring B together form In another more specific embodiment, Ring A and Ring B together form In another more specific embodiment, Ring A and Ring B together form In another more specific embodiment, ring A and ring B together form In another more specific embodiment, ring A and ring B together form

[0160] R1

[0161] In one embodiment, R1 is O; in another embodiment, R1 is S; in another embodiment, R1 is NR a , for example NH.

[0162] R2 and R3

[0163] In one embodiment, R2 is OR b ; in another embodiment, R2 is OH; in another embodiment, R2 is C 1-6 alkoxy; in another embodiment, R2 is C 1-4 alkoxy, for example OCH3 or OCH2CH3; in another embodiment, R2 is C 1- 6haloalkoxy; in another embodiment, R2 is SR c .

[0164] In one embodiment, R2 is OP2; in another embodiment, R2 is SP2.

[0165] In one embodiment, R3 is OR b ; in another embodiment, R3 is OH; in another embodiment, R3 is C 1-6 alkoxy; in another embodiment, R3 is C 1-4 alkoxy, for example OCH3 or OCH2CH3; in another embodiment, R3 is C 1- 6haloalkoxy; in another embodiment, R3 is SR c .

[0166] In one embodiment, R3 is OP2; in another embodiment, R3 is SP2.

[0167] X

[0168] In one embodiment, X is -O-; in another embodiment, X is -S-; in another embodiment, X is -NR d -; in another embodiment, X is -CR e R f -, for example -CH2-; in another embodiment, X is -CR e R f -CR e R f-CH2-CH2-; in another embodiment, X is -CH=CH-.

[0169] Y1, Y2, Y3and Y4

[0170] In one embodiment, Y1is H; in another embodiment, Y1is halogen; in another embodiment, Y1is OH; in another embodiment, Y1is CN; in another embodiment, Y1is NH2; in another embodiment, Y1is C 1-6 alkyl; in another embodiment, Y1is C 2-6 alkenyl; in another embodiment, Y1is C 2-6 alkynyl; in another embodiment, Y1is C 1-6 haloalkyl; in another embodiment, Y1is C 1-6 alkoxy; in another embodiment, Y1is C 1-6 haloalkoxy; in another embodiment, Y1is C 3-7 cycloalkyl, preferably C 3-5 cycloalkyl; in another embodiment, Y1is 3-7 membered heterocyclyl.

[0171] In one embodiment, Y2is H; in another embodiment, Y2is halogen; in another embodiment, Y2is OH; in another embodiment, Y2is CN; in another embodiment, Y2is NH2; in another embodiment, Y2is C 1-6 alkyl; in another embodiment, Y2is C 2-6 alkenyl; in another embodiment, Y2is C 2-6 alkynyl; in another embodiment, Y2is C 1-6 haloalkyl; in another embodiment, Y2is C 1-6 alkoxy; in another embodiment, Y2is C 1-6 haloalkoxy; in another embodiment, Y2is C 3-7 cycloalkyl, preferably C 3-5 cycloalkyl; in another embodiment, Y2is 3-7 membered heterocyclyl.

[0172] In one embodiment, Y3is H; in another embodiment, Y3is halogen; in another embodiment, Y3is OH; in another embodiment, Y3is CN; in another embodiment, Y3is NH2; in another embodiment, Y3is C 1-6 alkyl; in another embodiment, Y3is C 2-6 alkenyl; in another embodiment, Y3is C 2-6alkynyl; in another embodiment, Y3is C 1-6 haloalkyl; in another embodiment, Y3is C 1-6 alkoxy; in another embodiment, Y3is C 1-6 haloalkoxy; in another embodiment, Y3is C 3-7 cycloalkyl, preferably C 3-5 cycloalkyl; in another embodiment, Y3is 3-7 membered heterocyclyl.

[0173] in another embodiment, Y4is OH; in another embodiment, Y4is CN; in another embodiment, Y4is NH2; in another embodiment, Y4is C 1-6 alkyl; in another embodiment, Y4is C 2-6 alkenyl; in another embodiment, Y4is C 2-6 alkynyl; in another embodiment, Y4is C 1-6 haloalkyl; in another embodiment, Y4is C 1-6 alkoxy; in another embodiment, Y4is C 1-6 haloalkoxy; in another embodiment, Y4is C 3-7 cycloalkyl, preferably C 3-5 cycloalkyl; in another embodiment, Y4is 3-7 membered heterocyclyl.

[0174] Z

[0175] in another embodiment, Z is H; in another embodiment, Z is halogen; in another embodiment, Z is OR j , for example OH or OMe; in another embodiment, Z is NR k R l , for example NH2; in another embodiment, Z is C 1-18 alkyl; in another embodiment, Z is C 1-10 alkyl; in another embodiment, Z is C 1-6 alkyl; in another embodiment, Z is C 2-18 alkenyl; in another embodiment, Z is C 2-18 alkynyl; in another embodiment, Z is C 1-18 haloalkyl; in another embodiment, Z is C 1-10 haloalkyl; in another embodiment, Z is C 1-6 haloalkyl.

[0176] In one embodiment, Z is not substituted; in another embodiment, Z is further substituted with 1, 2, or 3 R#.

[0177] Q

[0178] In one embodiment, Q is -0-; in another embodiment, Q is -S-; in another embodiment, Q is -NR g -, for example -NH-; in another embodiment, Q is -CR h R i -, for example -CH2-.

[0179] Base

[0180] In one embodiment, Base is H; in another embodiment, Base is an unmodified base; in another embodiment, Base is a modified base, for example a base modified with C 1-6 alkyl, C 1-6 haloalkyl (such as C 1-4 alkyl, C 1-4 haloalkyl) modification.

[0181] In one particular embodiment, Base is H; in another embodiment, Base is In another embodiment, Base is In another embodiment, Base is In another embodiment, Base is In another embodiment, Base is

[0182] R*

[0183] In one embodiment, R* is H; in another embodiment, R* is halogen; in another embodiment, R* is OH; in another embodiment, R* is CN; in another embodiment, R* is NH2; in another embodiment, R* is C 1-6 alkyl; in another embodiment, R* is C 2-6 alkenyl; in another embodiment, R* is C 2-6 alkynyl; in another embodiment, R* is C 1-6 haloalkyl; in another embodiment, R* is C 1-6 alkoxy; in another embodiment, R* is C 1-6 haloalkoxy.

[0184] R#

[0185] In one embodiment, R# is H; in another embodiment, R# is halogen; in another embodiment, R# is OH; in another embodiment, R# is NH2; in another embodiment, R# is CN; in another embodiment, R# is C 1-6 alkyl; in another embodiment, R# is C 1-4 alkyl; in another embodiment, R# is C 1-6 haloalkyl; in another embodiment, R# is C 1-4 haloalkyl.

[0186] R a , R b , and R c

[0187] In one embodiment, R a is H; in another embodiment, R a is C 1-6 alkyl; in another embodiment, R a is C 1- 6 haloalkyl.

[0188] In one embodiment, R b is H; in another embodiment, R b is C 1-6 alkyl; in another embodiment, R b is C 1- 6 haloalkyl; in another embodiment, R b is C 3-7 cycloalkyl; in another embodiment, R b is 3-7 membered heterocyclyl.

[0189] In one embodiment, R c is H; in another embodiment, R c is C 1-6 alkyl; in another embodiment, R c is C 1- 6 haloalkyl; in another embodiment, R c is C 3-7 cycloalkyl; in another embodiment, R c is 3-7 membered heterocyclyl.

[0190] R d , R e , and R f

[0191] In one embodiment, R d is H; in another embodiment, R d is halogen; in another embodiment, Rd C 1-6 Alkyl; in another embodiment, R d C 1-6 Halogenated alkyl groups.

[0192] In one implementation, R e For H; in another implementation, R e For halogen; in another embodiment, R e C 1-6 Alkyl; in another embodiment, R e C 1-6 Halogenated alkyl groups.

[0193] In one implementation, R f For H; in another implementation, R f For halogen; in another embodiment, R f C 1-6 Alkyl; in another embodiment, R f C 1-6 Halogenated alkyl groups.

[0194] R g R h and R i

[0195] In one implementation, R g For H; in another implementation, R g For halogen; in another embodiment, R g C 1-6 Alkyl; in another embodiment, R g C 1-6 Halogenated alkyl groups.

[0196] In one implementation, R h For H; in another implementation, R h For halogen; in another embodiment, R h C 1-6 Alkyl; in another embodiment, R h C 1-6 Halogenated alkyl groups.

[0197] In one implementation, R i For H; in another implementation, R i For halogen; in another embodiment, R i C 1-6 Alkyl; in another embodiment, R i C 1-6 Halogenated alkyl groups.

[0198] Rj , R k , and R l

[0199] In one embodiment, R j is H; in another embodiment, R j is halo; in another embodiment, R j is C 1-6 alkyl; in another embodiment, R j is C 1-6 haloalkyl.

[0200] In one embodiment, the R j is unsubstituted; in another embodiment, the R j is optionally substituted with 1 or 2 R#.

[0201] In one particular embodiment, R j is CH3; in another embodiment, R j is CH2CH2OCH3.

[0202] In one embodiment, R k is H; in another embodiment, R k is halo; in another embodiment, R k is C 1-6 alkyl; in another embodiment, R k is C 1-6 haloalkyl.

[0203] In one embodiment, R l is H; in another embodiment, R l is halo; in another embodiment, R l is C 1-6 alkyl; in another embodiment, R l is C 1-6 haloalkyl.

[0204] m

[0205] In one embodiment, m is 0; in another embodiment, m is 1; in another embodiment m is 2.

[0206] Base’

[0207] In one embodiment, Base’ is H; in another embodiment, Base’ is an unmodified base; in another embodiment, Base’ is a modified base, for example, substituted with C 1-6 alkyl, C 1-6 haloalkyl (e.g., C 1-4 alkyl, C1-4 a halogenated alkyl group)-modified base, and the like, and a base modified by a protecting group (e.g., an amino-protecting group, a hydroxyl-protecting group, and the like); in another embodiment, Base' is a methyl pivalate group a modified base; in another embodiment, Base' is a leaving group.

[0208] in a particular embodiment, Base' is H; in another particular embodiment, Base' is in another particular embodiment, Base' is in another particular embodiment, Base' is in another particular embodiment, Base' is in another particular embodiment, Base' is in another particular embodiment, Base' is in another particular embodiment, Base' is in another particular embodiment, Base' is in another particular embodiment, Base' is in another particular embodiment, Base' is in another particular embodiment, Base' is in another particular embodiment, Base' is

[0209] P1

[0210] in one embodiment, P1 is a reactive phosphorus group.

[0211] in a preferred embodiment, P1 is -P(OCH2CH2CN)(N(iPr)2).

[0212] P2

[0213] in one embodiment, P2 is a protecting group.

[0214] in one embodiment, P2 is a hydroxyl protecting group.

[0215] in a particular embodiment, P2 is a methyl pivalate group

[0216] Any of the technical solutions in any of the above specific embodiments or any combination thereof can be combined with any of the technical solutions in other specific embodiments or any combination thereof. For example, any of the technical solutions of Ring A or any combination thereof can be combined with any of the technical solutions of Ring B, R1-R3, X, Y1-Y4, Z, Q, m, Base (or Base’), R*, R#, R a -R l (and P1-P2), etc. or any combination thereof. The present application is intended to include all combinations of these technical solutions, which are not listed one by one due to the length of the specification.

[0217] In one specific embodiment, the present application relates to the above oligonucleotide or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the nucleotide monomers have the following structure:

[0218] wherein m is selected from 0, 1, or 2;

[0219] each variable is as defined herein.

[0220] In one specific embodiment, the present application relates to the above oligonucleotide or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein,

[0221] represents a single or double bond;

[0222] Ring A is selected from C 3-10 cycloalkylene, 3-10 membered heterocyclylene, C 6-10 arylene, or 5-10 membered heteroarylene;

[0223] Ring B is selected from C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl;

[0224] each of Ring A and Ring B is optionally substituted with 1, 2, or 3 R*;

[0225] R* is selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, or C 1-6 haloalkoxy;

[0226] R1is selected from O, S, or NR a ;

[0227] R2and R3are independently selected from OR b or SR c ;

[0228] Ra H, C 1-6 alkyl or C 1-6 haloalkyl;

[0229] each R b and R c is independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl;

[0230] X is selected from -0-, -S-, -NR d - or -CR e R f -;

[0231] R d , R e and R f are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0232] Y1, Y2, Y3and Y4are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy;

[0233] Z is selected from H, halogen, OR j , C 1-6 alkyl or C 1-6 haloalkyl, said Z being optionally substituted with 1, 2 or 3 R#;

[0234] R j is selected from H, C 1-6 alkyl or C 1-6 haloalkyl;

[0235] R#is selected from H, halogen, OH, C 1-6 alkoxy or C 1-6 haloalkoxy;

[0236] Q is selected from -0-, -S-, -NR g - or -CR h R i -;

[0237] R g , R h and R i are independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl;

[0238] Base is selected from H, a modified or unmodified base.

[0239] In a particular embodiment, the present application relates to the above oligonucleotide or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein,

[0240] represents a single or double bond, preferably a single bond;

[0241] Ring A is selected from C 3-7 cycloalkylene or 3-7 membered heterocyclylene;

[0242] Ring B is selected from C 3-7 cycloalkyl or 3-7 membered heterocyclyl;

[0243] Ring A and Ring B are each optionally substituted with 1, 2, or 3 R*;

[0244] R* is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0245] R1is selected from O or S, preferably O;

[0246] R2and R3are independently selected from OH, C 1-6 alkoxy or C 1-6 haloalkoxy;

[0247] X is selected from -O-, -S-, -NH- or -CH2-;

[0248] Y1, Y2, Y3, and Y4are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0249] Z is selected from C 1-6 alkoxy or C 1-6 haloalkoxy, said Z is optionally substituted with 1 or 2 R#;

[0250] R#is selected from H, halogen, OH, C 1-6 alkoxy or C 1-6 haloalkoxy;

[0251] Q is selected from -O-, -S-, -NH- or -CH2-;

[0252] Base is selected from H, a modified or unmodified base.

[0253] In a particular embodiment, the present application relates to the above oligonucleotide or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein,

[0254] represents a single or double bond, preferably a single bond;

[0255] Ring A is selected from C 3-5 Cycloalkylene or 3-5 membered heterocyclylene, preferably C 3-5 Cycloalkylene, for example cyclopropylene, cyclobutylene or cyclopentylene;

[0256] Ring B is selected from C 3-5 Cycloalkyl or 3-5 membered heterocyclyl, preferably C 3-5 Cycloalkyl, for example cyclopropyl, cyclobutyl or cyclopentyl;

[0257] Preferably, Ring A and Ring B together form the following structure: for example

[0258] R1is selected from O or S, preferably O;

[0259] R2and R3are independently selected from OH or C 1-4 Alkoxy, for example OCH3or OCH2CH3;

[0260] X is selected from -O-, -S- or -CH2-, preferably O;

[0261] Y1, Y2, Y3and Y4are independently selected from H or C 1-4 Alkyl, preferably H;

[0262] Z is C 1-4 Alkoxy, said Z is optionally substituted with 1 or 2 R#;

[0263] R#is selected from H, halogen or C 1-4 Alkoxy;

[0264] Z is preferably -OCH3or -OCH2CH2OCH3;

[0265] Q is selected from -O-, -S- or -CH2-;

[0266] Base is selected from H or a base, preferably

[0267] In one particular embodiment, the present application relates to the above-mentioned oligonucleotide, or a pharmaceutically acceptable salt, isotopic variant, tautomer or stereoisomer thereof, wherein the oligonucleotide comprises nucleotide monomers having the following structure:

[0268] wherein,

[0269] Ring B is selected from C 3-7 Cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-7 Cycloalkyl;

[0270] each Rb independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl;

[0271] each R j independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl, said R j is optionally substituted with 1 or 2 R#;

[0272] R#is selected from H, halo, C 1-6 alkoxy or C 1-6 haloalkoxy;

[0273] Base is selected from

[0274] Preferably,

[0275] Ring B is selected from C 3-5 cycloalkyl or 3-5 membered heterocyclyl, preferably C 3-5 cycloalkyl, for example cyclopropyl, cyclobutyl or cyclopentyl;

[0276] each R b independently selected from H or C 1-6 alkyl, for example CH3or CH2CH3;

[0277] each R j independently selected from H or C 1-6 alkyl, said R j is optionally substituted with 1 or 2 R#;

[0278] R#is selected from H, halo or C 1-6 alkoxy;

[0279] R j is preferably selected from CH3or CH2CH2OCH3;

[0280] Base is selected from

[0281] In one particular embodiment, the present application relates to the above-mentioned oligonucleotide, or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer or stereoisomer thereof, wherein the oligonucleotide comprises nucleotide monomers having the following structure:

[0282] wherein Base is selected from

[0283] In a specific embodiment, the present application relates to the above-mentioned oligonucleotide, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the oligonucleotide comprises nucleotide monomers having the following structure:

[0284] In a specific embodiment, the present application relates to the above-mentioned oligonucleotide, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the oligonucleotide comprises nucleotide monomers having the following structure:

[0285] wherein Base is selected from

[0286] In a specific embodiment, the present application relates to the above-mentioned oligonucleotide, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the oligonucleotide comprises nucleotide monomers having the following structure:

[0287] In a specific embodiment, the present application relates to the above-mentioned oligonucleotide, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the oligonucleotide comprises nucleotide monomers having the following structure:

[0288] In a specific embodiment, the present application relates to the above-mentioned oligonucleotide, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the oligonucleotide comprises nucleotide monomers having the following structure:

[0289] In a specific embodiment, the present application relates to the above-mentioned oligonucleotide, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the oligonucleotide comprises nucleotide monomers having the following structure:

[0290] In a specific embodiment, the present application relates to the above-mentioned oligonucleotide, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the oligonucleotide comprises nucleotide monomers having the following structure:

[0291] In a specific embodiment, the present application relates to the above-mentioned oligonucleotide, or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the oligonucleotide is single-stranded and has 14 to 30 nucleotides.

[0292] In a particular embodiment, the present application relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the oligonucleotide is a double-stranded RNA comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides.

[0293] In a particular embodiment, the present application relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the double-stranded RNA comprises a nucleotide monomer described herein in the sense strand and / or in the antisense strand.

[0294] In a particular embodiment, the present application relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the double-stranded RNA comprises a nucleotide monomer described herein in the antisense strand;

[0295] In a particular embodiment, the present application relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the double-stranded RNA comprises a nucleotide monomer described herein in the 3’-end and / or in the 5’-end of the antisense strand, more preferably in the 5’-end of the antisense strand.

[0296] In a particular embodiment, the present application relates to the above-mentioned oligonucleotide or a pharmaceutically acceptable salt, isotopic variant, tautomer, or stereoisomer thereof, wherein the double-stranded RNA comprises one or more targeting ligand or delivery vehicle; preferably, the delivery vehicle is selected from a polypeptide delivery vehicle or an antibody delivery vehicle; preferably, the targeting ligand is an N-acetylgalactosamine (GalNAc) targeting ligand; preferably, the targeting ligand comprises or is selected from the conjugation groups and L96 shown in Table A.

[0297] In a particular embodiment, the present application provides an RNAi agent, characterized in that the RNAi agent comprises one or more nucleotide monomers described herein.

[0298] In a particular embodiment, the present application provides the above-mentioned RNAi agent, wherein the ends of the RNAi agent are modified by one or more nucleotide monomers described herein.

[0299] In a particular embodiment, the present application provides the above-mentioned RNAi agent, wherein the RNAi agent is double-stranded comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides.

[0300] In a specific embodiment, the present application provides the above-mentioned RNAi agent, wherein the nucleotide monomer described herein is linked to the 5' end of the antisense strand of the RNAi agent.

[0301] In a specific embodiment, the present application provides the above-mentioned RNAi agent, wherein the nucleotide monomer described herein is linked to the 5' end of the antisense strand of the RNAi agent.

[0302] In a specific embodiment, the present application provides the above-mentioned RNAi agent, wherein the RNAi agent comprises a targeting ligand or a delivery vehicle.

[0303] In a specific embodiment, the present application provides the above-mentioned RNAi agent, wherein the RNAi agent comprises a delivery vehicle selected from a polypeptide delivery vehicle or an antibody delivery vehicle.

[0304] In a specific embodiment, the present application provides the above-mentioned RNAi agent, wherein the RNAi agent comprises a targeting ligand, which is an N-acetylgalactosamine (GalNAc) targeting ligand; preferably, the GalNAc targeting ligand can be selected from the GalNAc targeting ligands known in the art, or the GalNAc targeting ligands described herein.

[0305] In a more specific embodiment, the present application provides the above-mentioned RNAi agent, wherein the RNAi agent comprises a targeting ligand as follows:

[0306] Preferably, the L125 is linked to the 3' end of the sense strand of the RNAi agent.

[0307] N-acetylgalactosamine (GalNAc) targeting ligand

[0308] The targeting ligands suitable for use in the targeted RNAi agents comprising 5'-spiro phosphonate modified nucleotides are known in the art, for example, see U.S. Patent Application Serial Nos. 14 / 452,626, 15 / 452,324, 15 / 452,423, and 62 / 415,752, the entire contents of which are hereby incorporated by reference in their entireties.

[0309] In particular, in one embodiment, the GalNAc delivery ligand described herein comprises a conjugation group represented by Formula (X):

[0310] wherein,

[0311] the R G by attached to a biomolecule;

[0312] each W is independently selected from H, D,

[0313] W0is selected from a bond, -OCH2-, -OCH2CH2-, -OCH2OCH2-, -OCH2CH2CH2-, -OCH2CH2CH2CH2-, -CH2OCH2CH2CH2-, -CH2CH2OCH2CH2-, -CH2CH2CH2OCH2-, -CH2CH2OCH2CH2O-, C 1-6 alkylene, C 1-6 haloalkylene, C 1-6 alkenylene, or C 1-6 alkynylene, which is optionally deuterated, up to complete deuteration;

[0314] W1is selected from -O-, -O-L1-, -C(O)-, -C(O)-L1-, -C(O)O-, or -C(O)O-L1-, which is optionally deuterated, up to complete deuteration;

[0315] L1is -(CR Ga R Gb ) m , 1, 2, 3, 4, or 5 CR Ga R Gb in said L1may be replaced with a heteroatom selected from -O-, -S-, and -NR Ga -;

[0316] each R Ga and R Gb is independently H, D, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 alkenyl, or C 1-6 alkynyl, or R Ga , R Gb on any one or more carbon atoms in L1together with the same or different carbon atom to which they are attached form a C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl;

[0317] W2is selected from -L2-NH-, -L2-O-, -L2-C(O)-, or -L2-OC(O)-, which is optionally deuterated, up to complete deuteration;

[0318] L2is -(CR Gc R Gd ) n, 1, 2, 3, 4, or 5 CR Gc R Gd in L2may be replaced with a heteroatom selected from the group consisting of -O-, -S-, and -NR Gc ;

[0319] each R Gc and R Gd is independently H, D, halo, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 alkenyl, or C 1-6 alkynyl, or any one or more of the R Gc , R Gd together with the same or different carbon atom to which they are attached form a C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl;

[0320] W3is selected from -L3-C(O)NH-, -L3-NHC(O)-, -L3-OC(O)NH-, or -L3-NHC(O)O-, which is optionally deuterated, up to fully deuterated;

[0321] L3is -(CR Ge R Gf ) h , 1, 2, 3, 4, or 5 CR Ge R Gf in L3may be replaced with a heteroatom selected from the group consisting of -O-, -S-, and -NR Ge ;

[0322] each R Ge and R Gf is independently H, D, halo, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 alkenyl, or C 1-6 alkynyl, or any one or more of the R Ge , R Gf together with the same or different carbon atom to which they are attached form a C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl;

[0323] W4is selected from -L4-NHC(O)-, -L4-NHC(O)-(CH2) 1-6 -, -L4-NHC(O)O-, -L4-NHC(O)O-(CH2)1-6 -, -L4-C(O)NH-, -L4-C(O)NH-(CH2) 1-6 -, -L4-OC(O)NH- or -L4-OC(O)NH-(CH2) 1-6 -, which is optionally deuterated, up to complete deuteration;

[0324] L4is selected from -(CR Gg R Gh ) k -, -(CR Gg R Gh ) 1-6 -(OCR Gg R Gh -CR Gg R Gh ) k - or -(CR Gg R Gh ) 1-6 -(NH-CR Gg R Gh -CR Gg R Gh ) k -, wherein 1, 2, 3, 4, or 5 non-adjacent CR Gg R Gh in said L4can be replaced with a heteroatom selected from the group consisting of -O-, -S-, and -NR Gg -;

[0325] each R Gg and R Gh is independently H, D, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 alkenyl, or C 1-6 alkynyl, or, R Gg , R Gh on any one or more carbon atoms of L4together with the same or different carbon atom to which they are attached form a C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl;

[0326] m, n, h, and k are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0327] L5is selected from a bond, C 1-6 alkylene, C 1-6 alkenylene, or -C 1-6 alkylene-O-C 1-6 alkylene-, which is optionally deuterated, up to complete deuteration;

[0328] A is selected from a chemical bond, -C(O)-, or -NHC(O)-;

[0329] T is -(CR Gi R Gj ) q , 1, 2, 3, 4, or 5 CR Gi R Gj in T can be replaced with a heteroatom selected from -O-, -S-, and -NR Gi -; T is optionally deuterated, up to fully deuterated;

[0330] each R Gi and R Gj is independently H, D, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 alkenyl, or C 1-6 alkynyl, or R Gi , R Gj on the same or different carbon atom to which they are attached, together with that carbon atom, form a C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl;

[0331] q is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15;

[0332] B is selected from -C(O)-(CR Gk R Gm ) 0-6 -, -NH-(CR Gk R Gm ) 0-6 -, -OC(O)-(CR Gk R Gm ) 0-6 -, -NHC(O)-(CR Gk R Gm ) 0-6 -, or -C(O)NH-(CR Gk R Gm ) 0-6 , 1, 2, or 3 CR Gk R Gm in B can be replaced with a heteroatom selected from -O-, -S-, and -NR Gk -; B is optionally deuterated, up to fully deuterated;

[0333] each R Gk and R Gm is independently H, D, halogen, C1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, C 1-6 alkenyl or C 1-6 The alkynyl group, or the R group on any one or more carbon atoms in B. Gk R Gm Together with the same or different carbon atoms connected to them, they form C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-10 heteroaryl groups;

[0334] P is selected from chemical bonds or 1 to 3 amino acid residues;

[0335] R G Selected from

[0336] Ring S is selected from C 3-10 Cycloalkyl, 3-10 membered heterocyclic groups, C 6-10 Aryl or 5-10 heteroaryl groups;

[0337] R G1 Selected from groups containing hydroxyl groups, such as -OR G0 -C 1-6 Alkylene-OR G0 -C 1-6 imide-OR G0 or -C 1-6 acetylenic-OR G0 , where R G0 It can be a H, D, or hydroxyl protecting group or a solid support;

[0338] the hydroxyl protecting group is selected from trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), methoxymethyl (MOM), phenoxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), p-methoxybenzyloxymethyl (PMBM), -C(O)CH2CH2C(O)OH, or 4,4'-dimethoxytrityl (DMTr), preferably -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl (DMTr);

[0339] R G2 and R G3 are independently selected from H, D, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 alkenyl, or C 1-6 alkynyl; or, R G2 , R G3 together with the same carbon atom to which they are attached form a C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl, R G2 , R G3 and the ring formed by both are optionally deuterated, up to complete deuteration;

[0340] R G4 is selected from H, D, halogen, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 alkenyl, or C 1-6 alkynyl, R G4 is optionally deuterated, up to complete deuteration;

[0341] Z is selected from O, NH, or CH2;

[0342] R G and R G #independently selected from H, D, OH, CN, NH2, NO2, oxo, thioxo, halo, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 cyanoalkyl, C1-C4 aminoalkyl, C1-C4 alkylcarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 haloalkylaminocarbonyl, C1-C4 dialkylaminocarbonyl, C1-C4 cycloalkyl, 3- to 10-membered heterocyclyl, aryl, and 5- to 10-membered heteroaryl; 1-6 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 cyanoalkyl, C1-C4 aminoalkyl, C1-C4 alkylcarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 haloalkylaminocarbonyl, C1-C4 dialkylaminocarbonyl, C1-C4 cycloalkyl, 3- to 10-membered heterocyclyl, aryl, and 5- to 10-membered heteroaryl; 1-6 haloalkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 cyanoalkyl, C1-C4 aminoalkyl, C1-C4 alkylcarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 haloalkylaminocarbonyl, C1-C4 dialkylaminocarbonyl, C1-C4 cycloalkyl, 3- to 10-membered heterocyclyl, aryl, and 5- to 10-membered heteroaryl; 1-6 alkoxy, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 cyanoalkyl, C1-C4 aminoalkyl, C1-C4 alkylcarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 haloalkylaminocarbonyl, C1-C4 dialkylaminocarbonyl, C1-C4 cycloalkyl, 3- to 10-membered heterocyclyl, aryl, and 5- to 10-membered heteroaryl; 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 cyanoalkyl, C1-C4 aminoalkyl, C1-C4 alkylcarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 haloalkylaminocarbonyl, C1-C4 dialkylaminocarbonyl, C1-C4 cycloalkyl, 3- to 10-membered heterocyclyl, aryl, and 5- to 10-membered heteroaryl; 6-10 aryl or 5- to 10-membered heteroaryl.

[0343] x, y are independently selected from 0, 1, 2, 3, 4, or 5;

[0344] the aforementioned W, A, T, B, P, and R G each is optionally further substituted with 1, 2, 3, 4, 5, 6, 7, 8, or more substituents selected from H, D, OH, CN, NH2, NO2, oxo, thioxo, halo, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 cyanoalkyl, C1-C4 aminoalkyl, C1-C4 alkylcarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 haloalkylaminocarbonyl, C1-C4 dialkylaminocarbonyl, C1-C4 cycloalkyl, 3- to 10-membered heterocyclyl, aryl, and 5- to 10-membered heteroaryl; 1-6 alkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 cyanoalkyl, C1-C4 aminoalkyl, C1-C4 alkylcarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 haloalkylaminocarbonyl, C1-C4 dialkylaminocarbonyl, C1-C4 cycloalkyl, 3- to 10-membered heterocyclyl, aryl, and 5- to 10-membered heteroaryl; 1-6 haloalkyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 cyanoalkyl, C1-C4 aminoalkyl, C1-C4 alkylcarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 haloalkylaminocarbonyl, C1-C4 dialkylaminocarbonyl, C1-C4 cycloalkyl, 3- to 10-membered heterocyclyl, aryl, and 5- to 10-membered heteroaryl; 1-6 alkoxy, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 cyanoalkyl, C1-C4 aminoalkyl, C1-C4 alkylcarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 haloalkylaminocarbonyl, C1-C4 dialkylaminocarbonyl, C1-C4 cycloalkyl, 3- to 10-membered heterocyclyl, aryl, and 5- to 10-membered heteroaryl; 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 cyanoalkyl, C1-C4 aminoalkyl, C1-C4 alkylcarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 haloalkylaminocarbonyl, C1-C4 dialkylaminocarbonyl, C1-C4 cycloalkyl, 3- to 10-membered heterocyclyl, aryl, and 5- to 10-membered heteroaryl; 6-10 aryl or 5- to 10-membered heteroaryl.

[0345] In one embodiment, the GalNAc delivery ligand described herein comprises a conjugate group represented by the preceding formula (X), wherein R G is R Ga is Gb R Gc is Gd R Ge is Gf R Gg is Gh R Gi is Gj R Gk is Gm at least one of the groups R 3-10 cycloalkyl, 3- to 10-membered heterocyclyl, C1-C4 haloalkyl, C1-C4 hydroxyalkyl, C1-C4 cyanoalkyl, C1-C4 aminoalkyl, C1-C4 alkylcarbonyl, C1-C4 alkylaminocarbonyl, C1-C4 haloalkylaminocarbonyl, C1-C4 dialkylaminocarbonyl, C1-C4 cycloalkyl, 3- to 10-membered heterocyclyl, aryl, and 5- to 10-membered heteroaryl; 6-10 aryl or 5- to 10-membered heteroaryl.

[0346] In one embodiment, the GalNAc delivery ligand described herein comprises a conjugate group represented by the preceding formula (X), wherein the conjugate group is selected from the conjugate groups represented by Table A:

[0347] Table A. Representative Conjugate Groups

[0348] Compounds of Formula (I')

[0349] As used herein, "compounds of Formula (I')" refer to compounds of Formula (I') (including sub-formulae, such as Formula (I'-1), (II'), (II'-1), (II'-2), (III'), (III'-1), (III'-2), (IV'), (IV'-1), or (IV'-2), etc.), pharmaceutically acceptable salts, isotopologues, tautomers, or stereoisomers thereof, and mixtures thereof.

[0350] In one embodiment, the present application provides a compound of Formula (I'), or a pharmaceutically acceptable salt, isotopologue, tautomer, or stereoisomer thereof, or mixture thereof:

[0351] wherein,

[0352] represents a single or double bond;

[0353] Ring A is selected from C 3-10 cycloalkylene, 3-10 membered heterocyclylene, C 6-10 arylene, or 5-10 membered heteroarylene;

[0354] Ring B is selected from C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, or 5-10 membered heteroaryl;

[0355] each of Ring A and Ring B is optionally substituted with 1, 2, 3, 4, or 5 R*;

[0356] R* is selected from H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, or C 1- 6haloalkoxy;

[0357] R1is selected from O, S, or NR a ;

[0358] R2and R3are selected from OR b , OP2, SR c , or SP2;

[0359] R a is selected from H, C 1-6 alkyl, or C 1-6 haloalkyl;

[0360] R b and Rc independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 3-7 cycloalkyl or 3-7 membered heterocyclyl;

[0361] P1is selected from reactive phosphorus groups, preferably -P(OCH2CH2CN)(N(iPr)2);

[0362] P2is selected from protecting groups, preferably hydroxyl protecting groups;

[0363] X is selected from -0-, -S-, -NR d -, -CR e R f -, -CR e R f -CR e R f - or -CH=CH-;

[0364] R d , R e and R f are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0365] Y1, Y2, Y3and Y4are independently selected from H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-7 cycloalkyl or 3-7 membered heterocyclyl;

[0366] Z is selected from H, halogen, OR j , NR k R l , C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or C 1-18 haloalkyl, preferably from H, halogen, OR j , NR k R l , C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl or C 1-10 haloalkyl, said Z being optionally substituted with 1, 2 or 3 R#;

[0367] R j , R k and Rl independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0368] R#is selected from H, halogen, OH, NH2, CN, C 1-6 alkoxy or C 1-6 haloalkoxy;

[0369] Q is selected from -0-, -S-, -NR g - or -CR h R i -;

[0370] R g , R h and R i are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl;

[0371] Base' is selected from H, a modified or unmodified base or a leaving group;

[0372] said compound of formula (I') is optionally further substituted with 1, 2, 3, 4 or 5 substituents selected from:

[0373] H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy.

[0374] specific embodiments of ring A, ring B, R1-R3, X, Y1-Y4, Z, Q, m, Base', R*, R#, R a - R l and P1-P2 are as described above, wherein any technical solution in any specific embodiment or any combination thereof can be combined with any technical solution in other specific embodiments or any combination thereof. For example, any technical solution of ring A or any combination thereof can be combined with any technical solution of ring B, R1-R3, X, Y1-Y4, Z, Q, m, Base', R*, R#, R a - R l and P1-P2, etc. or any combination thereof. The present application is intended to include all combinations of these technical solutions, which are not listed one by one due to the limited space.

[0375] In one embodiment, the present application provides a compound of formula (I), or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof, or a mixture thereof, as described above, wherein the compound has the structure of formula (I’):

[0376] wherein m is selected from 0, 1, or 2;

[0377] R2, R3, P1, R* and Base’ are as defined herein;

[0378] R1, Ring A, Ring B, X, Y1, Y2, Y3, Y4, Z, and Q are as defined herein.

[0379] In one embodiment, the present application provides a compound of formula (I), or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof, or a mixture thereof, as described above, wherein:

[0380] R1is selected from O or S;

[0381] R2and R3are selected from OR b , OP2, SR c , or SP2;

[0382] R b and R c are independently selected from H, C 1-6 alkyl, or C 1-6 haloalkyl;

[0383] P1is selected from a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0384] P2is selected from a protecting group, preferably a hydroxyl protecting group;

[0385] Base’ is selected from H, an unmodified base, or C 1-4 alkyl or a protecting group modified base;

[0386] The other variables, such as Ring A, Ring B, X, Y1, Y2, Y3, Y4, Z, and Q, are as defined herein.

[0387] In one embodiment, the present application provides a compound of formula (I), or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof, or a mixture thereof, as described above, wherein:

[0388] R1is selected from O or S, preferably O;

[0389] R2and R3are selected from OR b , or OP2;

[0390] P1is selected from a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0391] P2is selected from a protecting group, preferably a hydroxyl protecting group;

[0392] Base' is selected from H, an unmodified base, or C 1-4 an alkyl or protecting group modified base, for example

[0393] The other variables, such as Ring A, Ring B, X, Y1, Y2, Y3, Y4, Z, and Q, are as defined herein.

[0394] In one specific embodiment, the present application provides a compound of the above formula (I’), or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof, or a mixture thereof, wherein the compound has the following structure:

[0395] wherein,

[0396] Ring B is selected from C 3-7 cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-7 cycloalkyl;

[0397] each R b is independently selected from H, P2, C 1-6 alkyl or C 1-6 haloalkyl;

[0398] P1is selected from a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0399] P2is selected from a protecting group, preferably a hydroxyl protecting group;

[0400] each R j is independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl, said R j is optionally substituted with 1 or 2 R#;

[0401] R#is selected from H, halogen, or C 1-6 alkoxy;

[0402] Base' is selected from

[0403] Preferably,

[0404] Ring B is selected from C 3-5 cycloalkyl or 3-5 membered heterocyclyl, preferably C3-5 cycloalkyl, for example cyclopropyl, cyclobutyl or cyclopentyl;

[0405] each R b is independently selected from H, P2or C 1-6 alkyl, for example CH3or CH2CH3;

[0406] P1is selected from a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0407] P2is selected from a protecting group, preferably a hydroxyl protecting group;

[0408] each R j is independently selected from H or C 1-6 alkyl, said R j is optionally substituted with 1 or 2 R#;

[0409] R#is selected from H, halo or C 1-6 alkoxy;

[0410] R j is preferably selected from CH3or CH2CH2OCH3;

[0411] Base’ is selected from

[0412] In one specific embodiment, the present application provides a compound of the above formula (I’), or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer or stereoisomer thereof, or a mixture thereof, wherein the compound has the following structure:

[0413] wherein P1is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2);

[0414] Base’ is selected from

[0415] In one specific embodiment, the present application provides a compound of the above formula (I’), or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer or stereoisomer thereof, or a mixture thereof, wherein the compound is selected from the following structures:

[0416] The compounds of the present application can include one or more asymmetric centers and can thus occur as individual enantiomers, diastereomers, or as mixtures of stereoisomers, including racemates. The isolated isomers can be separated by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or the preferred isomers can be prepared by asymmetric synthesis.

[0417] The compounds of the present application can also exist as tautomers. Where compounds exist in different tautomeric forms, one of the compounds is not limited to any particular tautomer, but is intended to encompass all tautomers.

[0418] Those skilled in the art will appreciate that organic compounds can form complexes with solvents, in which the solvent is present in fixed stoichiometric amounts. These complexes are in essence stoichiometric hydrates or solvates. Such complexes are often formed by solvent molecules physically binding to the compound or by the compound incorporating solvent molecules as part of its crystal lattice. These complexes are "solvates". When the solvent is water, the solvate is a "hydrate". The present application encompasses all solvates of the compounds of the present application.

[0419] The present application also includes isotopically-labelled compounds (isotopic variants) which are identical to those recited by Formula (I') but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulphur, fluorine and chlorine, such as 2 H, 3 H, 13 C, 11 C, 14 C, 15 N, 18 O, 17 O, 31 P, 32 P, 35 S, 18 F and 36 Cl. Compounds of the present application, prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the present application. Certain isotopically-labelled compounds of the present application, for example those into which radioactive isotopes such as 3 H and 14 C) can be useful in drug and / or substrate tissue distribution assays. Tritiated, i.e., 3 H, and carbon-14, i.e., 14C isotopes are particularly preferred because of ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e. 2 H, can afford therapeutically equivalent, but pharmacologically preferable or advantageous, compounds. Isotopically labeled compounds of formula (A) of the present application and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes and / or in the examples and

[0420] Pharmaceutical compositions

[0421] The present application provides pharmaceutical compositions comprising one or more oligonucleotides described herein, or an RNAi agent described herein, and a pharmaceutically acceptable carrier or excipient.

[0422] The oligonucleotides or RNAi agents of the present application (also referred to herein as "active compounds") can be incorporated into a pharmaceutical composition suitable for administration. Such compositions typically comprise one or more oligonucleotides described herein, or an RNAi agent described herein, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. Such vehicles and solvents are well known in the art. Unless otherwise specified, techniques used to formulate and administer the active compounds are believed to be well within the capability of those skilled in the art. All such techniques are included in the present application. Additional active compounds can also be incorporated into the composition.

[0423] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0424] The pharmaceutical compositions of the present application can be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0425] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating such as lecithin, by the maintenance of required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0426] A sterile injectable solution can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of a sterile powder, methods of preparation are vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0427] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, in which the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0428] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or a nebulizer or a dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide.

[0429] Systemic administration can also be by way of transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into a

[0430] The active compounds can also be prepared in the form of suppositories (e.g., using conventional suppository base such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0431] In one embodiment, the active compound is prepared with an agent that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations are apparent to those skilled in the art.

[0432] The present application provides therapeutic compositions comprising the oligonucleotides or RNAi agents of the present application. The therapeutic compositions according to the present application will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, etc. Many suitable formulations are known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, vesicles (cationic or anionic) such as LIPOFECTIN TM ), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions with montanide®, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. See also, Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0433] Methods of treatment and uses

[0434] The present application provides a method of inhibiting expression of a target nucleic acid, the method comprising administering to the subject an effective amount of an oligonucleotide, RNAi agent disclosed herein, or a pharmaceutical composition disclosed herein.

[0435] The present application also provides the use of an oligonucleotide, RNAi agent disclosed herein, or a pharmaceutical composition disclosed herein, in the manufacture of a medicament for inhibiting expression of a target nucleic acid.

[0436] The present application also provides an oligonucleotide, RNAi agent disclosed herein, or a pharmaceutical composition disclosed herein, for use in inhibiting expression of a target nucleic acid.

[0437] The present application provides a method for treating a disease in a subject, comprising administering to the subject an effective amount of an oligonucleotide, RNAi agent disclosed herein, or a pharmaceutical composition disclosed herein.

[0438] The present application also provides the use of an oligonucleotide, an RNAi agent disclosed herein, or a pharmaceutical composition disclosed herein in the manufacture of a medicament for treating a disease in a subject.

[0439] The present application also provides an oligonucleotide, an RNAi agent disclosed herein, or a pharmaceutical composition disclosed herein for use in treating a disease in a subject.

[0440] In some embodiments, the disease is a cancer, an autoimmune disease, an inflammatory disease, a metabolic disease, a genetic disease, or a rare disease.

[0441] In some embodiments, the disease is a cancer, which can be selected from a neuroendocrine tumor, a stomach cancer, a colon cancer, a rectal cancer, a small intestine cancer, a pancreatic cancer, a breast cancer, an ovarian cancer, a prostate cancer, a squamous cell carcinoma, a basal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous gland carcinoma, a papillary carcinoma, a papillary adenocarcinoma, a cystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, an esophageal carcinoma, a renal cell carcinoma, a hepatoma, a bile duct carcinoma, a choriocarcinoma, a seminoma, a embryonal carcinoma, a Wilms’ tumor, a cervical cancer, a uterine cancer, a testicular cancer, a lung cancer, a small cell lung cancer, a non-small cell lung cancer, a bladder cancer, or an epithelial carcinoma.

[0442] In some embodiments, the disease is an autoimmune disease or an inflammatory disease, which can be selected from sepsis, septic shock, Crohn’s disease, rheumatoid arthritis, asthma, allergy, atopic disorders, multiple sclerosis, pertussis, gonorrhea, inflammatory bowel disease, scleroderma, lupus, polymyositis, dermatomyositis, fibromyalgia, psoriatic arthritis, ankylosing spondylitis, Reiter’s syndrome, or juvenile rheumatoid arthritis.

[0443] In some embodiments, the disease is a metabolic disease, which can be selected from type I diabetes, type II diabetes, impaired glucose tolerance, impaired fasting glucose, hyperglycemia, postprandial hyperglycemia, overweight, obesity, hypertension, insulin resistance, and / or metabolic syndrome.

[0444] In some embodiments, the disease is a genetic disease, which can be selected from cystic fibrosis (CF), Duchenne muscular dystrophy (DMD), ataxia telangiectasia, Hurler syndrome, hemophilia A, hemophilia B, Usher syndrome, Tay-Sachs disease, Becker muscular dystrophy (BMD), congenital muscular dystrophy (CMD), familial atrial fibrillation, Huntington’s disease, Macleod’s disease, mucopolysaccharidosis, nephropathic cystinosis, polycystic kidney disease, Rett syndrome, spinal muscular atrophy (SMA), X-linked nephrogenic diabetes insipidus (XNDI), or X-linked retinitis pigmentosa.

[0445] In some embodiments, the disease is an orphan disease, which can be selected from the group consisting of albinism, acromegaly, idiopathic pulmonary arterial hypertension, phenylketonuria, or a mitochondrial disease.

[0446] In some embodiments, a therapeutically effective amount of an RNAi agent of one (or more different classes) described herein is administered to a subject, thereby inhibiting expression of a target gene in the subject (e.g., an amount effective to inhibit expression of a target gene in the subject).

[0447] In some embodiments, the dose administered to a subject can vary with the embodiment, the drug used, the method of administration, and the site and subject to be treated. However, the dose should be sufficient to provide a therapeutic response. The clinician can determine the effective amount for administration to a human or other subject to treat a medical condition. The precise amount required can depend on many factors such as the activity of the oligonucleotide or RNAi agent and the route of administration.

[0448] An oligonucleotide, RNAi agent, or composition described herein can be administered to a mammal in a single dose or in a series of sub-doses over a suitable period of time, e.g., as needed, daily, semi-weekly, weekly, bi-weekly, semi-monthly, bi-monthly, semi-annually, or annually. Dose units comprising an effective amount of an oligonucleotide, RNAi agent, or composition can be administered as a single daily dose, or the total daily dose can be administered in two, three, four, or more divided doses administered daily as needed.

[0449] A suitable mode of administration can be selected by a physician. The route of administration can be parenteral administration, e.g., administration by injection, nasal administration, pulmonary administration, or transdermal administration. Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the oligonucleotide, RNAi agent, or composition is selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, e.g., orally. The dose and method of administration can vary depending on the weight, age, condition, etc. of the subject, and can be appropriately selected.

[0450] In some embodiments, the method further comprises administering to the subject a second therapeutic agent. In some embodiments, the oligonucleotide, RNAi agent, or pharmaceutical composition disclosed herein is used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug. In certain embodiments, the oligonucleotide, RNAi agent, or composition disclosed herein is administered prior to, substantially simultaneously with, or following administration of the second therapeutic agent.

[0451] Kit / dosing device

[0452] The present disclosure provides a kit or dosing device comprising an oligonucleotide, RNAi agent disclosed herein, or a pharmaceutical composition disclosed herein.

[0453] In some embodiments, the kit or drug delivery device includes one or more containers with one or more of the components of a pharmaceutical composition described herein, such as an oligonucleotide, an RNAi agent disclosed herein.

[0454] In particular embodiments, the kit includes a first container containing an oligonucleotide or RNAi agent disclosed herein. In particular embodiments, the kit includes a first container that is a vial containing an oligonucleotide or RNAi agent as a lyophilized sterile powder under vacuum, and the kit further includes a second container containing a pharmaceutically acceptable fluid.

[0455] In particular embodiments, provided herein are injection devices containing an oligonucleotide or RNAi agent. In particular embodiments, the injection device contains an oligonucleotide or RNAi agent in a sterile solution. In particular embodiments, the injection device is a syringe.

[0456] In one embodiment, the kit includes instructional materials that disclose the manner in which the oligonucleotide or RNAi agent of the application is to be used. The instructional materials can be written, electronic (e.g., computer diskette or CD-ROM), or visual (e.g., video cassette or DVD) and can take the form of a package insert. The kit can further include additional components to facilitate the use of the kit for which it is designed. Thus, for example, the kit can additionally contain tools for detecting a label (e.g., enzyme substrates for enzymatic labels, filter sets for detecting fluorescent labels, appropriate secondary labels such as a second antibody, etc.). The kit can also include buffers and other reagents commonly used to practice the particular method for which the kit is designed. Such kits and suitable contents are well known to those of skill in the art.

[0457] Examples

[0458] The following examples are given for the purpose of illustrating various embodiments of the application and are not meant to limit the present application in any way. This example, as well as the methods described herein, are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the application. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the application as defined by the scope of the claims.

[0459] Example 1. Compound Examples

[0460] The structure of the compounds was determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). Chemical shifts δ are given in 10 -6 (ppm) units. NMR measurements were made on a Bruker NMR spectrometer using deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCI3), deuterated methanol (CD3OD) as the solvent and tetramethylsilane (TMS) as the internal standard.

[0461] LCMS was measured by Agilent 1260 Infinity II (ESI) mass spectrometer, Waters UPLC H Class plus (ESI) or Shimadzu LCMS-2020 (ESI).

[0462] High performance liquid chromatography (HPLC) analysis used Agilent 1260 or Shimadzu LC-20AD.

[0463] Preparative high performance liquid chromatography (pre-HPLC) used GILSON GX-281 or Agilent 1260 Infinity II preparative liquid.

[0464] Chiral preparation used critical fluid chromatography (SFC), and the instrument used Shimadzu LC-30Adsf or Shimadzu LC-20AD.

[0465] Thin layer chromatography silica gel plate used Anhui Liangchen Silicon Source Material Co., Ltd. GF254 acrylic adhesive silica gel plate, and the silica gel plate used in thin layer chromatography (TLC) adopted a specification of 0.2 mm silica gel plate, and the thin layer chromatography separation and purification product adopted a specification of 0.5 mm silica gel plate.

[0466] Column chromatography generally used 200-300 mesh silica gel as a carrier from Anhui Liangchen Silicon Source Material Co., Ltd.

[0467] Determination of average inhibition rate and IC 50 of kinases used SpectraMax i3X microplate reader (MD, USA).

[0468] Known starting materials of the present disclosure can be synthesized or purchased from companies such as Bide Pharmaceutical, Leyan, Shaoyuan Chemical Technology, and Anning Chemicals, etc. according to methods known in the art.

[0469] Unless otherwise specified, the reactions in the following examples were carried out under an argon or nitrogen atmosphere.

[0470] An argon or nitrogen atmosphere refers to a reaction bottle connected to an argon or nitrogen balloon with a volume of about 1 L.

[0471] A hydrogen atmosphere refers to a reaction bottle connected to a hydrogen balloon with a volume of about 1 L.

[0472] Hydrogenation reactions were usually vacuumed and filled with hydrogen, and the operation was repeated 3 times.

[0473] An oxygen atmosphere refers to a reaction bottle connected to an oxygen balloon with a volume of about 1 L.

[0474] In the following examples, unless otherwise specified, the solution refers to an aqueous solution, and the reaction temperature is room temperature, which is 20-30°C.

[0475] The monitoring of the reaction progress in the examples was carried out by thin layer chromatography (TLC), and the developing agent used in the reaction, the eluent system used in the column chromatography for purifying the compounds, and the developing agent system of thin layer chromatography included: A: dichloromethane / methanol system, B: petroleum ether / ethyl acetate system, the volume ratio of the solvents was adjusted according to the polarity of the compounds, and a small amount of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0476] Example 1-1: Preparation of compound 1

[0477] First step

[0478] 1a (280 mg, 1.40 mmol, prepared by the method disclosed in Example 2 on page 24 of the patent application “WO2016155545 A1”) and 1b (200 mg, 425 μmol, prepared by the method disclosed in Example 4 on page 512 of the patent application “WO2014179620 A1”) were dissolved in 10 mL of dichloromethane, 2-(7-azobenzo triazole)-N,N,N',N'-tetramethyl urea hexafluorophosphate (532 mg, 1.40 mmol) and N,N-diisopropyl ethylamine (197 mg, 1.53 mmol) were added, and stirred at 20°C for 2 hours. The reaction solution was concentrated by distillation under reduced pressure, and the obtained residue was purified by reverse phase liquid chromatography (separation conditions: mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 20%-80%), to obtain the title product 1c (400 mg, yield: 93%) in the form of a white solid.

[0479] Second step

[0480] 1c (400 mg, 393 μmol) was dissolved in 1 mL of methanol, and a hydrochloric acid solution in dioxane (4N, 20 mL) was added, and stirred at 20°C for 6 hours. The reaction solution was concentrated by distillation under reduced pressure, and the residue was adjusted to neutral with a sodium carbonate solution, and the obtained mixture was purified by reverse phase liquid chromatography (separation conditions: mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 5%-50%), to obtain the title product 1d (270 mg, yield: 96%) in the form of a white solid.

[0481] MS m / z (ESI): 359.83 [1 / 2 (M+2)].

[0482] Third step

[0483] Dissolve 1e (144 mg, 322 μηιοΐ, prepared according to the procedure disclosed in patent application "WO2009073809 A2", page 161, example 1) and 1d (70 mg, 97 μηιοΐ) in 5 mL of dichloromethane, add 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (122 mg, 322 μηιοΐ) and N,N-diisopropylethylamine (88 mg, 682 μηιοΐ) and stir at 20 °C for 2 hours. Concentrate the reaction under reduced pressure, purify the residue obtained by reverse phase liquid chromatography (separation conditions: mobile phase: A - water (0.05% ammonium bicarbonate) : B - acetonitrile, gradient elution: B%: 20% - 70%) to obtain the title product 1f (120 mg, yield: 61%) as a white solid.

[0484] MS m / z (ESI): 1004.37 [1 / 2 (M+2)].

[0485] Fourth step

[0486] Dissolve 1f (120 mg, 60 μηιοΐ) and acetic acid (17 mg, 283 μηιοΐ) in 10 mL of methanol and 10 mL of ethyl acetate, add wet palladium on carbon (60 mg, 10%), displace three times with hydrogen and stir the reaction under hydrogen (15 Psi) at 25 °C for 6 hours. Filter the reaction over celite, concentrate the filtrate by distillation under reduced pressure, purify the residue obtained by reverse phase liquid chromatography (separation conditions: mobile phase: A - water (0.1% formic acid) : B - acetonitrile, gradient elution: B%: 10% - 50%) to obtain the title product 1g (90 mg, yield: 76%) as a white solid.

[0487] MS m / z (ESI): 937.12 [1 / 2 (M+2)].

[0488] Fifth step

[0489] Dissolve 1 g (15 mg, 8.01 μmol) and 1 h (5.06 mg, 8.01 μmol, prepared by the method disclosed in patent application "WO2012037254 A1" page 90, example 2) in 2 mL of dichloromethane, add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.57 mg, 12.02 μmol) and N,N-diisopropylethylamine (5.18 mg, 40.08 μmol) and react at 25 °C for 2 hours. Concentrate the reaction under reduced pressure, purify the residue obtained by preparative high-performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 μm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 20% - 70%) and obtain the title product 1i (12 mg, yield: 57%) as a white solid.

[0490] Sixth step

[0491] Dissolve 1i (10 mg, 4.02 μmol) in 1 mL of dichloromethane, under nitrogen protection, add 4-dimethylaminopyridine (0.09 mg, 0.8 μmol), succinic anhydride (2.01 mg, 20.12 μmol) and N,N-diisopropylethylamine (5.19 mg, 40.2 μmol), stir at 25 °C for 15 hours. Concentrate the reaction under reduced pressure, purify the residue obtained by preparative high-performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 μm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 20% - 60%) and obtain the title product 1 (4 mg, yield: 35%) as a white solid.

[0492] MS m / z (ESI): 1142.9 [1 / 2 (M-301)].

[0493] 1H NMR (400 MHz, CD3OD) δ 7.37-7.36 (m, 1H), 7.29-7.23 (m, 7H), 6.89-6.82 (m, 5H), 5.33-5.32 (m, 3H), 5.07-5.03 (m, 4H), 4.61-4.58 (m, 2H), 4.56-4.53 (m, 3H), 4.19-4.04 (m, 12H), 4.02-3.97 (m, 4H), 3.90-3.84 (m, 4H), 3.79-3.76 (m, 6H), 3.70-3.65 (m, 12H), 3.56-3.49 (m, 5H), 3.14-3.11 (m, 10H), 2.57-2.53 (m, 4H), 2.46-2.42 (m, 6H), 2.25-2.21 (m, 6H), 2.14-2.12 (m, 9H), 2.02-2.00 (m, 9H), 1.95-1.91 (m, 18H), 1.70-1.56 (m, 16H), 1.33-1.24 (m, 16H), 0.50-0.45 (m, 12H).

[0494] Example 1-2: Preparation of compound 2

[0495] First step

[0496] Dissolve 2a (5.00 g, 34.9 mmol) in 20 mL of ethanol, add ethyl 2-bromoacetate (5.83 g, 34.9 mmol), stir at 80 °C for 15 hours. Concentrate the reaction solution by distillation under reduced pressure, add 25 mL of isopropyl alcohol to the obtained residue, purify by recrystallization to obtain the title product 2b (7.34 g, yield: 91%) in the form of a yellow solid.

[0497] MS m / z (ESI): 230.1 [M+1].

[0498] Second step

[0499] Dissolve 2b (6.84 g, 29.7 mmol) in 50 mL of acetonitrile, protect under nitrogen, add 2-cyclopentenone (11.7 g, 142 mmol) and triethylamine (3.31 g, 32.6 mmol), stir at 25 °C for 24 hours under nitrogen protection. Add 50 mL of water to the reaction solution, extract the reaction solution with ethyl acetate (80 mL x 3), wash the organic phase with saturated sodium chloride solution (60 mL x 2), then dry over anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system B to obtain the title product 2c (5.81 g, yield: 61%) in the form of a yellow solid.

[0500] MS m / z (ESI): 312.1 [M+1].

[0501] Third step

[0502] Dissolve 2c (5.31 g, 16.5 mmol) in 60 mL of toluene, under nitrogen protection, add tributyltin hydride (11.9 g, 40.9 mmol) and azobisisobutyronitrile (543 mg, 3.31 mmol), under nitrogen protection, react at 120 °C for 6 hours. Concentrate the reaction solution by distillation under reduced pressure, add 80 mL of ethyl acetate and hydrochloric acid (1 N, 40 mL) to the obtained residue, let the obtained liquid stand at 25 °C for 14 hours, suck out the organic phase, wash the remaining aqueous phase with ethyl acetate (100 mL x 3), obtain the crude product of the title compound 2d (3.27 g) as a yellow liquid without purification, and directly use the product for the next step reaction.

[0503] Fourth step

[0504] Add sodium bicarbonate solution (25 mL), acetonitrile (25 mL), 9-fluorenylmethyl-N-succinimidyl carbonate (6.71 g, 19.9 mmol) to 2d (3.27 g, 16.6 mmol), under nitrogen protection, react at 25 °C for 2 hours. Extract the reaction solution with ethyl acetate (80 mL x 3), wash the organic phase with saturated sodium chloride solution (100 mL x 2), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with the developing system B, and obtain the title product 2e (2.01 g, yield: 27%) as a yellow oil.

[0505] Separate 2e (2.01 g, 4.80 mmol) by SFC (separation conditions: column: DAICEL CHIRALPAK AD (250 mm x 30 mm, 10 μm); mobile phase: A-carbon dioxide: B-methanol, isocratic elution: B: 35%), to obtain single configuration compound 2e-1 and single configuration compound 2e-2.

[0506] Single configuration compound 2e-1 (shorter retention time)

[0507] Yellow gum, 775 mg, yield: 39%. SFC analysis: retention time 1.830 minutes. (column: Chiralpak AD-3 50 x 4.6 mm I.D., 3 μm, mobile phase: A-carbon dioxide, B-methanol (0.05% diethylamine), gradient elution: B: 5%-40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30AD sf).

[0508] MS m / z (ESI): 420.1 [M+1].

[0509] 2e single configuration compound one (retention time longer)

[0510] Yellow gum, 750 mg, yield: 37%. SFC analysis: retention time 2.193 min. (column: Chiralpak AD-3 50 x 4.6 mm I.D., 3 pm, mobile phase: A-carbon dioxide, B-methanol (0.05% diethylamine), gradient elution: B: 5%-40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30ADsf).

[0511] MS m / z (ESI): 420.1 [M+1].

[0512] Fifth step

[0513] (R)-5,5-biphenyl-2-methyl-3,4-propanol-1,3,2-oxazaborolidine (79.3 mg, 286 pmol) was dissolved in 2 mL of toluene, protected by nitrogen, and cooled to 0 °C. Borane dimethyl sulfide complex (10 M, 171 pL) was added dropwise, and the reaction was allowed to proceed at 0 °C for 15 min. A toluene solution (8 mL) of 2e single configuration compound one (600 mg, 1.43 mmol) was then added, and the reaction was allowed to proceed at 0 °C for 30 min. 5 mL of methanol and 8 mL of water were added to the reaction at 0 °C, and the reaction was extracted with dichloromethane (10 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation. The resulting residue was purified by silica gel column chromatography with developing system A to obtain the title product 2f (500 mg, yield: 81%) in the form of yellow oil.

[0514] MS m / z (ESI): 422.0 [M+1].

[0515] Sixth step

[0516] 2f (500 mg, 1.15 mmol) was dissolved in 10 mL of tetrahydrofuran, protected by nitrogen, and cooled to 0 °C. A tetrahydrofuran solution (2 M, 864 pL) of lithium borohydride was added dropwise, and the reaction was allowed to proceed at 25 °C for 1 h. 1 mL of ammonium chloride solution and 5 mL of water were added at 0 °C, and the reaction was extracted with dichloromethane (8 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation. The resulting residue was purified by silica gel column chromatography with developing system A to obtain the title product 2g (189 mg, yield: 63%) in the form of white solid.

[0517] MS m / z (ESI): 380.0 [M+1].

[0518] Seventh step

[0519] To a solution of 2g (25 mg, 64.57 pmol) in 3 mL of dry pyridine, under nitrogen protection, was cooled to below 5 °C, then 4,4'-dimethoxybenzhydryl chloride (21.88 mg, 64.57 pmol) was dissolved in 5 mL of dichloromethane and slowly added dropwise to the reaction solution, and then naturally raised to 25 °C and stirred for 12 h. 1 mL of methanol was added to quench the reaction solution, and the reaction solution was concentrated by reduced pressure distillation. The obtained residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 20%-100%), to obtain the title product 2h (27 mg, yield: 58%) in the form of a white solid.

[0520] Eighth step

[0521] 2h (26 mg, 38 pmol) and piperidine (6 mg, 73 pmol) were dissolved in 3 mL of N,N-dimethylformamide, and reacted at 25 °C for 12 h. The reaction solution was purified by reverse phase liquid chromatography (mobile phase: A-water (0.05% ammonium bicarbonate), B-acetonitrile; gradient elution: B%: 10%-80%), without purification, to obtain the title product 2i (16 mg, yield: 91%) in the form of a white solid.

[0522] Ninth step

[0523] 2i (15 mg, 31.00 pmol) and dodecanedioic acid monomethyl ester (7.58 mg, 31.02 pmol) were dissolved in 2 mL of dichloromethane, and 2-(7-azobenzo-triazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (14.15 mg, 37.21 pmol) and N,N-diisopropyl ethylamine (20.03 mg, 155.0 pmol) were added, and reacted at 25 °C for 2 h. The reaction solution was concentrated by reduced pressure distillation, and the obtained residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 30%-100%), to obtain the title product 2j (14 mg, yield: 62%) in the form of a white solid.

[0524] Tenth step

[0525] Dissolve 2j (12 mg, 16.62 pmol) in 2 mL tetrahydrofuran and 2 mL water, add lithium hydroxide (1.99 mg, 83.09 pmol), react at 25 °C for 12 h. Concentrate the reaction solution by distillation under reduced pressure, the obtained residue is purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 10% - 70%) to give the title product 2k (10 mg, yield: 85%) as a white solid.

[0526] Tenth step

[0527] Dissolve 2k (5 mg, 7.07 pmol) and 1g (14.71 mg, 7.07 pmol) in 2 mL dichloromethane, add 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.03 mg, 10.6 pmol) and N,N-diisopropylethylamine (4.57 mg, 35.36 pmol), react at 25 °C for 2 h. Concentrate the reaction solution by distillation under reduced pressure, the obtained residue is purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 20% - 70%) to give the title product 2l (11 mg, yield: 55%) as a white solid.

[0528] Twelfth step

[0529] Dissolve 2l (10 mg, 3.94 pmol) in 3 mL dichloromethane, protect under nitrogen, add 4-dimethylaminopyridine (0.1 mg, 0.79 pmol), succinic anhydride (1.97 mg, 19.7 pmol) and N,N-diisopropylethylamine (5.09 mg, 39.4 pmol), stir at 25 °C for 15 h. Concentrate the reaction solution by distillation under reduced pressure, the obtained residue is purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 10% - 90%) to give the title product 2 (4 mg, yield: 36%) as a beige solid.

[0530] MS m / z (ESI): 1162.9 [1 / 2 (M-301)].

[0531] 1H NMR (400 MHz, CD3OD) δ 7.39-7.36 (m, 2H), 7.30-7.28 (m, 1H), 7.27-7.24 (m, 5H), 7.21-7.18 (m, 1H), 6.87-6.82 (m, 4H), 5.33-5.31 (m, 3H), 5.08-5.03 (m, 4H), 4.57-4.54 (m, 3H), 4.17-4.04 (m, 12H), 4.02-3.97 (m, 4H), 3.89-3.84 (m, 4H), 3.78-3.77 (m, 6H), 3.70-3.65 (m, 12H), 3.56-3.50 (m, 4H), 3.48-3.46 (m, 1H), 3.33-3.31 (m, 2H), 3.15-3.11 (m, 12H), 2.46-2.42 (m, 8H), 2.25-2.21 (m, 6H), 2.13-2.11 (m, 9H), 2.02-2.00 (m, 9H), 1.94-1.91 (m, 18H), 1.71-1.49 (m, 22H), 1.48-1.42 (m, 2H), 1.31-1.25 (m, 12H), 0.50-0.46 (m, 12H).

[0532] Example 1-3: Preparation of compound 3

[0533] First step

[0534] (S)-5,5-diphenyl-2-methyl-3,4-propanol-1,3,2-oxazaborolidine (85.9 mg, 310 μmol) was dissolved in 10 mL of toluene, protected by nitrogen, cooled to 0 °C, and borane dimethyl sulfide complex (10 M, 186 μL) was added dropwise. The reaction was carried out at 0 °C for 15 min, then 2e single compound di (650 mg, 1.55 mmol) was added, and the reaction was carried out at 0 °C for 30 min. 5 mL of methanol and 8 mL of water were added to the reaction solution at 0 °C, and the reaction solution was extracted with dichloromethane (10 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation. The resulting residue was purified by silica gel column chromatography with developing system A to obtain the title product 3a (503 mg, yield: 74%) in the form of yellow oil.

[0535] MS m / z (ESI): 422.1 [M+1].

[0536] Second step

[0537] Dissolve 3a (503 mg, 1.15 mmol) in 8 mL of tetrahydrofuran, protect with nitrogen, cool to 0 °C, and then drop in lithium borohydride tetrahydrofuran solution (2 M, 862 μL). Stir at 25 °C for 1 hour. Add 1 mL of ammonium chloride solution and 5 mL of water at 0 °C, extract the reaction solution with dichloromethane (8 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by reduced pressure distillation, and purify the obtained residue by silica gel column chromatography with the developing system A to obtain the title product 3b (51.83 mg, yield: 38%) in the form of white solid.

[0538] MS m / z (ESI): 380.1 [M+1].

[0539] Third step

[0540] Dissolve 3b (20 mg, 52.71 μmol) in 1 mL of dry pyridine, protect with nitrogen, cool to below 5 °C, then dissolve 4,4'-dimethoxytrityl chloride (27 mg, 79.07 μmol) in 1 mL of dichloromethane and slowly drop into the reaction solution, naturally raise to room temperature and stir for 15 hours. Quench the reaction solution by adding 1 mL of methanol, concentrate the reaction solution by reduced pressure distillation, and purify the obtained residue by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 μm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 20%-100%) to obtain the title product 3c (21 mg, yield: 58%) in the form of light yellow oil.

[0541] Fourth step

[0542] Dissolve 3c (21 mg, 30.8 μmol) in 1 mL of N,N-dimethylformamide, then add piperidine (13 mg, 153.96 μmol), and stir the reaction at room temperature for 1 hour. Concentrate the reaction solution by reduced pressure distillation, and purify the obtained residue by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 μm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 20%-100%) to obtain the title product 3d (12 mg, yield: 85%) in the form of light yellow oil.

[0543] Fifth step

[0544] To a solution of 3d (12 mg, 26.11 pmol) and dodecanedioic acid monomethyl ester (7.02 mg, 28.73 pmol) in 1 mL of dichloromethane was added 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.91 mg, 31.31 pmol) and N,N-diisopropylethylamine (13.50 mg, 104.46 pmol) and the reaction was stirred at 25 °C for 2 h. The reaction was concentrated by distillation under reduced pressure and the residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 20% - 100%) to give the title product 3e (15 mg, yield: 84%) as a light yellow oil.

[0545] Sixth step

[0546] To a solution of 3e (15 mg, 21.87 pmol) in 1 mL of methanol and 0.3 mL of water was added lithium hydroxide (2.62 mg, 109.39 pmol) and the reaction was stirred at 25 °C for 2 h. The reaction was concentrated by distillation under reduced pressure and the residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 5% - 80%) to give the title product 3f (9 mg, yield: 61%) as a beige solid.

[0547] Seventh step

[0548] To a solution of 3f (8 mg, 11.9 pmol) and 1g (22.48 mg, 12.0 pmol) in 1 mL of dichloromethane was added 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.43 mg, 14.28 pmol) and N,N-diisopropylethylamine (6.16 mg, 47.66 pmol) and the reaction was stirred at 25 °C for 2 h. The reaction was concentrated by distillation under reduced pressure and the residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 5% - 90%) to give the title product 3g (17 mg, yield: 56%) as a white solid.

[0549] MS m / z (ESI): 2224.8 [M-301].

[0550] Eighth step

[0551] To a solution of 3 g (17 mg, 6.73 pmol) in 1 mL of dichloromethane, 4-dimethylaminopyridine (0.16 mg, 1.35 pmol), succinic anhydride (3.37 mg, 33.68 pmol) and N,N-diisopropylethylamine (8.7 mg, 67.31 pmol) were added under nitrogen protection, and stirred at 25 °C for 15 hours. The reaction solution was concentrated by distillation under reduced pressure, and the obtained residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 10%-90%) to obtain the title product 3 (16 mg, yield: 90%) as a white solid.

[0552] MS m / z (ESI): 2324.8 [M-301].

[0553] 1H NMR (400 MHz, CDC13) d 7.51 - 7.49 (m, 1H), 7.32 - 7.30 (m, 1H), 7.30 - 7.28 (m, 3H), 7.20 - 7.16 (m, 4H), 6.86 - 6.84 (m, 2H), 6.83 - 6.82 (m, 2H), 5.39 - 5.34 (m, 3H), 5.21 - 5.17 (m, 3H), 5.09 - 4.97 (m, 1H), 4.66 - 4.58 (m, 3H), 4.55 - 4.44 (m, 1H), 4.21 - 4.05 (m, 10H), 3.99 - 3.90 (m, 6H), 3.82 - 3.80 (m, 6H), 3.72 - 3.67 (m, 12H), 3.55 - 3.49 (m, 4H), 3.19 - 3.05 (m, 12H), 2.63 - 2.55 (m, 4H), 2.50 - 2.45 (m, 6H), 2.38 - 2.21 (m, 10H), 2.20 - 2.17 (m, 2H), 2.17 - 2.15 (m, 9H), 2.13 - 2.08 (m, 2H), 2.07 - 2.05 (m, 9H), 2.01 - 1.99 (m, 9H), 1.98 - 1.95 (m, 9H), 1.85 - 1.77 (m, 4H), 1.76 - 1.67 (m, 6H), 1.66 - 1.61 (m, 6H), 1.57 - 1.53 (m, 2H), 1.39 - 1.33 (m, 2H), 1.31 - 1.29 (m, 2H), 1.28 - 1.24 (m, 8H), 0.92 - 0.82 (m, 2H), 0.54 - 0.47 (m, 12H).

[0554] Example 1-4: Preparation of compound 4

[0555] First step

[0556] To a solution of 1 g (200 mg, 0.11 mmol) and dodecanedioic acid monobenzyl ester (41.08 mg, 1.28 μmol) in 10 mL of dichloromethane, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (60.93 mg, 0.16 mmol) and N,N-diisopropylethylamine (41.42 mg, 0.32 mmol) were added and the reaction was stirred at 25 °C for 2 h. The reaction was concentrated by distillation under reduced pressure and the residue was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 μm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to give the title product 4a (115 mg, yield: 48%) as a white solid.

[0557] MS m / z (ESI): 1088.3 [1 / 2 M+1].

[0558] Second step

[0559] To a solution of 4a (115 mg, 52.89 μmol) in 10 mL of methanol and 10 mL of ethyl acetate, one drop of acetic acid was added and wet palladium on carbon (100 mg, 10%) was added. The reaction was stirred under hydrogen (15 Psi) at 25 °C for 3 h. The reaction was filtered over celite and the filtrate was concentrated by distillation under reduced pressure. The residue was purified by preparative high performance liquid chromatography (separation conditions: column: Phenomenex luna C18 150 x 25 mm x 4 μm; mobile phase: A-water (0.1% formic acid): B-acetonitrile, gradient elution: B%: 20%-55%) to give the title product 4b (85 mg, yield: 77%) as a white solid.

[0560] MS m / z (ESI): 1043.4 [1 / 2 M+1].

[0561] Third step

[0562] To a solution of 4c (500 mg, 1.55 mmol, prepared using the method disclosed in patent application “WO2023109938 A1” page 99, example 6) and tetraisopropyl titanate (659.16 mg, 2.32 mmol) in 20 mL of super dry tetrahydrofuran, under nitrogen protection, was cooled to below -78°C, then ethyl magnesium bromide (2M, 2.32 mL) was slowly added dropwise into the reaction solution, and then allowed to naturally rise to room temperature and stirred for 1 hour. Then trifluoroboron diethyl ether (614.43 mg, 4.33 mmol) was slowly added dropwise into the reaction solution, and stirred at 25°C for 0.5 hour, the reaction solution was quenched by adding 10 mL of saturated ammonium chloride, then the reaction solution was diluted with 100 mL of water, and the reaction solution was extracted with ethyl acetate (100 mL x 3), the organic phase was washed with saturated sodium chloride solution (100 mL x 2), then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation, and the obtained residue was purified by preparative high performance liquid chromatography (separation conditions: column: Phenomenex luna C18 150 x 25 mm x 4 μm; mobile phase: A-water (0.1% formic acid):B-acetonitrile, gradient elution: B%: 5%-80%), to obtain the title product 4d (350 mg, yield: 64%) in the form of a milky white solid.

[0563] MS m / z (ESI): 354.3 [M+1].

[0564] Fourth step

[0565] 4d (350 mg, 0.99 mmol) was dissolved in 10 mL of 1,4-dioxane and 10 mL of water, sodium bicarbonate (415.94 mg, 4.95 mmol) and 9-fluorenylmethyl-N-succinimidyl carbonate (434.24 mg, 1.29 mmol) were added, and the reaction was carried out at 25°C for 1 hour. The reaction solution was added to 50 mL of water, and the reaction solution was extracted with ethyl acetate (50 mL x 3), the organic phase was washed with saturated sodium chloride solution (50 mL x 2), then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation, and the obtained residue was purified by silica gel column chromatography with developing system B to obtain the title product 4e (437 mg, yield: 76%) in the form of a white solid.

[0566] MS m / z (ESI): 576.5 [M+1].

[0567] Fifth step

[0568] To a solution of 4e (425 mg, 0.74 mmol) in 2 mL of dry dichloromethane, under nitrogen atmosphere, was cooled to below -70 °C and then boron trichloride (1 M, 7.38 mL) was added slowly drop wise to the reaction mixture, maintaining below -70 °C for 1 h. To the reaction mixture was added 2 mL of methanol slowly at -70 °C and the reaction mixture was concentrated under reduced pressure. The residue obtained was purified by reverse phase liquid chromatography (Separation condition: Column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; Mobile phase: A - Water (0.1% formic acid): B - Acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to get the title product 4f (160 mg, yield: 52%) as a colorless oil.

[0569] MS m / z (ESI): 396.3 [M+1].

[0570] Sixth step

[0571] To a solution of 4f (160 mg, 0.4 mmol) in 3 mL of dry pyridine, under nitrogen atmosphere, was cooled to below 5 °C and then 4,4'-dimethoxytrityl chloride (165 mg, 0.49 mmol) was dissolved in 3 mL of dichloromethane and added slowly drop wise to the reaction mixture, allowed to warm to room temperature and stirred for 2 h. The reaction was quenched by adding 1 mL of methanol and the reaction mixture was concentrated under reduced pressure. The residue obtained was purified by reverse phase liquid chromatography (Separation condition: Column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; Mobile phase: A - Water (0.05% ammonium bicarbonate): B - Acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to get the title product 4g (210 mg, yield: 74%) as a colorless oil.

[0572] Seventh step

[0573] To a solution of 4g (210 mg, 0.3 mmol) in 2 mL of N,N-dimethylformamide, 1,5-diazabicyclo[5.4.0]-5-undecene (46.89 mg, 0.3 mmol) was added and stirred at 25 °C for 1 h. The reaction mixture was purified by reverse phase liquid chromatography (Separation condition: Column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; Mobile phase: A - Water (0.05% ammonium bicarbonate): B - Acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) as such to get the title product 4h (141 mg, yield: 98%) as a colorless oil.

[0574] MS m / z (ESI): 476.4 [M+1].

[0575] Eighth step

[0576] Dissolve 4h (3.01 mg, 6.33 pmol) and 4b (11 mg, 5.28 pmol) in 1 mL of N,N- dimethylformamide, add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.41 mg, 6.34 pmol) and N,N-diisopropylethylamine (2.72 mg, 21.12 pmol), and react at 25°C for 0.5 hours. Purify the reaction solution directly by preparative high-performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 20% - 100%) to obtain the title product 4i (8 mg, yield: 56%) as a white solid.

[0577] MS m / z (ESI): 2240.7 [M-301].

[0578] Ninth step

[0579] Dissolve 4i (8 mg, 3.15 pmol) in 1 mL of dichloromethane, add 4-dimethylaminopyridine (0.08 mg, 0.63 pmol), succinic anhydride (1.57 mg, 15.69 pmol), and N,N- diisopropylethylamine (4.07 mg, 31.49 pmol), and stir at 25°C for 15 hours under nitrogen protection. Concentrate the reaction solution by distillation under reduced pressure, and purify the obtained residue by preparative high-performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 10% - 90%) to obtain the title product 4 (6.75 mg, yield: 80%) as a beige solid.

[0580] MS m / z (ESI): 2339.9 [M-301].

[0581] 1H NMR (400 MHz, CDC13) δ 7.59 - 7.57 (m, 1H), 7.34 - 7.33 (m, 1H), 7.32 - 7.31 (m, 1H), 7.30 - 7.29 (m, 1H), 7.29 - 7.29 (m, 1H), 7.20 - 7.16 (m, 4H), 6.95 - 6.89 (m, 2H), 6.86 - 6.82 (m, 4H), 6.74 - 6.71 (m, 1H), 6.64 - 6.59 (m, 1H), 6.57 - 6.54 (m, 1H), 5.37 - 5.35 (m, 3H), 5.23 - 5.18 (m, 3H), 5.16 - 5.12 (m, 1H), 5.09 - 5.02 (m, 1H), 4.66 - 4.61 (m, 3H), 4.56 - 4.49 (m, 1H), 4.21 - 4.08 (m, 10H), 4.07 - 4.01 (m, 2H), 4.00 - 3.90 (m, 8H), 3.82 - 3.80 (m, 6H), 3.73 - 3.72 (m, 2H), 3.71 - 3.69 (m, 6H), 3.67 - 3.64 (m, 2H), 3.56 - 3.49 (m, 4H), 3.20 - 3.14 (m, 4H), 3.13 - 3.11 (m, 4H), 3.10 - 3.06 (m, 2H), 2.67 - 2.64 (m, 2H), 2.61 - 2.57 (m, 2H), 2.50 - 2.46 (m, 6H), 2.31 - 2.27 (m, 4H), 2.25 - 2.22 (m, 2H), 2.19 - 2.18 (m, 2H), 2.17 - 2.15 (m, 9H), 2.12 - 2.10 (m, 2H), 2.06 - 2.05 (m, 9H), 2.02 - 2.00 (m, 9H), 1.98 - 1.96 (m, 9H), 1.83 - 1.80 (m, 2H), 1.80 - 1.77 (m, 2H), 1.76 - 1.73 (m, 2H), 1.72 - 1.70 (m, 2H), 1.69 - 1.67 (m, 2H), 1.65 - 1.62 (m, 4H), 1.61 - 1.55 (m, 4H), 1.30 - 1.24 (m, 12H), 0.99 - 0.76 (m, 6H), 0.53 - 0.50 (m, 10H).

[0582] Example 1-5: Preparation of compound 5

[0583] First step

[0584] Dissolve 5a (10.2 g, 44.8 mmol) in 360 mL of toluene, add p-toluenesulfonic acid monohydrate (853 mg, 4.48 mmol) and ethylene glycol (5.57 g, 89.7 mmol), stir at 135 °C for 12 hours. Add 200 mL of saturated sodium bicarbonate solution to the reaction solution, extract the reaction solution with dichloromethane (200 mL x 2), wash the organic phase with saturated sodium chloride solution (200 mL x 2), dry the organic phase over anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, and purify the obtained residue by silica gel column chromatography with developing system B to obtain the title product 5b (3.67 g, yield: 30%) as a yellow oil.

[0585] MS m / z (ESI): 214.0 [M-55].

[0586] Second step

[0587] Dissolve 5b (9.5 g, 35.3 mmol) and (+)-sparteine (8.27 g, 35.3 mmol) in 190 mL of tetrahydrofuran, cool to below -78 °C under nitrogen protection, then slowly drop sec-butyllithium (1.3 M, 40.7 mL) into the reaction solution, stir at -78 °C for 1.5 hours. Then slowly drop methyl chloroformate (4.01 g, 42.4 mmol) into the reaction solution, stir at -78 °C for 1 hour, then naturally raise to room temperature and stir for 12 hours. Slowly pour the reaction solution into 200 mL of saturated ammonium chloride solution at 0 °C, extract the reaction solution with dichloromethane (100 mL x 2), wash the organic phase with saturated sodium chloride solution (100 mL x 2), dry the organic phase over anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, and purify the obtained residue by silica gel column chromatography with developing system B to obtain the title product 5c (2.54 g, yield: 16%) as a yellow oil.

[0588] MS m / z (ESI): 228.1 [M-99].

[0589] Third step

[0590] Dissolve 5c (2.54 g, 5.65 mmol) in 12 mL of tetrahydrofuran, add hydrochloric acid (4 M, 12 mL), and stir at 25 °C for 2 hours. Concentrate the reaction solution by distillation under reduced pressure, and purify the obtained residue without purification to obtain the crude title product 5d (1.04 g) as a yellow oil. The product is directly used in the next step reaction without purification.

[0591] MS m / z (ESI): 184.0 [M+1].

[0592] Fourth step

[0593] Dissolve 5d (1.04 g, 5.68 mmol) in 10 mL of tetrahydrofuran and 10 mL of saturated sodium bicarbonate solution, add 9-fluorenylmethyl-N-succinimidyl carbonate (2.3 g, 6.81 mmol), stir at 25 °C for 1 hour. Add 10 mL of water to the reaction solution, extract the reaction solution with dichloromethane (20 mL x 3), wash the organic phase with saturated sodium chloride solution (20 mL x 2), then dry over anhydrous sodium sulfate, filter, concentrate the filtrate by reduced pressure distillation, purify the obtained residue by silica gel column chromatography with the developing system B to obtain the title product 5e (1.24 g, yield: 51%) in the form of yellow oil.

[0594] Separate 5e (1.24 g, 2.91 mmol) by SFC (separation conditions: column: DAICEL CHIRALCEL OJ (250 mm*30 mm, 10 μm); mobile phase: A-carbon dioxide: B-ethanol, isocratic elution: B: 35%), to obtain single configuration compound 5e-1 and single configuration compound 5e-2.

[0595] 5e single configuration compound one (shorter retention time)

[0596] Colorless oil, 201 mg, yield: 52%. SFC analysis: retention time 1.636 minutes. (column: Chiralpak AD-3 50x4.6mm I.D., 3μm, mobile phase: A-carbon dioxide, B-ethanol (0.05% diethylamine), gradient elution: B: 5%-40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30ADsf).

[0597] MS m / z (ESI): 406.0 [M+1].

[0598] 5e single configuration compound two (longer retention time)

[0599] Colorless oil, 197 mg, yield: 16%. SFC analysis: retention time 1.830 minutes. (column: Chiralpak AD-3 50x4.6mm I.D., 3μm, mobile phase: A-carbon dioxide, B-ethanol (0.05% diethylamine), gradient elution: B: 5%-40%, flow rate: 3 mL / min, instrument: Shimadzu LC-30ADsf).

[0600] MS m / z (ESI): 406.0 [M+1].

[0601] Fifth step

[0602] Dissolve 5e single configuration compound one (472 mg, 1.16 mmol) in 5 mL of tetrahydrofuran, under nitrogen protection, cool to below 0°C, drop in lithium borohydride tetrahydrofuran solution (2M, 2.32 mL), stir at 25°C for 1 hour. Add 10 mL of saturated ammonium chloride solution at 0°C, extract the reaction solution with dichloromethane (10 mL x 3), wash the organic phase with saturated sodium chloride solution (10 mL x 2), then dry over anhydrous sodium sulfate, filter, concentrate the filtrate by reduced pressure distillation, purify the obtained residue by silica gel column chromatography with developing system A to obtain the title product 5f (297 mg, yield: 66%) in the form of colorless sticky substance.

[0603] MS m / z (ESI): 380.0 [M+1].

[0604] Sixth step

[0605] Dissolve 5f (15 mg, 38.73 μmol) in 3 mL of pyridine, under nitrogen protection, then dissolve 4,4'-bismethoxybenzhydryl chloride (13.13 mg, 38.75 μmol) in 3 mL of dichloromethane, drop into the reaction solution at 0°C, stir at 25°C for 12 hours. Concentrate the reaction solution by reduced pressure distillation, purify the obtained residue by reverse phase liquid chromatography (separation condition: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 μm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to obtain the title product 5g (9 mg, yield: 30%) in the form of white solid.

[0606] Seventh step

[0607] Dissolve 5g (9 mg, 11.88 μmol) and piperidine (3.03 mg, 35.65 μmol) in 1 mL of N,N-dimethylformamide, stir the reaction at 25°C for 6 hours. Concentrate the reaction solution by reduced pressure distillation, purify the obtained residue by reverse phase liquid chromatography (separation condition: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 μm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to obtain the title product 5h (5 mg, yield: 87%) in the form of white solid.

[0608] Eighth step

[0609] Dissolve 5h (3.03 mg, 5.93 pmol) and 4b (13 mg, 5.93 pmol) in 2 mL of dichloromethane, add 2-(7-azobenzo-triazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.51 mg, 11.86 pmol) and N,N-diisopropylethylamine (3.83 mg, 29.63 pmol), and react at 25 °C for 0.5 hours. Concentrate the reaction solution by distillation under reduced pressure, and purify the obtained residue by preparative high-performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate) : B - acetonitrile, gradient elution: B%: 20% - 70%) to obtain the title product 5i (10 mg, yield: 60%) as a white solid.

[0610] Ninth step

[0611] Dissolve 5i (8 mg, 3.01 pmol) in 1 mL of dichloromethane, add 4-dimethylaminopyridine (0.07 mg, 0.57 pmol), succinic anhydride (1.51 mg, 15.09 pmol), and N,N-diisopropylethylamine (3.88 mg, 30.02 pmol), and stir at 25 °C for 15 hours under nitrogen protection. Concentrate the reaction solution by distillation under reduced pressure, and purify the obtained residue by preparative high-performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate) : B - acetonitrile, gradient elution: B%: 20% - 60%) to obtain the title product 5 (3.8 mg, yield: 43%) as a white solid.

[0612] MS m / z (ESI): 2325.1 [M-301].

[0613] 1H NMR (400 MHz, CD3OD) δ 7.48 - 7.45 (m, 1H), 7.39 - 7.33 (m, 4H), 7.27 - 7.26 (m, 2H), 7.25 - 7.24 (m, 2H), 6.88 - 6.86 (m, 1H), 6.86 - 6.84 (m, 2H), 6.83 - 6.82 (m, 1H), 5.33 - 5.31 (m, 3H), 5.07 - 5.03 (m, 4H), 4.57 - 4.54 (m, 3H), 4.17 - 4.05 (m, 12H), 4.02 - 3.97 (m, 4H), 3.90 - 3.84 (m, 5H), 3.78 - 3.77 (m, 6H), 3.70 - 3.68 (m, 6H), 3.67 - 3.65 (m, 4H), 3.55 - 3.50 (m, 4H), 3.48 - 3.45 (m, 2H), 3.15 - 3.11 (m, 12H), 2.48 - 2.42 (m, 10H), 2.26 - 2.20 (m, 8H), 2.14 - 2.11 (m, 9H), 2.02 - 1.99 (m, 9H), 1.96 - 1.90 (m, 18H), 1.72 - 1.64 (m, 8H), 1.62 - 1.57 (m, 8H), 1.32 - 1.25 (m, 18H), 0.50 - 0.45 (m, 12H).

[0614] Example 1-6: Preparation of compound 6

[0615] First step

[0616] The 5e single configuration compound di (469 mg, 1.09 mmol) was dissolved in 5 mL of tetrahydrofuran, cooled to below 0°C under nitrogen protection, and a lithium borohydride tetrahydrofuran solution (2M, 2.19 mL) was added dropwise, and stirred at 25°C for 1 hour. 10 mL of saturated ammonium chloride solution was added at 0°C, and the reaction solution was extracted with dichloromethane (10 mL x 3), and the organic phase was washed with saturated sodium chloride solution (10 mL x 2), then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by reduced pressure distillation. The obtained residue was purified by silica gel column chromatography with developing system A to obtain the title product 6a (278 mg, yield: 66%) in the form of a colorless sticky substance.

[0617] MS m / z (ESI): 380.0 [M+1].

[0618] Second step

[0619] To a solution of 6a (46 mg, 0.12 mmol) and 2,4,6-trimethylpyridine (58.76 mg, 0.48 mmol) in 3 mL of dichloromethane under nitrogen protection, 4,4'-dimethoxybenzhydryl chloride (61.61 mg, 0.18 mmol) and silver nitrate (41.18 mg, 0.24 mmol) were added and stirred at 25 °C for 0.5 h. 1 mL of methanol was added to quench the reaction, and the reaction was concentrated under reduced pressure. The residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 20%-100%) to give the title product 6b (47 mg, yield: 55%) as a light yellow oil.

[0620] MS m / z (ESI): 704.3 [M+23].

[0621] Third step

[0622] To a solution of 6b (47 mg, 68.93 pmol) in 1 mL of N,N-dimethylformamide, 1,5- diazabicyclo[5.4.0]-5-undecene (10.49 mg, 68.90 pmol) was added and stirred at room temperature for 1 h. The reaction was concentrated under reduced pressure. The residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 20%-90%) to give the title product 6c (22 mg, yield: 68%) as a colorless oil.

[0623] MS m / z (ESI): 460.4 [M+1].

[0624] Fourth step

[0625] Dissolve 6c (3.83 mg, 8.33 pmol) and 4b (14 mg, 6.72 pmol) in 1 mL of N,N- dimethylformamide, add 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.06 mg, 8.05 pmol) and N,N-diisopropylethylamine (3.47 mg, 26.85 pmol), and react at 25 °C for 0.5 hours. Concentrate the reaction solution by distillation under reduced pressure, and purify the obtained residue by preparative high-performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate) : B - acetonitrile, gradient elution: B%: 20% - 100%) to obtain the title product 6d (11 mg, yield: 50%) as a white solid.

[0626] MS m / z (ESI): 2224.8 [M-301].

[0627] Fifth step

[0628] Dissolve 6d (11 mg, 4.35 pmol) in 1 mL of dichloromethane, add 4-dimethylaminopyridine (0.53 mg, 4.34 pmol), succinic anhydride (4.36 mg, 43.57 pmol), and N,N- diisopropylethylamine (11.26 mg, 87.12 pmol), and stir at 25 °C for 15 hours. Concentrate the reaction solution by distillation under reduced pressure, and purify the obtained residue by preparative high-performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate) : B - acetonitrile, gradient elution: B%: 10% - 90%) to obtain the title product 6 (10.64 mg, yield: 92%) as a white solid.

[0629] MS m / z (ESI): 2324.8 [M-301].

[0630] 1H NMR (400 MHz, CDC13) δ 7.56-7.54 (m, 1H), 7.34-7.31 (m, 2H), 7.30-7.30 (m, 1H), 7.29-7.29 (m, 1H), 7.20-7.18 (m, 2H), 7.17-7.16 (m, 2H), 6.94-6.91 (m, 1H), 6.91-6.89 (m, 1H), 6.86-6.84 (m, 2H), 6.84-6.82 (m, 2H), 6.74-6.70 (m, 1H), 5.37-5.34 (m, 3H), 5.22-5.17 (m, 3H), 4.66-4.60 (m, 3H), 4.26-4.22 (m, 1H), 4.21-4.18 (m, 2H), 4.17-4.15 (m, 2H), 4.13-4.12 (m, 2H), 4.11-4.08 (m, 2H), 4.00-3.97 (m, 2H), 3.96-3.94 (m, 2H), 3.93-3.91 (m, 2H), 3.82-3.80 (m, 6H), 3.73-3.72 (m, 2H), 3.71-3.70 (m, 4H), 3.69-3.67 (m, 4H), 3.64-3.59 (m, 2H), 3.57-3.50 (m, 4H), 3.22-3.17 (m, 2H), 3.16-3.13 (m, 2H), 3.13-3.10 (m, 4H), 3.09-3.05 (m, 2H), 2.62-2.59 (m, 2H), 2.57-2.54 (m, 2H), 2.50-2.46 (m, 6H), 2.38-2.34 (m, 2H), 2.33-2.31 (m, 2H), 2.31-2.28 (m, 4H), 2.27-2.25 (m, 2H), 2.25-2.21 (m, 2H), 2.20-2.17 (m, 2H), 2.17-2.15 (m, 9H), 2.12-2.10 (m, 2H), 2.06-2.05 (m, 9H), 2.02-2.00 (m, 9H), 1.98-1.95 (m, 9H), 1.91-1.87 (m, 2H), 1.86-1.82 (m, 2H), 1.82-1.76 (m, 4H), 1.74-1.69 (m, 4H), 1.67-1.61 (m, 8H), 1.57-1.53 (m, 2H), 1.33-1.31 (m, 2H), 1.29-1.24 (m, 10H), 0.54-0.48 (m, 12H).

[0631] Example 1-7: Preparation of compound 7

[0632] First step

[0633] To a solution of 4c (2.2 g, 6.8 mmol) and iodomethane (1.4 g, 9.86 mmol) in 12 mL of tetrahydrofuran under nitrogen protection, potassium bis(trimethylsilyl)amide (1 M, 33 mL) was added dropwise slowly, and the mixture was allowed to warm to 25 °C and stirred for 0.5 h. The reaction was quenched by the addition of 200 mL of saturated ammonium chloride solution at 0 °C, and the reaction was extracted with ethyl acetate (70 mL x 3). The organic phase was washed with saturated sodium chloride solution (50 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure to give the title product 7a (998 mg) as a white solid. 7a (998 mg, 2.8 mmol) was purified by silica gel column chromatography with the developing system B to give the title product 7a-1 (570 mg, yield: 57%) and the title product 7a-2 (420 mg, yield: 42%) as white solids, respectively.

[0634] Second step

[0635] To a solution of 7a-1 (500 mg, 1.4 mmol) in 35 mL of tetrahydrofuran under nitrogen protection, lithium aluminum hydride (225.52 mg, 5.94 mmol) was added slowly, and the mixture was stirred at 70 °C for 2 h. The reaction was quenched by the addition of 225 μL of water, 225 μL of 15% aqueous sodium hydroxide solution, and 675 μL of water, successively, and dried over anhydrous sodium sulfate. The reaction was filtered through celite, and the filtrate was concentrated by distillation under reduced pressure. The residue was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 μm; mobile phase: A-water (0.1% formic acid):B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to give the title product 7b (380 mg, yield: 78%) as a white solid.

[0636] MS m / z (ESI): 342.2 [M+1].

[0637] Third step

[0638] To a solution of 7b (360 mg, 1.0 mmol) in 5 mL of tetrahydrofuran and 5 mL of water, 9-fluorenylmethyl-N-succinimidyl carbonate (426.93 mg, 1.27 mmol) and sodium bicarbonate (319.25 mg, 3.8 mmol) were added, and the mixture was stirred at 25 °C for 1 h. The reaction was extracted with ethyl acetate (30 mL x 3), and the organic phase was washed with saturated sodium chloride solution (50 mL x 2) and then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The residue was purified by silica gel column chromatography with the developing system B to give the title product 7c (398 mg, yield: 69%) as a yellow solid.

[0639] MS m / z (ESI): 564.5 [M+1].

[0640] Fourth step

[0641] Dissolve 7c (338 mg, 0.59 mmol) in 35 mL of super dry dichloromethane, under nitrogen protection, cool to below -70 °C, then slowly drop 1 M boron trichloride (6.89 mL) into the reaction solution, keep stirring below -70 °C for 15 minutes. Slowly add 30 mL of methanol into the reaction solution, concentrate the reaction solution under reduced pressure, the obtained residue is purified by reverse phase liquid chromatography (separation condition: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 μm; mobile phase: A-water (0.1% formic acid):B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage), to obtain the title product 7d (67 mg, yield: 29%) in the form of colorless oil.

[0642] MS m / z (ESI): 384.3 [M+1].

[0643] Fifth step

[0644] Dissolve 7d (48 mg, 125.18 μmol) and 2,4,6-trimethylpyridine (37.91 mg, 312.84 μmol) in 5 mL of super dry dichloromethane, under nitrogen protection, then add 4,4'-dimethoxybenzhydryl chloride (38.21 mg, 112.77 μmol) and silver nitrate (21.28 mg, 125.27 μmol), stir at 25 °C for 0.5 hours. Add 20 mL of methanol to quench the reaction solution, filter, concentrate the filtrate under reduced pressure, the obtained residue is purified by reverse phase liquid chromatography (separation condition: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 μm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage), to obtain the title product 7e (54 mg, yield: 61%) in the form of white solid.

[0645] Sixth step

[0646] To a solution of 7e (53 mg, 77.29 pmol) in 1 mL of N,N-dimethylformamide was added 1,5-diazobicyclo[5.4.0]-5-undecene (11.83 mg, 77.71 pmol) and the reaction was stirred at room temperature for 1 h. The reaction was concentrated under reduced pressure and the residue was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to give the title product 7f (30 mg, yield: 84%) as a colorless oil.

[0647] MS m / z (ESI): 464.4 [M+1].

[0648] Seventh step

[0649] To a solution of 7f (30 mg, 64.72 pmol) and dodecanedioic acid monomethyl ester (17.39 mg, 71.09 pmol) in 2 mL of N,N-dimethylformamide was added 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (29.53 mg, 77.66 pmol) and N,N-diisopropylethylamine (33.46 mg, 258.9 pmol) and the reaction was stirred at 25 °C for 0.5 h. The reaction was concentrated under reduced pressure and the residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution: B%: 30%-100%) to give the title product 7g (36 mg, yield: 77%) as a white solid.

[0650] MS m / z (ESI): 712.7 [M+23].

[0651] Eighth step

[0652] To a solution of 7g (36 mg, 49.87 pmol) in 2 mL of methanol and 1 mL of water, lithium hydroxide (12.5 mg, 521.92 pmol) was added and the reaction mixture was stirred at 25 °C for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 5% - 80%) to afford the title product 7h (26 mg, yield: 76%) as a white solid.

[0653] MS m / z (ESI): 698.7 [M+23].

[0654] Ninth step

[0655] To a solution of 7h (12 mg, 17.76 pmol) and 1g (33.24 mg, 17.76 pmol) in 1 mL of N,N-dimethylformamide, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.1 mg, 21.3 pmol) and N,N-diisopropylethylamine (9.18 mg, 71.03 pmol) were added and the reaction mixture was stirred at 25 °C for 0.5 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 10% - 90%) to afford the title product 7i (16 mg, yield: 35%) as a white solid.

[0656] MS m / z (ESI): 2228.8 [M-301].

[0657] Tenth step

[0658] Dissolve 7i (16 mg, 6.31 pmol) in 2 mL of dichloromethane, protect under nitrogen, add 4-dimethylaminopyridine (0.77 mg, 6.3 pmol), succinic anhydride (6.33 mg, 63.26 pmol) and N,N-diisopropylethylamine (16.35 mg, 126.51 pmol), stir at 25 °C for 15 hours. Concentrate the reaction solution by distillation under reduced pressure, purify the obtained residue by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 10%-90%), to obtain the title product 7 (16 mg, yield: 96%) as a beige solid.

[0659] MS m / z (ESI): 2329.0 [M-301].

[0660] 1H NMR (400 MHz, CDC13) δ 7.57 - 7.55 (m, 1H), 7.34 - 7.32 (m, 1H), 7.32 - 7.31 (m, 1H), 7.30 - 7.29 (m, 1H), 7.29 - 7.29 (m, 1H), 7.20 - 7.16 (m, 4H), 6.97 - 6.91 (m, 1H), 6.90 - 6.88 (m, 1H), 6.86 - 6.85 (m, 2H), 6.84 - 6.82 (m, 2H), 6.71 - 6.67 (m, 1H), 5.37 - 5.34 (m, 3H), 5.22 - 5.18 (m, 3H), 4.65 - 4.61 (m, 3H), 4.22 - 4.09 (m, 10H), 4.08 - 4.03 (m, 2H), 4.01 - 3.87 (m, 8H), 3.82 - 3.80 (m, 6H), 3.74 - 3.72 (m, 2H), 3.71 - 3.69 (m, 6H), 3.68 - 3.66 (m, 2H), 3.55 - 3.49 (m, 4H), 3.38 - 3.30 (m, 2H), 3.21 - 3.16 (m, 2H), 3.16 - 3.14 (m, 2H), 3.13 - 3.11 (m, 4H), 3.09 - 3.06 (m, 2H), 2.66 - 2.60 (m, 4H), 2.50 - 2.46 (m, 6H), 2.37 - 2.32 (m, 2H), 2.32 - 2.28 (m, 4H), 2.27 - 2.25 (m, 2H), 2.25 - 2.23 (m, 2H), 2.19 - 2.18 (m, 2H), 2.17 - 2.15 (m, 9H), 2.12 - 2.10 (m, 2H), 2.06 - 2.05 (m, 9H), 2.02 - 2.00 (m, 9H), 1.98 - 1.96 (m, 9H), 1.83 - 1.80 (m, 2H), 1.79 - 1.77 (m, 2H), 1.76 - 1.73 (m, 2H), 1.72 - 1.70 (m, 2H), 1.69 - 1.67 (m, 2H), 1.66 - 1.61 (m, 6H), 1.59 - 1.52 (m, 4H), 1.29 - 1.28 (m, 3H), 1.28 - 1.25 (m, 8H), 0.54 - 0.48 (m, 12H).

[0661] Example 1-8: Preparation of compound 8

[0662] First step

[0663] Dissolve 7a-2 (420 mg, 1.24 mmol) in 30 mL of tetrahydrofuran, protect with nitrogen, slowly add lithium aluminum hydride (189.44 mg, 4.99 mmol), stir at 70 °C for 2 hours. Add 193 μL of water, 193 μL of 15% aqueous sodium hydroxide solution, and 579 μL of water to the reaction solution at 0 °C in sequence, quench, add anhydrous sodium sulfate to dry, filter the reaction solution with diatomite, concentrate the filtrate by reduced pressure distillation, purify the obtained residue by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 μm; mobile phase: A-water (0.1% formic acid):B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage), to obtain the title product 8a (357 mg, yield: 82%) in the form of a white solid.

[0664] MS m / z (ESI): 342.3 [M+1].

[0665] Second step

[0666] Dissolve 8a (337 mg, 0.99 mmol) in 5 mL of tetrahydrofuran and 5 mL of water, add 9-fluorenylmethyl-N-succinimidyl carbonate (399.66 mg, 1.18 mmol) and sodium bicarbonate (249.04 mg, 2.96 mmol), stir at 25 °C for 1 hour. Add 20 mL of water to the reaction solution, extract the reaction solution with ethyl acetate (30 mL x 3), wash the organic phase with saturated sodium chloride solution (50 mL x 2), then dry with anhydrous sodium sulfate, filter, concentrate the filtrate by reduced pressure distillation, purify the obtained residue by silica gel column chromatography with developing system B, to obtain the title product 8b (420 mg, yield: 74%) in the form of a yellow solid.

[0667] MS m / z (ESI): 564.5 [M+1].

[0668] Third step

[0669] To a solution of 8b (410 mg, 0.71 mmol) in 35 mL of dry dichloromethane, under nitrogen atmosphere, was cooled to below -70 °C and then boron trichloride (1 M, 7.28 mL) was added slowly drop wise to the reaction mixture, maintaining below -70 °C for 15 minutes. To the reaction mixture was added 20 mL of methanol slowly and the reaction mixture was concentrated under reduced pressure. The residue obtained was purified by reverse phase liquid chromatography (Separation conditions: Column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; Mobile phase: A - Water (0.1% formic acid): B - Acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to get the title product 8c (120 mg, yield: 43%) as a colorless oil.

[0670] MS m / z (ESI): 384.3 [M+1].

[0671] Fourth step

[0672] To a solution of 8c (120 mg, 0.31 mmol) in 2 mL of dry pyridine, under nitrogen atmosphere, was added 4,4'-dimethoxytrityl chloride (159.23 mg, 0.47 mmol) in 2 mL of dichloromethane slowly drop wise and stirred at 25 °C for 4 hours. The reaction mixture was concentrated under reduced pressure. The residue obtained was purified by reverse phase liquid chromatography (Separation conditions: Column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; Mobile phase: A - Water (0.05% ammonium bicarbonate): B - Acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to get the title product 8d (70 mg, yield: 32%) as a white solid.

[0673] Fifth step

[0674] To a solution of 8d (62 mg, 89.5 pmol) in 1 mL of N,N-dimethylformamide, under nitrogen atmosphere, was added 1,5-diazabicyclo[5.4.0]-5-undecene (6.8 mg, 44.67 pmol) and stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue obtained was purified by preparative high performance liquid chromatography (Separation conditions: Column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; Mobile phase: A - Water (0.05% ammonium bicarbonate): B - Acetonitrile, gradient elution: B%: 10% - 70%) to get the title product 8e (30 mg, yield: 71%) as a white solid.

[0675] Sixth step

[0676] Dissolve 8e (6 mg, 12.81 pmol) and dodecanedioic acid monomethyl ester (4.7 mg, 19.24 pmol) in 3.5 mL of N,N-dimethylformamide, add 2-(7-azabenzotriazol-1- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (7.31 mg, 19.22 pmol) and N,N- diisopropylethylamine (8.29 mg, 64.14 pmol), and react at 25 °C for 0.5 h. Concentrate the reaction under reduced pressure, and purify the resulting residue by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate) : B - acetonitrile, gradient elution: B%: 20% - 95%) to obtain the title product 8f (7 mg, yield: 75%) as a white solid.

[0677] Seventh step

[0678] Dissolve 8f (7 mg, 9.64 pmol) in 1.5 mL of methanol and 0.5 mL of water, add lithium hydroxide (20 mg, 0.84 mmol), and react at 25 °C for 2 h. Concentrate the reaction under reduced pressure, and purify the resulting residue by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate) : B - acetonitrile, gradient elution: B%: 20% - 95%) to obtain the title product 8g (5 mg, yield: 73%) as a white solid.

[0679] MS m / z (ESI): 675.6 [M+1].

[0680] Eighth step

[0681] Dissolve 8g (5 mg, 7.03 pmol) and 1g (13.16 mg, 7.03 pmol) in 1 mL of N,N- dimethylformamide, add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.94 mg, 7.73 pmol) and N,N-diisopropylethylamine (5.45 mg, 42.17 pmol), and react at 25 °C for 0.5 h. Concentrate the reaction under reduced pressure, and purify the resulting residue by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate) : B - acetonitrile, gradient elution: B%: 15% - 95%) to obtain the title product 8h (5 mg, yield: 26%) as a white solid.

[0682] MS m / z (ESI): 2228.8 [M-301].

[0683] Ninth step

[0684] Dissolve 8h (5 mg, 1.88 pmol) in 1 mL of dichloromethane, protect under nitrogen, add 4-dimethylaminopyridine (2.29 mg, 18.74 pmol), succinic anhydride (3.76 mg, 37.57 pmol) and N,N-diisopropylethylamine (7.28 mg, 56.33 pmol), stir at 25 °C for 16 hours. Concentrate the reaction solution by distillation under reduced pressure, and the obtained residue is purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 15%-95%), to obtain the title product 8 (2.9 mg, yield: 58%) in off-white solid.

[0685] MS m / z (ESI): 2328.9 [M-301].

[0686] Example 1-9: Preparation of compound 9

[0687] First step

[0688] Dissolve 9a (20 g, 133.22 mmol) in 400 mL of N,N-dimethylformamide, protect under nitrogen, slowly add imidazole (10.88 g, 159.86 mmol) and tert-butyl diphenylchlorosilane (43.94 g, 159.86 mmol), stir at 25 °C for 12 hours. Concentrate the reaction solution by distillation under reduced pressure, add 200 mL of water to the obtained residue, extract the reaction solution with ethyl acetate (200 mL x 3), wash the organic phase with saturated sodium chloride solution (200 mL x 2), dry the organic phase over anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, and purify the obtained residue by silica gel column chromatography with developing system A to obtain the title product 9b (35 g, yield: 60%) in colorless oil.

[0689] Second step

[0690] Dissolve 9b (35 g, 81.07 mmol) in 400 mL of acetone, protect with nitrogen, slowly add copper sulfate (19.41 g, 121.61 mmol) and sulfuric acid (12 M, 2.16 mL), stir at 25 °C for 12 hours. Filter the reaction solution, add 50 mL of saturated sodium bicarbonate solution to the reaction solution, remove acetone by concentration under reduced pressure, add 200 mL of water to the obtained residue, extract the reaction solution with ethyl acetate (200 mL x 3), wash the organic phase with saturated sodium chloride solution (200 mL x 2), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system B, and obtain the title product 9c (20 g, yield: 52%) in the form of colorless oil.

[0691] Third step

[0692] Dissolve 9c (35 g, 73.49 mmol) in 200 mL of dichloromethane, protect with nitrogen, add pyridine (15.12 g, 191.09 mmol) and N,N-dimethylaniline (890.64 mg, 7.35 mmol), dissolve phenyl chlorothioformate (19.03 g, 110.24 mmol) in 50 mL of super dry dichloromethane, slowly drop into the reaction solution, stir at 25 °C for 12 hours. Add 100 mL of ice water to the reaction solution, extract the reaction solution with dichloromethane (200 mL x 3), wash the organic phase with saturated sodium chloride solution (200 mL x 2), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system B, and obtain the title product 9d (22 g, yield: 52%) in the form of colorless oil.

[0693] Fourth step

[0694] Dissolve 9d (12 g, 19.12 mmol) in 100 mL of toluene, protect with nitrogen, add azobisisobutyronitrile (314.02 mg, 1.91 mmol), stir at 25 °C for 15 minutes. Then slowly drop tributyltin hydride (1 M, 5.66 mL), stir at 80 °C for 1 hour. Concentrate the reaction solution by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system B, and obtain the title product 9e (7.95 g, yield: 90%) in the form of colorless oil.

[0695] Fifth step

[0696] Dissolve 9e (7.95 g, 17.34 mmol) in 100 mL of super dry tetrahydrofuran, protect with nitrogen, slowly add tetrabutylammonium fluoride (1 M, 52.02 mL), stir at 25 °C for 12 hours. Concentrate the reaction solution by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system B, and obtain the title product 9f (2.56 g, yield: 76%) in the form of colorless oil.

[0697] Sixth step

[0698] Dissolve 9f (2.56 g, 13.23 mmol) in 30 mL of super dry N,N-dimethylformamide, protect with nitrogen, add sodium hydride (793.52 mg, 19.84 mmol, 60%), stir at 0 °C for 0.5 hours, then add benzyl bromide (2.71 g, 15.87 mmol), stir at 25 °C for 2 hours. Add 50 mL of ice water to the reaction solution, extract the reaction solution with ethyl acetate (50 mL x 3), wash the organic phase with saturated sodium chloride solution (50 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system B, and obtain the title product 9g (2.86 g, yield: 74%) in the form of colorless oil.

[0699] Seventh step

[0700] Dissolve 9g (2.86 g, 9.74 mmol) in 30 mL of anhydrous methanol, add 1 mL of concentrated hydrochloric acid, stir at 68 °C for 2 hours. Concentrate the reaction solution by distillation under reduced pressure, dilute the obtained residue with 50 mL of water, adjust the pH of the reaction solution to be greater than 7 by adding saturated sodium bicarbonate solution, extract the reaction solution with ethyl acetate (50 mL x 3), wash the organic phase with saturated sodium chloride solution (50 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by silica gel column chromatography with developing system B, and obtain the title product 9h (2.16 g, yield: 75%) in the form of colorless oil.

[0701] Eighth step

[0702] Dissolve 9h (2.16 g, 8.16 mmol) in 10 mL of super dry N,N-dimethylformamide, protect with nitrogen, add sodium hydride (489.46 mg, 12.24 mmol, 60%), stir for 0.5 hours at 0°C, then add benzyl bromide (1.67 g, 9.79 mmol), stir for 2 hours at 25°C. Add 30 mL of ice water to the reaction solution, extract the reaction solution with ethyl acetate (50 mL x 3), wash the organic phase with saturated sodium chloride solution (50 mL x 3), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by reduced pressure distillation, purify the obtained residue by silica gel column chromatography with developing system B, and obtain the title product 9i (2.4 g, yield: 80%) in the form of colorless oil.

[0703] Ninth step

[0704] Dissolve 9i (2.4 g, 6.58 mmol) in 12 mL of acetic acid and 8 mL of water, add concentrated sulfuric acid (12 M, 100 μL), stir for 12 hours at 70°C. Add 100 mL of saturated sodium bicarbonate solution to the reaction solution, extract the reaction solution with ethyl acetate (100 mL x 3), wash the organic phase with saturated sodium chloride solution (100 mL x 2), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by reduced pressure distillation, purify the obtained residue by silica gel column chromatography with developing system B, and obtain the title product 9j (1.59 g, yield: 69%) in the form of colorless oil.

[0705] Tenth step

[0706] Dissolve hydroxylamine hydrochloride (91.51 mg, 1.32 mmol) and sodium bicarbonate (96.2 mg, 1.15 mmol) in 4 mL of ethanol and 4 mL of water, stir for 15 minutes at 25°C, then add 9j (100 mg, 0.29 mmol), stir for 2 hours at 25°C. Add 10 mL of water to the reaction solution, extract the reaction solution with ethyl acetate (50 mL x 3), wash the organic phase with saturated sodium chloride solution (50 mL x 2), dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate by reduced pressure distillation, and obtain the crude title product 9k (90 mg) in the form of colorless oil without purification. The product is directly used in the next reaction without purification.

[0707] Eleventh step

[0708] Methylsulfonyl chloride (281.79 mg, 2.46 mmol) was dissolved in 2 mL of pyridine, 9k (90 mg, 245.9 μmol) was dissolved in 2 mL of pyridine, and the reaction solution was added dropwise at 0°C. The reaction solution was stirred at 25°C for 3 hours. The reaction solution was added to 10 mL of water, and the reaction solution was extracted with ethyl acetate (30 mL x 3). The organic phase was washed with saturated sodium chloride solution (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The obtained residue was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 μm; mobile phase: A-water (0.05% sodium bicarbonate):B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to obtain the title product 9l (83 mg, yield: 78%) as a colorless oil.

[0709] Twelfth step

[0710] 9l (300 mg, 0.77 mmol) and tetraisopropyl titanate (328.39 mg, 1.16 mmol) were dissolved in 7 mL of super dry tetrahydrofuran, and the reaction solution was cooled to below -78°C under nitrogen protection, and then ethylmagnesium bromide (2M, 1.16 mL) was slowly added dropwise. The reaction solution was naturally raised to 25°C and stirred for 1 hour. Then, boron trifluoride etherate (306.11 mg, 2.31 mmol) was slowly added dropwise, and the reaction solution was stirred at 25°C for 0.5 hours. The reaction solution was quenched by adding 3 mL of saturated ammonium chloride, and then diluted with 30 mL of water. The reaction solution was extracted with ethyl acetate (30 mL x 3), and the organic phase was washed with saturated sodium chloride solution (30 mL x 2), and then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The obtained residue was purified by preparative high performance liquid chromatography (separation conditions: column: Phenomenex luna C18 150 x 25 mm x 4 μm; mobile phase: A-water (0.1% formic acid):B-acetonitrile, gradient elution: B%: 20%-80%) to obtain the title product 9m (50 mg, yield: 20%) as a colorless oil.

[0711] MS m / z (ESI): 324.3 [M+1].

[0712] Thirteenth step

[0713] To a solution of 9m (50 mg, 154.59 pmol) in 3 mL of 1,4-dioxane and 3 mL of water was added sodium bicarbonate (64.93 mg, 772.88 pmol) and 9-fluorenylmethyl-N-succinimidyl carbonate (67.79 mg, 200.96 pmol) and stirred at 25 °C for 1 h. The reaction solution was diluted with 20 mL of water and extracted with ethyl acetate (20 mL x 3). The organic phase was washed with saturated sodium chloride solution (20 mL x 2), then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated by distillation under reduced pressure. The obtained residue was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A-water (0.05% sodium bicarbonate):B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to give the title product 9n (74 mg, yield: 86%) as a colorless oil.

[0714] MS m / z (ESI): 546.5 [M+1].

[0715] Fourteenth step

[0716] To a solution of 9n (74 mg, 135.61 pmol) in 3 mL of super dry dichloromethane was slowly added boron trichloride (1 M, 1.36 mL) dropwise under nitrogen protection at a temperature below -70 °C, and stirred at a temperature below -70 °C for 1 h. To the reaction solution was slowly added 2 mL of methanol at -70 °C, and the reaction solution was concentrated by distillation under reduced pressure. The obtained residue was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to give the title product 90 (15 mg, yield: 29%) as a colorless oil.

[0717] MS m / z (ESI): 366.3 [M+1].

[0718] Fifteenth step

[0719] To a solution of 9o (15 mg, 41.05 pmol) and 2,4,6-trimethylpyridine (19.9 mg, 164.22 pmol) in 2 mL of dichloromethane under nitrogen protection, 4,4'-dimethoxybenzhydryl chloride (20.86 mg, 61.56 pmol) and silver nitrate (13.95 mg, 82.12 pmol) were added and stirred at 25 °C for 0.5 h. 1 mL of methanol was added to quench the reaction, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by preparative high performance liquid chromatography (separation condition: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 20%-100%) to give the title product 9p (11 mg, yield: 40%) as a light yellow oil.

[0720] MS m / z (ESI): 690.6 [M+23].

[0721] Sixteenth step

[0722] To a solution of 9p (11 mg, 16.47 pmol) in 1 mL of N,N-dimethylformamide, 1,5-diazabicyclo[5.4.0]-5-undecene (2.51 mg, 16.49 pmol) was added and stirred at 25 °C for 1 h. The reaction was purified by reverse phase liquid chromatography without treatment (separation condition: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to give the title product 9q (6.2 mg, yield: 81%) as a colorless oil.

[0723] MS m / z (ESI): 446.3 [M+1].

[0724] Seventeenth step

[0725] Dissolve 9q (1.28 mg, 2.87 pmol) and 4b (6 mg, 2.87 pmol) in 1 mL of N,N- dimethylformamide, add 2-(7-azobenzo-triazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.64 mg, 4.31 pmol) and N,N-diisopropylethylamine (1.49 mg, 11.53 pmol), and react at 25 °C for 0.5 hours. The reaction solution is purified by preparative high performance liquid chromatography without treatment (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 20% - 100%) to obtain the title product 9r (5 mg, yield: 69%) as a white solid.

[0726] MS m / z (ESI): 2210.8 [M-301].

[0727] Eighth step

[0728] Dissolve 9r (5 mg, 1.99 pmol) in 1 mL of dichloromethane, add 4-dimethylaminopyridine (0.24 mg, 1.96 pmol), succinic anhydride (3.98 mg, 39.77 pmol), and N,N- diisopropylethylamine (7.72 mg, 59.73 pmol), and stir at 25 °C for 15 hours under nitrogen protection. Concentrate the reaction solution by distillation under reduced pressure, and purify the obtained residue by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 10% - 90%) to obtain the title product 9 (4.85 mg, yield: 92%) as a white solid.

[0729] MS m / z (ESI): 2310.9 [M-301].

[0730] Example 1-10: Preparation of compound 10

[0731] First step

[0732] To a solution of 10a (300 mg, 0.996 mmol, prepared using the method disclosed in patent application “WO2022161452 A1” page 65 example 4) in 20 mL of tetrahydrofuran, 1-cyclopropyl bromomethane carbonitrile (1.1 g, 6.97 mmol) and potassium tert-butoxide (1.01 g, 8.96 mmol) were added and stirred at 25 °C for 12 h. The tetrahydrofuran was removed by distillation under reduced pressure and the residue obtained was added with 20 mL of water and the reaction was extracted with ethyl acetate (50 mL x 3). The organic phase was washed with saturated sodium chloride solution (60 mL x 2) and then dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated by distillation under reduced pressure. The residue obtained was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to give the title product 10b (453 mg, yield: 83%) as a yellow oil.

[0733] MS m / z (ESI): 596.5 [M+1].

[0734] Second step

[0735] To a solution of 10b (453 mg, 0.83 mmol) in 7 mL of hydrochloric acid and 3.5 mL of water, was added and stirred at 100 °C for 12 h. The reaction was added with 20 mL of water and extracted with ethyl acetate (50 mL x 3). The organic phase was washed with saturated sodium chloride solution (60 mL x 2) and then dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated by distillation under reduced pressure. The residue obtained was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A-water (0.1% formic acid): B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to give the title product 10c (283 mg, yield: 55%) as a white solid.

[0736] MS m / z (ESI): 596.5 [M+1].

[0737] Third step

[0738] To a solution of 10c (129 mg, 0.21 mmol) in 1 mL of dichloromethane was added N-tert-butoxycarbonyl-1,3-propanediamine (292.84 mg, 1.68 mmol), 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (559.49 mg, 1.47 mmol) and N,N-diisopropylethylamine (190.18 mg, 1.47 mmol) and the reaction was allowed to proceed at 25 °C for 2 h. The reaction was concentrated under reduced pressure and the residue was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A - water (0.05% ammonium bicarbonate) : B - acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to afford the title product 10d (60 mg, yield: 24%) as a white solid.

[0739] MS m / z (ESI): 1065.1 [M+1].

[0740] Fourth step

[0741] To a solution of 10d (50 mg, 42.31 pmol) in 0.5 mL of methanol was added 5 mL of hydrochloric acid in ethyl acetate solution and the reaction was allowed to proceed at 25 °C for 1 h. The reaction was concentrated under reduced pressure and the residue was used as such without purification to afford the crude title product 10e (50 mg) as a colorless oil. The product was used as such without purification for the next reaction.

[0742] MS m / z (ESI): 764.8 [M+1].

[0743] Fifth step

[0744] To a solution of 10e (50 mg, 65.48 pmol) in 1 mL of dichloromethane was added N,N-diisopropylethylamine (59.25 mg, 0.46 mmol) and the reaction was monitored for 2 min until the pH of the reaction was equal to 8. Then 1e (117.14 mg, 0.26 mmol) and 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (224.11 mg, 0.59 mmol) were added and the reaction was allowed to proceed at 25 °C for 2 h. The reaction was concentrated under reduced pressure and the residue was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A - water (0.05% ammonium bicarbonate) : B - acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to afford the title product 10f (56 mg, yield: 42%) as a white solid.

[0745] MS m / z (ESI): 1027.4 [1 / 2 M+1].

[0746] Sixth step

[0747] Dissolve 10f (50 mg, 23.96 pmol) in 2 mL of methanol, add one drop of acetic acid, add wet palladium on carbon (50 mg, 10%), replace with hydrogen gas three times, stir the reaction under hydrogen gas (15 Psi) at 25 °C for 1 hour. Filter the reaction over celite, concentrate the filtrate by distillation under reduced pressure, purify the obtained residue by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A - water (0.1% formic acid): B - acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to obtain the title product 10g (30 mg, yield: 66%) as a white solid.

[0748] MS m / z (ESI): 937.2 [1 / 2 M+1].

[0749] Seventh step

[0750] Dissolve 10g (30 mg, 16.03 pmol) in 1 mL of N,N-dimethylformamide, add 1h (15.19 mg, 24.04 pmol), 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (13.72 mg, 24.04 pmol) and N,N-diisopropylethylamine (13.99 mg, 48,08 pmol), react at 25 °C for 2 hours. Concentrate the reaction under reduced pressure by distillation, purify the obtained residue by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 20% - 80%), to obtain the title product 10h (29 mg, yield: 73%) as a white solid.

[0751] MS m / z (ESI): 1244.6 [1 / 2 M+1].

[0752] Eighth step

[0753] Compound 10 was prepared according to the procedure described in Example 1-10, using 10h (29 mg, 11.67 pmol) dissolved in 1 mL of dichloromethane, nitrogen protection, addition of 4-dimethylaminopyridine (0.29 mg, 2.33 pmol), succinic anhydride (5.84 mg, 58.33 pmol) and N,N-diisopropylethylamine (15.09 mg, 116.66 pmol), stirring at 25 °C for 36 hours. The reaction solution was concentrated by distillation under reduced pressure, and the obtained residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A-water (0.05% ammonium bicarbonate):B-acetonitrile, gradient elution: B%: 20%-80%), to obtain the title product 10 (15 mg, yield: 49%) as a white solid.

[0754] MS m / z (ESI): 1294.1 [1 / 2 M+1].

[0755] 1H NMR (400 MHz, CDC13) d 7.41 - 7.40 (m, 1H), 7.32 - 7.30 (m, 2H), 7.29 - 7.28 (m, 2H), 7.20 - 7.18 (m, 2H), 7.17 - 7.16 (m, 2H), 6.86 - 6.85 (m, 2H), 6.84 - 6.82 (m, 2H), 5.38 - 5.36 (m, 3H), 5.27 - 5.21 (m, 4H), 4.68 - 4.65 (m, 3H), 4.22 - 4.16 (m, 4H), 4.15 - 4.09 (m, 4H), 4.09 - 4.01 (m, 4H), 4.00 - 3.89 (m, 8H), 3.81 - 3.80 (m, 6H), 3.79 - 3.77 (m, 4H), 3.74 - 3.66 (m, 4H), 3.55 - 3.48 (m, 10H), 3.33 - 3.25 (m, 12H), 2.66 - 2.58 (m, 5H), 2.30 - 2.28 (m, 2H), 2.27 - 2.24 (m, 4H), 2.19 - 2.18 (m, 2H), 2.17 - 2.15 (m, 9H), 2.12 - 2.09 (m, 2H), 2.07 - 2.05 (m, 9H), 2.02 - 2.00 (m, 9H), 1.98 - 1.95 (m, 9H), 1.69 - 1.59 (m, 20H), 1.29 - 1.21 (m, 18H), 0.71 - 0.67 (m, 6H).

[0756] Example 1-11: Preparation of compound 11

[0757] First step

[0758] Dissolve 11a (0.92 g, 9.2 mmol, prepared using the method disclosed in patent application “WO2019154261 A1” page 40 example 16) in 30 mL dichloromethane, add 5-pentenolide (0.92 g, 4.6 mmol), stir at 25 °C for 12 h. Concentrate the reaction mixture by distillation under reduced pressure, the obtained residue was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to afford the title product 11b (900 mg, yield: 64%) as a white solid.

[0759] MS m / z (ESI): 301.2 [M+1].

[0760] Second step

[0761] Dissolve 11b (350 mg, 1.05 mmol) in 20 mL methanolic hydrochloric acid, stir at 25 °C for 2 h. Concentrate the reaction mixture by distillation under reduced pressure, the obtained residue was used as crude without purification to afford the title product 11c (239 mg) as a white solid, the product was used directly in the next step without purification.

[0762] MS m / z (ESI): 201.1 [M+1].

[0763] Third step

[0764] To a solution of 11c (133 mg, 0.53 mmol) and 11d (700 mg, 2.12 mmol, prepared using the method disclosed in patent application "WO2009082607 A2" page 96 example 1) in 5 mL of 1,2-dichloroethane, 4A molecular sieves (140 mg) were added, it was stirred for 0.5 h at 25 °C under nitrogen atmosphere, trimethylsilyl trifluoromethanesulfonate (118.11 mg, 0.53 mmol) was added, it was stirred for 12 h at 25 °C, the reaction mixture was filtered, to the filtrate 20 mL of saturated sodium bicarbonate solution was added, the reaction mixture was extracted with dichloromethane (50 mL x 3), the organic phase was washed with saturated sodium chloride solution (60 mL x 2), then dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure, the residue was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A-water (0.1% formic acid): B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to afford the title product 11e (140 mg, yield: 40%) as a yellow solid.

[0765] MS m / z (ESI): 530.5 [M+1].

[0766] Fourth step

[0767] To a solution of 11e (140 mg, 0.21 mmol) and 1b (35.61 mg, 0.06 mmol, prepared using the method disclosed in patent application "CN106255755 A" page 182 example 4) in 5 mL of dichloromethane, 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (114.81 mg, 0.3 mmol) and N,N-diisopropylethylamine (187.07 mg, 1.45 mmol) were added, it was reacted for 2 h at 25 °C. The reaction mixture was concentrated under reduced pressure, the residue was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A-water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to afford the title product 11f (48 mg, yield: 36%) as a white solid.

[0768] MS m / z (ESI): 1004.6 [1 / 2 M+1].

[0769] Fifth step

[0770] To a solution of 11f (20 mg, 9.98 pmol) in 0.5 mL of methanol and 0.5 mL of ethyl acetate, one drop of acetic acid was added, wet palladium on carbon (20 mg, 10%) was added, hydrogen gas was purged three times and the reaction was stirred at 25 °C under hydrogen gas (15 Psi) for 1 h. The reaction was filtered over celite and the filtrate was concentrated by distillation under reduced pressure. The residue was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB CI 8 20-40 pm; mobile phase: A - water (0.1% formic acid) : B - acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to give the title product 11g (10 mg, yield: 53%) as a white solid.

[0771] MS m / z (ESI): 937.2 [1 / 2 M+1].

[0772] Sixth step

[0773] To a solution of 11g (10 mg, 5.34 pmol) in 1 mL of N,N-dimethylformamide, 1h (5.06 mg, 8.01 pmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.05 mg, 8.02 pmol) and N,N-diisopropylethylamine (2.07 mg, 16.02 pmol) were added and the reaction was stirred at 25 °C for 2 h. The reaction was concentrated by distillation under reduced pressure and the residue was purified by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate) : B - acetonitrile, gradient elution: B%: 20% - 80%) to give the title product 11h (10 mg, yield: 75%) as a white solid.

[0774] MS m / z (ESI): 2183.7 [M-301].

[0775] Seventh step

[0776] Dissolve 11h (10 mg, 3.98 pmol) in 1 mL of dichloromethane, protect with nitrogen, add 4-dimethylaminopyridine (0.1 mg, 0.80 pmol), succinic anhydride (2.01 mg, 20.09 pmol) and N,N-diisopropylethylamine (5.2 mg, 40.23 pmol), stir at 25 °C for 36 h. Concentrate the reaction solution by distillation under reduced pressure, purify the obtained residue by preparative high performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 20% - 65%) to obtain the title product 11 (4 mg, yield: 34%) as a white solid.

[0777] MS m / z (ESI): 2284.4 [M-301].

[0778] 1H NMR (400 MHz, CDC13) δ 7.41 - 7.40 (m, 1H), 7.32 - 7.31 (m, 1H), 7.30 - 7.30 (m, 1H), 7.29 - 7.28 (m, 2H), 7.20 - 7.18 (m, 2H), 7.18 - 7.16 (m, 2H), 6.86 - 6.84 (m, 2H), 6.84 - 6.82 (m, 2H), 5.38 - 5.35 (m, 3H), 5.26 - 5.21 (m, 3H), 4.69 - 4.64 (m, 3H), 4.21 - 4.08 (m, 8H), 4.07 - 4.01 (m, 3H), 3.99 - 3.92 (m, 6H), 3.82 - 3.80 (m, 6H), 3.79 - 3.77 (m, 3H), 3.73 - 3.69 (m, 2H), 3.61 - 3.56 (m, 2H), 3.54 - 3.51 (m, 6H), 3.50 - 3.47 (m, 2H), 3.32 - 3.25 (m, 10H), 2.66 - 2.58 (m, 4H), 2.31 - 2.23 (m, 8H), 2.20 - 2.18 (m, 2H), 2.18 - 2.14 (m, 9H), 2.12 - 2.09 (m, 2H), 2.07 - 2.04 (m, 9H), 2.02 - 2.00 (m, 9H), 1.98 - 1.95 (m, 9H), 1.86 - 1.75 (m, 10H), 1.69 - 1.55 (m, 22H), 1.28 - 1.22 (m, 14H), 0.71 - 0.66 (m, 6H).

[0779] Example 1-12: Preparation of compound 12

[0780] First Step

[0781] To a solution of 1b (200 mg, 0.41 mmol) and tert-butyl N-[1-(2- aminoethyl)cyclopropyl]carbamate (347.25 mg, 1.65 mmol) in 8 mL of dichloromethane was added 2-(7-azobenzotriazol)-N,N,N',N'- tetramethyluronium hexafluorophosphate (635.91 mg, 1.65 mmol) and N,N- diisopropylethylamine (1.06 g, 8.24 mmol) and the reaction mixture was stirred at 25 °C for 2 h. The reaction mixture was added to 10 mL of water and the reaction mixture was extracted with dichloromethane (20 mL x 3), the organic phase was washed with saturated sodium chloride solution (50 mL x 2) and then dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated by distillation under reduced pressure, the residue obtained was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 μm; mobile phase: A- water (0.05% ammonium bicarbonate): B-acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to give the title product 12a (310 mg, yield: 76%) as a white solid.

[0782] MS m / z (ESI): 1019.0 [M+1].

[0783] Second Step

[0784] To a solution of 12a (320 mg, 0.31 mmol) in 10 mL of methanolic hydrochloric acid was stirred at 25 °C for 2 h. The reaction mixture was concentrated by distillation under reduced pressure and the residue obtained was used as such without purification to give the crude title product 12b (500 mg) as a yellow oil, which was used as such without purification for the next step.

[0785] MS m / z (ESI): 718.9 [M+1].

[0786] Third Step

[0787] To a solution of 12b (165 mg, 0.19 mmol) and 1e (350 mg, 0.76 mmol) in 5 mL of dichloromethane was added 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (360.67 mg, 0.95 mmol) and N,N-diisopropylethylamine (587.7 mg, 4.56 mmol) and the reaction was stirred at 25 °C for 2 h. The reaction was added to 10 mL of water and the reaction was extracted with dichloromethane (20 mL x 3), the organic phase was washed with saturated sodium chloride solution (50 mL x 2) and then dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated by distillation under reduced pressure, the residue obtained was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A- water (0.05% ammonium bicarbonate): B- acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to obtain the title product 12c (197 mg, yield: 46%) as a white solid.

[0788] MS m / z (ESI): 1004.3 [1 / 2 M+1].

[0789] Fourth step

[0790] To a solution of 12c (50 mg, 23.93 pmol) in 0.5 mL of methanol and 0.5 mL of ethyl acetate was added one drop of acetic acid, palladium on carbon (50 mg, 10%) was added and the reaction was purged with hydrogen gas three times, the reaction was stirred under hydrogen gas (15 Psi) at 25 °C for 1 h. The reaction was filtered through celite and the filtrate was concentrated by distillation under reduced pressure, the residue obtained was purified by reverse phase liquid chromatography (separation conditions: column: 40 g Flash Coulmn Welch Ultimate XB_C18 20-40 pm; mobile phase: A- water (0.1% formic acid): B- acetonitrile, gradient elution, flow rate: 30 mL / min, instrument: Biotage) to obtain the title product 12d (40 mg, yield: 80%) as a white solid.

[0791] MS m / z (ESI): 937.2 [1 / 2 M+1].

[0792] Fifth step

[0793] Dissolve 12d (20 mg, 10.68 pmol) in 1 mL of dichloromethane, add 1h (6.75 mg, 10.68 pmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (6.09 mg, 16.02 pmol) and N,N-diisopropylethylamine (6.89 mg, 53.31 pmol), and react at 25 °C for 2 hours. Concentrate the reaction solution by distillation under reduced pressure, and purify the obtained residue by preparative high-performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 20% - 70%) to obtain the title product 12e (13 mg, yield: 44%) as a white solid.

[0794] Sixth step

[0795] Dissolve 12e (10 mg, 4.02 pmol) in 1 mL of dichloromethane, add 4-dimethylaminopyridine (0.1 mg, 0.81 pmol), succinic anhydride (2.01 mg, 20.1 pmol) and N,N-diisopropylethylamine (5.2 mg, 40.23 pmol), and stir at 25 °C for 36 hours. Concentrate the reaction solution by distillation under reduced pressure, and purify the obtained residue by preparative high-performance liquid chromatography (separation conditions: column: InfinityLab Poroshell 120 SB-C18 21.2 x 250 mm, 4 pm; mobile phase: A - water (0.05% ammonium bicarbonate): B - acetonitrile, gradient elution: B%: 20% - 60%) to obtain the title product 12 (5.2 mg, yield: 41%) as a white solid.

[0796] MS m / z (ESI): 1142.8 [1 / 2 (M-301)].

[0797] 1H NMR (400 MHz, CD3OD) δ 7.42-7.38 (m, 2H), 7.34-7.31 (m, 1H), 7.30-7.26 (m, 5H), 7.25-7.20 (m, 1H), 6.91-6.85 (m, 4H), 5.59-5.48 (m, 2H), 5.37-5.34 (m, 3H), 5.10-5.06 (m, 3H), 4.65-4.60 (m, 2H), 4.59-4.55 (m, 3H), 4.36-4.28 (m, 2H), 4.20-4.07 (m, 10H), 4.06-3.99 (m, 4H), 3.96-3.86 (m, 5H), 3.82-3.79 (m, 6H), 3.72-3.66 (m, 12H), 3.64-3.49 (m, 8H), 2.59-2.53 (m, 4H), 2.46-2.41 (m, 6H), 2.40-2.33 (m, 3H), 2.22-2.16 (m, 6H), 2.16-2.15 (m, 9H), 2.15-2.14 (m, 2H), 2.05-2.02 (m, 9H), 1.99-1.92 (m, 18H), 1.75-1.56 (m, 22H), 1.35-1.29 (m, 10H), 0.76-0.66 (m, 12H).

[0798] Example 1-13: Preparation of compounds E1-1 and E1-2

[0799] First step

[0800] Dissolve 1b (3.25 g, 10.35 mmol) in dry dimethyl sulfoxide (20 mL), and add sodium hydride (410 mg, 10.35 mmol, 60%) under nitrogen atmosphere. After addition, stir the reaction at 25 °C for 0.5 h. Then dissolve 1a (1.7 g, 3.45 mmol, prepared by the method disclosed in Example 2, page 84 of the patent application “WO 2017 / 214112 Al”) in dry dimethyl sulfoxide (10 mL), and add dropwise into the reaction. Stir the resulting mixture at 25 °C under nitrogen atmosphere for 1 h. Quench the reaction by slowly adding saturated aqueous ammonium chloride solution (30 mL) into the reaction, then add water (160 mL), and extract with ethyl acetate (200 mL x 2). Wash the combined organic phase with saturated brine (200 mL), dry the organic phase over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure. Purify the resulting residue by silica gel chromatography column (mobile phase: A-petroleum ether, B-ethyl acetate; gradient elution: B%: 20%-80%), to obtain the title product 1c (701 mg, yield: 33%) as a yellow solid.

[0801] MS m / z (ESI): 561.4 [M+1].

[0802] Second step

[0803] The 1c (701 mg, 1.57 mmol) was dissolved in a mixture solution of tetrahydrofuran (15 mL) and water (15 mL), and cooled to below 5°C in an ice bath, then potassium peroxymonosulfate (1641 mg, 2.67 mmol) was added, after addition, it was naturally raised to 25°C and stirred for 2 hours. The reaction solution was poured into water (50 mL), then extracted with ethyl acetate (60 mL x 2), the organic phase was combined and washed with saturated brine (60 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by C18 reverse phase column (mobile phase: A-water (10 mmol / L ammonium bicarbonate), B-acetonitrile; gradient elution: B%: 20%-90%), to obtain the title product 1d (405 mg, yield: 46%) in the form of white solid.

[0804] MS m / z (ESI): 545.4 [M+1].

[0805] Third step

[0806] The 1d (405 mg, 0.74 mmol) was dissolved in a mixture solution of formic acid (3 mL), water (2 mL) and acetonitrile (2 mL), and stirred at 25°C for 15 hours. The reaction solution was directly purified by Prep-HPLC preparation (column: InfinityLab Poroshell 120SB-C18 21.2x150mm, 4um, mobile phase: A-water (0.1% TFA), B-acetonitrile, gradient elution, B%: 5%-40%), to obtain a pair of isomers of the title product in the form of white solid.

[0807] Single configuration compound 1e-1 (115 mg, 35%).

[0808] HPLC analysis: retention time 13.626 minutes. (column: Agilent ZORBAX BONUS RP, 4.6*150mm, 3.5um, mobile phase: A-water (0.05% trifluoroacetic acid), B-acetonitrile, gradient elution: B%: 5%-35%, 20min, flow rate: 1mL / min, instrument: Agilent 1260).

[0809] MS m / z (ESI): 431.3 [M+1].

[0810] 1H NMR (400 MHz, Chloroform-d) δ 9.45 (s, 1H), 7.48 (d, J = 8.1 Hz, 1H), 5.87 (d, J = 1.9 Hz, 1H), 5.79 (d, J = 8.1 Hz, 1H), 4.17 - 4.03 (m, 4H), 3.98 - 3.92 (m, 1H), 3.82 - 3.75 (m, 1H), 3.63 - 3.55 (m, 4H), 2.03 - 1.94 (m, 1H), 1.45 - 1.37 (m, 1H), 1.37 - 1.26 (m, 6H), 1.16 - 1.01 (m, 3H), 0.98 - 0.88 (m, 1H). 31 P NMR (400 MHz, Chloroform-d) δ 27.29.

[0811] Single configuration compound 1e-2 (173 mg, 53%).

[0812] HPLC analysis: Retention time 14.179 min. (Chromatography column: Agilent ZORBAX BONUS RP, 4.6*150 mm, 3.5 μm, mobile phase: A - water (0.05% trifluoroacetic acid), B - acetonitrile, gradient elution: B%: 5% - 35%, 20 min, flow rate: 1 mL / min, instrument: Agilent 1260).

[0813] MS m / z (ESI): 431.3 [M+1].

[0814] 1 H NMR (400 MHz, Chloroform-d) δ 9.96 (s, 1H), 7.39 (d, J = 8.1 Hz, 1H), 5.85 (d, J = 1.9 Hz, 1H), 5.79 (d, J = 8.1 Hz, 1H), 4.24 - 4.02 (m, 5H), 3.83 - 3.75 (m, 1H), 3.60 (s, 3H), 3.59 - 3.51 (m, 1H), 2.06 - 1.92 (m, 1H), 1.52 - 1.42 (m, 1H), 1.41 - 1.26 (m, 6H), 1.18 - 0.99 (m, 3H), 0.94 - 0.82 (m, 1H). 31 P NMR (400 MHz, Chloroform-d) δ 27.60.

[0815] Fourth step

[0816] To a solution of 1e-1 (115 mg, 0.27 mmol) in dry acetonitrile (5 mL) was added 4,5-dicyanoimidazole (31.56 mg, 0.27 mmol) and 1f (241.61 mg, 0.80 mmol) under nitrogen atmosphere. After addition, the reaction mixture was stirred at 25 °C for 1 h. The reaction mixture was diluted with dichloromethane (20 mL) and washed with aqueous sodium bicarbonate solution (0.5 mmol / mL, 10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residue was purified by Prep-HPLC (column: InfinityLab Poroshell 120 SB-C18 21.2 x 150 mm, 4 um, mobile phase: A- water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution, B%: 20-80%) to give the title product E1-1 (105 mg, 60%) as a white solid.

[0817] MS m / z (ESI): 631.3 [M+1].

[0818] 1 H NMR (400 MHz, Chloroform-d) δ 7.53 - 7.45 (m, 1H), 5.91 - 5.83 (m, 1H), 5.78 - 5.73 (m, 1H), 4.20 - 4.00 (m, 4H), 4.00 - 3.80 (m, 3H), 3.79 - 3.48 (m, 6H), 2.67 - 2.60 (m, 2H), 2.01 - 1.87 (m, 1H), 1.49 - 1.38 (m, 2H), 1.35 - 1.27 (m, 7H), 1.22 - 1.13 (m, 12H), 1.13 - 0.88 (m, 3H). 31 P NMR (400 MHz, Chloroform-d) δ

[0819] 149.66, 149.63, 27.23, 27.04.

[0820] Fifth step

[0821] To a solution of 1e-2 (173 mg, 0.40 mmol) in dry acetonitrile (7 mL) was added 4,5-dicyanoimidazole (47.47 mg, 0.40 mmol) and 1f (363.47 mg, 1.21 mmol) under nitrogen atmosphere. After addition, the reaction was stirred at 25 °C for 1 h. The reaction mixture was diluted with dichloromethane (30 mL) and washed with aqueous sodium bicarbonate solution (0.5 mmol / mL, 15 mL), then dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The obtained residue was purified by Prep-HPLC (column: InfinityLab Poroshell 120 SB-C18 21.2 x 150 mm, 4 um, mobile phase: A- water (10 mmol / L ammonium bicarbonate), B-acetonitrile, gradient elution, B%: 20-80%) to give the title product E1-2 (166 mg, 63%) as a white solid.

[0822] MS m / z (ESI): 631.3 [M+1].

[0823] 1 H NMR (400 MHz, Chloroform-d) δ 7.48 - 7.34 (m, 1H), 6.02 - 5.92 (m, 1H), 5.82 - 5.70 (m, 1H), 4.33 - 4.24 (m, 1H), 4.20 - 4.12 (m, 2H), 4.12 - 4.02 (m, 2H), 3.99 - 3.62 (m, 6H), 3.49 (d, J = 4.2 Hz, 3H), 2.73 - 2.61 (m, 2H), 2.11 - 1.98 (m, 1H), 1.45 - 1.34 (m, 3H), 1.34 - 1.25 (m, 5H), 1.25 - 1.15 (m, 12H), 1.14 - 0.83 (m, 4H). 31 P NMR (400 MHz, Chloroform-d) δ 150.16, 150.09, 27.74, 27.35.

[0824] Example 1-14: Preparation of compounds E2-1 and E2-2

[0825] E2-1 and E2-2 were prepared according to the procedure described in Reference Example 1-13.

[0826] Example 1-15: Preparation of compounds E3-1 and E3-2

[0827] E3-1 and E3-2 were prepared according to the procedure described in Reference Example 1-13.

[0828] Example 2: Preparation of SiRNA

[0829] The siRNAs of the application are prepared using the solid phase phosphoramidite method well known in the art. The specific method is outlined below.

[0830] 1. Synthesis of sense strand (SS strand)

[0831] The solid phase phosphoramidite synthesis method is used to link the nucleoside monomers one by one in the order of the nucleotide sequence of the sense strand from 3'-5' direction, using blank CPG solid phase carrier or GalNAc-attached solid phase carrier as the starting cycle. Each nucleoside monomer linkage includes four steps of deprotection, coupling, capping, oxidation or thiolation. The synthesis scale is 0.5 umol of oligonucleic acid, and the synthesis conditions are as follows:

[0832] The nucleoside monomers are provided in 0.05 mol / L acetonitrile solution, and each step of the reaction is under the same conditions, i.e. temperature of 25°C, 3% trichloroacetic acid-dichloromethane solution for deprotection, 4 times of deprotection; 0.30 mol / L 5-BTT-acetonitrile solution for coupling reaction, 3 times of coupling; 20% acetic anhydride-acetonitrile and 15% N-methylimidazole-acetonitrile for capping, 2 times of capping; 0.05 mol / L iodine-tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v) for oxidation, 2 times of oxidation; and 0.05 mol / L DTTT acetonitrile / pyridine (1 / 1, v / v) for thiolation, 3 times of thiolation.

[0833] 2. Synthesis of antisense strand (AS strand)

[0834] The solid phase phosphoramidite synthesis method is used to link the nucleoside monomers or the nucleotide dimers of the application one by one in the order of the nucleotide sequence of the antisense strand from 3'-5' direction, using blank CPG solid phase carrier as the starting cycle. Each nucleoside monomer linkage includes four steps of deprotection, coupling, capping, oxidation or thiolation. The synthesis scale is 0.5 umol of oligonucleic acid, and the synthesis conditions are the same as those of the sense strand.

[0835] 3. Hydrolysis of 5'-phosphonate-modified nucleotides

[0836] The synthesized solid phase carrier with 5'-phosphonate-modified nucleotides is added to a 3 mL centrifuge tube, and a mixture of trimethylsilyl iodide / pyridine / acetonitrile (1 mL, 1:13:40, v / v / v) is added. The reaction is carried out at 25°C in a constant temperature water bath for 1 hour, filtered, and the solid phase carrier is washed with acetonitrile / water (1 / 1, v / v) three times, 2 mL each time, and then collected.

[0837] 4. Aminolysis

[0838] The synthesized solid support (sense strand or antisense strand) was added to a 3 mL centrifuge tube, and ammonia / ethanol (0.5 mL, 3 / 1, v / v) was added. The reaction was performed in a 45 °C constant temperature water bath for 16 hours. After filtration, the filtrate was concentrated by centrifugation, and the crude product was purified.

[0839] 5. Purification

[0840] The methods of purification and desalting are well known to those skilled in the art. For example, a strong anion filler column can be used for purification, and a sodium chloride-sodium hydroxide system can be used for elution. The product can be collected and can be desalted using a gel filler purification column, and a pure water elution system.

[0841] 6. Annealing

[0842] The sense strand (SS strand) and the antisense strand (AS strand) were mixed in a molar ratio (SS strand / AS strand = 1 / 1) according to the following table, and a water bath was heated to 70-95 °C for 3-5 min. The system was naturally cooled to room temperature, and the product was obtained by lyophilization.

[0843] Table 1. The siRNA sequences used in the present application are as follows:

[0844] In this document, the meanings of the abbreviations are as follows:

[0845] A, U, G, and C represent natural adenine ribonucleotides, uracil ribonucleotides, guanine ribonucleotides, and cytosine ribonucleotides, respectively.

[0846] m represents that the nucleotide adjacent to the left thereof is a 2'-OCH3-modified nucleotide. For example, Am, Um, Gm, and Cm represent 2'-OCH3-modified A, U, G, and C, respectively.

[0847] f represents that the nucleotide adjacent to the left thereof is a 2'-F-modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-F-modified A, U, G, and C, respectively.

[0848] "s" or "s-" represents that the two nucleotides adjacent to the left and right thereof and / or the delivery carrier are connected by a phosphorothioate linkage.

[0849] L125 represents the structure formed after the deprotection of the GalNAc delivery carrier of Compound 2 described above in the siRNA. represents the position of connection to the siRNA by a phosphate group or a phosphorothioate group.

[0850] L96 represents the GalNAc delivery carrier of the following structure well known in the art, wherein denotes the position of attachment to the siRNA via a phosphonate or phosphorothioate group. See, e.g., PCT Publication Nos. WO2009073809A2 and WO2009082607A2.

[0851] SPP1a-Um, SPP1b-Um, respectively, denote nucleotide surrogates prepared from compounds E1-1 and E1-2, respectively, as described above, wherein denotes the position of attachment to the other nucleotide via a phosphonate or phosphorothioate group.

[0852] VP denotes that the nucleotide adjacent to the right of VP is a vinyl phosphonate modified nucleotide, which is well known in the art. See, e.g., PCT Publication Nos. WO2011139702A2, WO2013033230A1, and WO2019105419A1. In particular, VP-Um denotes the following structure, wherein, denotes the position of attachment to the other nucleotide via a phosphonate or phosphorothioate group.

[0853] Example 3 Testing of different siRNAs for inhibition activity of ApoB mRNA in Hep3b cells

[0854] The effect of siRNAs targeting apolipoprotein ApoB on the mRNA expression level of apolipoprotein ApoB was tested in vitro. The mRNA inhibition effect of different siRNAs on apolipoprotein ApoB was determined; for each siRNA, the concentrations determined were 10.0, 2.0, 0.4, 0.08, 0.016, 0.032, and 0.00064 nM, a total of 7 concentrations, 5-fold dilution, 2 biological replicates.

[0855] 1) Cell culture and plating: Hep3b cells were cultured in MEM medium containing 10% fetal bovine serum at 37°C, 5% CO2. 24 hours before transfection, Hep3b cells were collected and seeded in a 96-well plate at a density of 20,000 cells per well in 100 μL of culture medium.

[0856] 2) Transfection: siRNA was transfected using lipofectamine RNAiMAX (ThermoFisher, 13778150) according to the product manual. The final concentration of siRNA transfection was 10 nM, three replicate wells.

[0857] 3) RNA extraction: Continue culturing cells for 24 hours, and use Thermo Fisher's automated nucleic acid extractor in conjunction with Tiangen's magnetic bead-based tissue / cell / blood total RNA extraction kit (DP761) to extract RNA. Refer to the product instruction manual for specific steps.

[0858] 4) RNA reverse transcription to cDNA: using Novizan III. Reverse transcription was performed using the RT SuperMix for qPCR (+gDNA wiper) kit. A 10 μL reverse transcription reaction mixture was prepared according to the kit instructions to reverse transcribe the total RNA from the cells. The transcribed cDNA was then diluted 3-fold with 20 μL of DEPC water. The sample was used for subsequent experiments or stored at -20°C.

[0859] 5) qPCR: For each reverse transcription reaction system, take 4 μL of the diluted cDNA solution as a template and prepare 10 μL of qPCR reaction system using the reagents provided by the Novizan AceQ Universal U+Probe Master Mix V2 kit. The PCR primer sequences for amplifying the target gene ApoB and the internal reference gene GAPDH are shown in Table 2, and the TaqMan probe sequences for amplifying the target gene ApoB and the internal reference gene GAPDH are shown in Table 3. The final concentration of each primer and probe is 10 μM. The Ct values ​​of the target gene ApoB and the internal reference gene GAPDH are obtained using a real-time quantitative PCR system based on the TaqMan method.

[0860] Table 2. PCR primer sequences

[0861] Table 3. Taqman probe sequences used in real-time quantitative PCR

[0862] 6-FAM and VIC represent fluorescent groups, and BHQ1 represents a quenching group.

[0863] The relative quantitative calculation of the expression level of the target gene ApoB in each test group and control group was performed using the Ct (ΔΔCt) method. The calculation method is as follows:

[0864] ΔCt(test group) = Ct(target gene in test group) - ΔCt(internal reference gene in test group)

[0865] ΔCt(control group) = Ct(target gene in control group) - ΔCt(internal reference gene in control group)

[0866] ΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0867] ACt(control) = Ct(control) - Ct(control average)

[0868] wherein, ACt(control average) is the arithmetic mean of the respective ACt(control) of the three culture wells of the control group. Thus, each culture well of the test group and the control group corresponds to a value of ACt. The relative expression level of ApoB mRNA of the test group = 2-ACt(test group) x 100%.

[0869] Table 4. Inhibition activity of different siRNA on ApoB mRNA in Hep3b cells

[0870] The test results are recorded in Table 4 and Figure 1. The results show that the inhibition activity of ApoB-1-SPP1a and ApoB-1-SPP1b is significantly improved compared with ApoB-1.

[0871] Example 4: Pharmacodynamic verification of siRNA compounds of the present disclosure in mice

[0872] C57BL / 6 mice (male, 6-8 weeks) were bled to detect the expression amount of LDL 3 days before administration (counted as day -3), and then were grouped in a snake shape, with 4 mice in each group; after grouping was completed, the mice were administered once at day 0 (administration) by subcutaneous injection, and were administered with siRNA conjugates (compound information is shown in Table 5) at a dose of 10 mg / kg, with the administration volume being 100 μL.

[0873] The mice were bled to detect the expression amount of LDL at day 7, day 14, day 21 and day 35 after administration, respectively. Then, the serum LDL concentration was detected by using a Mindray biochemical instrument direct method (automatic biochemical instrument, model BS-240VET). The experimental results are shown in Table 6 and Figure 2.

[0874] Table 5: Compound information

[0875] Table 6. Pharmacodynamic of different siRNA compounds in C57BL / 6 mouse models

[0876] As can be seen from the results of Table 6 and Figure 2, in the mouse models of the present experiment, the siRNA conjugates of the present disclosure, the sequence modified by SPP1b has a better inhibition effect on LDL than the unmodified parent sequence and the sequence modified by VP.

[0877] Example 5: Pharmacodynamic verification of siRNA compounds of the present disclosure in mice

[0878] The humanized FXI mice used in this example were purchased from Jiangsu Jizu Pharmaceutical Biotechnology Co., Ltd., hereinafter referred to as TG mice. The purchased TG mouse strain was BALB / c-hF11, all were ♂ male mice, 6-8 weeks old. The expression amount of human FXI of all mice was determined on the day of administration (recorded as day 0), then the human hFXI protein content in the plasma of the above-mentioned mice was tested using Factor XI Human ELISA Kit kit (Abeam Company, item number ab108834), then the mice were divided into groups in a snake shape, 4 mice per group; after grouping was completed, the mice were administered once by subcutaneous injection, and the siRNA conjugate was administered at a dose of 3 mg / kg (compound information is shown in Table 7).

[0879] The siRNA conjugate was provided in the form of 0.9% saline solution for injection at 5 mg / mL of siRNA conjugate (0.9% sodium chloride solution for injection was used to configure the required solution concentration and volume before the experiment), and the administration volume was 5 mL / kg.

[0880] One group of mice was administered 0.9% saline solution, and the administration volume was 5 mL / kg, as a control group. The mice were bled from the orbital venous plexus on days 7, 14, 21, and 29 after administration, and the expression level of FXI protein in the plasma of the mice was detected at each time point by ELISA kit (Abeam Company, item number ab108834), and the ELISA detection results are shown in Table 8 and Figure 3.

[0881] Table 7: Compound information

[0882] Table 8. Pharmacodynamics of different siRNA compounds in TG mouse model

[0883] As can be seen from the results of Table 8 and Figure 3, the siRNA conjugate of the present disclosure can significantly improve the effect of inhibiting the expression of FXI protein in the TG mouse model, and the SPP1a and SPP1b modified sequences (dsRNA-F2, dsRNA-F3, dsRNA-F5 and dsRNA-F6) are compared with the unmodified parent sequence (dsRNA-F1 and dsRNA-F4).

[0884] Example 6: Stability study

[0885] The preparation method of Example 2 was used to prepare the siRNA single strands shown in Table 9 below, respectively. The crude single strands prepared before purification after the 4th step of ammonolysis were taken, respectively, and the IPRP method (chromatographic column: Agilent advanced bio oligonucleotide 2.1*50mm, 2.7um; mobile phase: A-10mM N,N-dimethylbutylamine + 100mM hexafluoroisopropanol, B-70% acetonitrile, gradient elution, B%: 10%~90%) was used to detect the purity in order to investigate the effect of different modifications on the stability of siRNA. The detection results are shown in Table 9 below.

[0886] Table 9. Single strand sequence information

[0887] The results show that the IPRP purity of the crude product prepared in the preparation process of the sequence modified by SPP1b is about 3%~18% higher than that of the sequence modified by VP, which indicates that the modification by SPP1b is more conducive to the stability of siRNA product.

[0888] Although the preferred embodiments of the present application have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided only as examples. Many variants, changes and substitutions will occur to those skilled in the art without departing from the present application. It should be understood that various alternatives to the embodiments described herein can be employed. The following claims are intended to define the scope of the present application and encompass the methods and structures within the scope of these claims and their equivalents.

Claims

An oligonucleotide or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof, the oligonucleotide comprising nucleotide monomers of the structure shown in Formula (I): wherein represents a single bond or a double bond; Ring A is selected from C 3-10 cycloalkylene, 3-10 membered heterocyclyl, C 6-10 arylene or 5-10 membered heteroarylene; Ring B is selected from C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl; each of ring A and ring B is optionally substituted with 1, 2, 3, 4, or 5 R*; R* is selected from H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1- 6haloalkoxy; R1is selected from O, S or NR a ; R2and R3are independently selected from OR b or SR c ; R a selected from H, C 1-6 alkyl or C 1-6 haloalkyl; R b and R c are independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 3-7 cycloalkyl or 3-7 membered heterocyclyl; X is selected from -0-, -S-, -NR d -, -CR e R f -, -CR e R f -CR e R f - or -CH=CH-; R d , R e , and R f are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; Y1, Y2, Y3and Y4are independently selected from H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-7 cycloalkyl or 3-7 membered heterocyclyl; Z is selected from H, halogen, OR j , NR k R l , C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or C 1-18 haloalkyl, preferably from H, halogen, OR j , NR k R l , C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl or C 1-10 haloalkyl, said Z being optionally further substituted with 1, 2 or 3 R#; R j , R k , and R l are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; R# is selected from H, halogen, OH, NH2, CN, C 1-6 alkyl, alkoxy or C 1-6 haloalkoxy; Q is selected from -0-, -S-, -NR g - or -CR h R i -; R g , R h , and R i are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; Base is selected from H, a modified or unmodified base; the compound of formula (I) is optionally further substituted with 1, 2, 3, 4, or 5 substituents selected from: H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy. The oligonucleotide or pharmaceutically acceptable salt, isotopolog, tautomer, or stereoisomer thereof of claim 1, wherein, The nucleotide monomer has the following structure: wherein m is selected from 0, 1, or 2; each variable is as defined in claim 1. The oligonucleotide or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof of claim 1 or 2, wherein, represents a single bond or a double bond; Ring A is selected from C 3-10 cycloalkylene, 3-10 membered heterocyclyl, C 6-10 arylene or 5-10 membered heteroarylene; Ring B is selected from C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl; each of ring A and ring B is optionally substituted with 1, 2, or 3 R*; R* is selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy; R1is selected from O, S or NR a ; R2and R3are independently selected from OR b or SR c ; R a selected from H, C 1-6 alkyl or C 1-6 haloalkyl; Each R b and R c Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; X is selected from -0-, -S-, -NR d - or -CR e R f -; R d , R e , and R f are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; Y1, Y2, Y3and Y4are independently selected from H, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy; Z is selected from H, halogen, OR j , C 1-6 alkyl or C 1-6 haloalkyl, said Z is optionally substituted with 1, 2 or 3 R#; R j selected from H, C 1-6 alkyl or C 1-6 haloalkyl; R# is selected from H, halogen, OH, C 1-6 alkoxy or C 1-6 haloalkoxy; Q is selected from -0-, -S-, -NR g - or -CR h R i -; R g , R h , and R i are independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl; Base is selected from H, a modified or unmodified base. The oligonucleotide or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof of any one of claims 1-3, wherein, represents a single bond or a double bond, preferably a single bond; Ring A is selected from C 3-7 cycloalkylene or 3-7 membered heterocyclylene; Ring B is selected from C 3-7 cycloalkyl or 3-7 membered heterocyclyl; each of ring A and ring B is optionally substituted with 1, 2, or 3 R*; R* is selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; R1is selected from O or S, preferably O; R2and R3are independently selected from OH, C 1-6 alkoxy or C 1-6 haloalkoxy; X is selected from -O-, -S-, -NH-, or -CH2-; Y1, Y2, Y3and Y4are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; Z is selected from C 1-6 alkyl, alkoxy or C 1-6 haloalkoxy, said Z is optionally substituted with 1 or 2 R#; R# is selected from H, halogen, OH, C 1-6 alkyl or C 1-6 haloalkoxy; Q is selected from -O-, -S-, -NH-, or -CH2-; Base is selected from H, a modified or unmodified base. The oligonucleotide or a pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof of any one of claims 1-4, wherein, represents a single bond or a double bond, preferably a single bond; Ring A is selected from C 3-5 Cycloalkylene or 3-5 membered heterocyclyl, preferably C 3-5 Cycloalkylene, for example cyclopropylene, cyclobutylene or cyclopentylene; Ring B is selected from C 3-5 cycloalkyl or 3-5 membered heterocyclyl, preferably C 3-5 cycloalkyl, for example cyclopropyl, cyclobutyl or cyclopentyl; Preferably, ring A and ring B together form the following structure: For example R1is selected from O or S, preferably O; R2and R3are independently selected from OH or C 1-4 alkoxy, for example OCH3or OCH2CH3; X is selected from -O-, -S-, or -CH2-, preferably O; Y1, Y2, Y3and Y4are independently selected from H or C 1-4 alkyl, preferably H; Z is C 1-4 alkoxy, said Z being optionally substituted by 1 or 2 R#; R# is selected from H, halo or C 1-4 alkoxy; Z is preferably -OCH3or -OCH2CH2OCH3; Q is selected from -O-, -S-, or -CH2-; Base is selected from H or a base, preferably The oligonucleotide or pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof of any one of claims 1-5, wherein, The oligonucleotide comprises a nucleotide monomer having the structure: wherein Ring B is selected from C 3-7 Cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-7 Cycloalkyl; each R is independently selected from H, C b is independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl; each R j is independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl, said R j optionally substituted with 1 or 2 R R# is selected from H, halo, C 1-6 alkyl or C 1-6 haloalkoxy; Base is selected from preferably, Ring B is selected from C 3-5 cycloalkyl or 3-5 membered heterocyclyl, preferably C 3-5 cycloalkyl, for example cyclopropyl, cyclobutyl or cyclopentyl; each R is independently selected from H or C b is independently selected from H or C 1-6 alkyl, for example CH3or CH2CH3; each R is independently selected from H or C1-6alkyl; j is independently selected from H or C1-6alkyl; 1-6 is independently selected from H or C1-6alkyl; j optionally substituted with 1 or 2 R#; R# is selected from H, halo or C 1-6 alkoxy; R j preferably selected from CH3or CH2CH2OCH3; Base is selected from The oligonucleotide or pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof of any one of claims 1-6, wherein, The oligonucleotide comprises a nucleotide monomer having the structure: wherein Base is selected from Preferably, the oligonucleotide comprises nucleotide monomers having the structure of: The oligonucleotide or pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof of any one of claims 1-6, wherein, The oligonucleotide comprises a nucleotide monomer having the structure: wherein Base is selected from Preferably, the oligonucleotide comprises nucleotide monomers having the structure of: The oligonucleotide or pharmaceutically acceptable salt, isotopically enriched variant, tautomer, or stereoisomer thereof of any one of claims 1-8, wherein, the oligonucleotide comprises a nucleotide monomer of any one of claims 1-8 at the 5’ terminus and / or the 3’ terminus; preferably, the oligonucleotide comprises a nucleotide monomer of any one of claims 1-8 at the 3’ terminus; preferably, the oligonucleotide comprises a nucleotide monomer of any one of claims 1-8 at the 5’ terminus; more preferably, the oligonucleotide comprises a nucleotide monomer of any one of claims 1-8 at both the 3’ terminus and the 5’ terminus. The oligonucleotide of any one of claims 1-9, wherein, The oligonucleotide is single-stranded, having 14 to 30 nucleotides. The oligonucleotide of any one of claims 1-10, wherein, The oligonucleotide is double-stranded RNA comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides. The oligonucleotide of claim 11, wherein, The double-stranded RNA comprises a nucleotide monomer of any one of claims 1-8 in the sense strand and / or the antisense strand; preferably, the double-stranded RNA comprises a nucleotide monomer of any one of claims 1-8 in the antisense strand; more preferably, the double-stranded RNA comprises a nucleotide monomer of any one of claims 1-8 in the 3’ terminus and / or the 5’ terminus of the antisense strand, more preferably in the 5’ terminus of the antisense strand. The oligonucleotide of claim 11 or 12, wherein the double stranded RNA comprises one or more targeting ligands or delivery vehicles; preferably, the delivery vehicle is selected from a polypeptide delivery vehicle or an antibody delivery vehicle; preferably, the targeting ligand is an N-acetylgalactosamine (GalNAc) targeting ligand. a compound of Formula (I’), or a pharmaceutically acceptable salt, isotopolog, tautomer, or stereoisomer thereof, or a mixture thereof: wherein, represents a single or double bond; Ring A is selected from C 3-10 cycloalkylene, 3-10 membered heterocyclyl, C 6-10 arylene or 5-10 membered heteroarylene; Ring B is selected from C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl or 5-10 membered heteroaryl; each of ring A and ring B is optionally substituted with 1, 2, 3, 4, or 5 R*; R* is selected from H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1- 6haloalkoxy; R1is selected from O, S or NR a ; R2and R3are selected from OR b , OP2, SR c or SP2; R a selected from H, C 1-6 alkyl or C 1-6 haloalkyl; R b and R c are independently selected from H, C 1-6 alkyl, C 1-6 haloalkyl, C 3-7 cycloalkyl or 3-7 membered heterocyclyl; P1is selected from a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); P2is selected from a protecting group, preferably a hydroxyl protecting group; X is selected from -0-, -S-, -NR d -, -CR e R f -, -CR e R f -CR e R f - or -CH=CH-; R d , R e and R f are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; Y1, Y2, Y3and Y4are independently selected from H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-7 cycloalkyl or 3-7 membered heterocyclyl; Z is selected from H, halogen, OR j , NR k R l , C 1-18 alkyl, C 2-18 alkenyl, C 2-18 alkynyl or C 1-18 haloalkyl, preferably from H, halogen, OR j , NR k R l , C 1-10 alkyl, C 2-10 alkenyl, C 2-10 alkynyl or C 1-10 haloalkyl, said Z being optionally substituted with 1, 2 or 3 R#; R j , R k and R l are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; R# is selected from H, halogen, OH, NH2, CN, C 1-6 alkyl, alkoxy or C 1-6 haloalkoxy; Q is selected from -0-, -S-, -NR g - or -CR h R i -; R g , R h and R i are independently selected from H, halogen, C 1-6 alkyl or C 1-6 haloalkyl; Base’ is selected from H, a modified or unmodified base, or a leaving group; the compound of formula (I’) is optionally further substituted with 1, 2, 3, 4, or 5 substituents selected from: H, halogen, OH, CN, NH2, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy or C 1-6 haloalkoxy. The compound of claim 14, or a pharmaceutically acceptable salt, isotopolog, tautomer, or stereoisomer thereof, or a mixture thereof, wherein, The compound has the following structure: wherein, m is selected from 0, 1, or 2; R2, R3, P1, R*, and Base’ are as defined in claim 14; R1, ring A, ring B, X, Y1, Y2, Y3, Y4, Z, and Q are as defined in any one of claims 1-6. The compound of claim 14 or 15, or a pharmaceutically acceptable salt, isotopologues, tautomer, or stereoisomer thereof, or a mixture thereof, wherein: R1is selected from O or S; R2and R3are selected from OR b , OP2, SR c or SP2; R b and R c are independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl; P1is selected from a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); P2is selected from a protecting group, preferably a hydroxyl protecting group; Base' is selected from H, an unmodified base, or C 1-4 an alkyl or a protecting group modified base; the other variables, such as ring A, ring B, X, Y1, Y2, Y3, Y4, Z, and Q, are as defined in any one of claims 3-5; preferably, R1is selected from O or S, preferably O; R2and R3are selected from OR b or OP2; P1is selected from a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); P2is selected from a protecting group, preferably a hydroxyl protecting group; Base' is selected from H, an unmodified base, or C 1-4 alkyl or a protecting group modified base, for example the other variables, such as ring A, ring B, X, Y1, Y2, Y3, Y4, Z, and Q, are as defined in any one of claims 3-5. The compound of any one of claims 14-16, or a pharmaceutically acceptable salt, isotopolog, tautomer, or stereoisomer thereof, or a mixture thereof, wherein, The compound has the following structure: wherein, Ring B is selected from C 3-7 Cycloalkyl or 3-7 membered heterocyclyl, preferably C 3-7 Cycloalkyl; each R is independently selected from H, P2, C b is independently selected from H, P2, C 1-6 alkyl or C 1-6 haloalkyl; P1is selected from a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); P2is selected from a protecting group, preferably a hydroxyl protecting group; each R j is independently selected from H, C 1-6 alkyl or C 1-6 haloalkyl, said R j optionally substituted with 1 or 2 R#; R# is selected from H, halo or C 1-6 alkoxy; Base' is selected from preferably, Ring B is selected from C 3-5 cycloalkyl or 3-5 membered heterocyclyl, preferably C 3-5 cycloalkyl, for example cyclopropyl, cyclobutyl or cyclopentyl; each R is independently selected from H, P2, or C b is independently selected from H, P2, or C 1-6 alkyl, for example CH3or CH2CH3; P1is selected from a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); P2is selected from a protecting group, preferably a hydroxyl protecting group; each R j is independently selected from H or C 1-6 alkyl, said R j is optionally substituted with 1 or 2 R#; R# is selected from H, halo or C 1-6 alkoxy; R j preferably selected from CH3or CH2CH2OCH3; Base' is selected from The compound of any one of claims 14-17, or a pharmaceutically acceptable salt, isotopolog, tautomer, or stereoisomer thereof, or a mixture thereof, wherein, The compound has the following structure: wherein, P1is a reactive phosphorus group, preferably -P(OCH2CH2CN)(N(iPr)2); Base' is selected from Preferably, the compound is selected from the following structures: An RNAi agent, characterized in that, the RNAi agent comprises one or more nucleotide monomers of any one of claims 1-8; preferably, the ends of the RNAi agent are modified with one or more nucleotide monomers of any one of claims 1-8. The RNAi agent of claim 19, characterized in that, the RNAi agent is double stranded comprising a sense strand and an antisense strand, wherein each strand has 14 to 30 nucleotides. The RNAi agent of claim 20, characterized in that, the nucleotide monomer of any one of claims 1-8 is linked to the end of the antisense strand of the RNAi agent, for example to the 3’ end and / or 5’ end of the antisense strand; preferably, the nucleotide monomer of any one of claims 1-8 is linked to the 5’ end of the antisense strand of the RNAi agent. The RNAi agent of any one of claims 19-21, characterized in that, The RNAi agent comprises a targeting ligand or a delivery vehicle; preferably, the delivery vehicle is selected from a polypeptide delivery vehicle or an antibody delivery vehicle; preferably, the targeting ligand is an N-acetylgalactosamine (GalNAc) targeting ligand. A composition comprising one or more oligonucleotides of any one of claims 1-13, or an RNAi agent of any one of claims 19-22. A kit comprising one or more oligonucleotides of any one of claims 1-13, or an RNAi agent of any one of claims 19-22. A method of inhibiting expression of a target nucleic acid, the method comprising administering to a subject a therapeutic amount of an oligonucleotide of any one of claims 1-13, an RNAi agent of any one of claims 19-22, or a composition of claim 23. A method of treating a disease, the method comprising administering to a subject in need thereof a therapeutic amount of an oligonucleotide of any one of claims 1-13, an RNAi agent of any one of claims 19-22, or a composition of claim 23. The method of claim 26, wherein, The disease is selected from a cancer, an autoimmune disease, an inflammatory disease, a metabolic disease, a genetic disease, or a rare disease.

Images