Ligands for extrahepatic delivery

Abstract

The present invention provides an oligonucleotide, wherein the oligonucleotide has at least 15 nucleotides sufficiently complementary to a target mRNA, one or more sites of the oligonucleotide are coupled to a hydrophobic group.

Description

[0001] This invention claims priority to Chinese invention patent applications CN202410015313.8 (filed January 4, 2024), CN202410643557.0 (filed May 22, 2024), CN202410705730.5 (filed May 31, 2024), and CN202411096110.2 (filed August 9, 2024), all of which are incorporated herein by reference in their entirety as a part of this disclosure.

[0002] Invention Field

[0003] This invention pertains to the pharmaceutical field and specifically relates to hydrophobic groups that enhance the extrahepatic delivery of oligonucleotides and double-stranded RNA, such as the -RT group shown in Formula I, or the R group in Formula (I), and a Formula I compound that attaches the hydrophobic group to a nucleotide, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. Background Technology

[0004] RNA interference is a phenomenon in which target mRNA is efficiently and specifically degraded by double-stranded RNA (dsRNA, also known as siRNA).

[0005] However, current siRNA therapies mostly focus on treating liver-related diseases through N-acetylgalactosamine (GalNAc)-based delivery vectors, making it difficult to deliver siRNA to extrahepatic tissues (such as the eye or central nervous system) to exert their effects, which limits the application of siRNA. Some attempts have been made in the field to deliver siRNA extrahepatically; for example, WO2004094595A2 discloses the delivery of siRNA using a single lipid ligand (e.g., cholesterol or long-chain alkanes) at the chain end, WO2019217459A1 discloses the delivery of siRNA using a single lipid ligand within the chain, and WO2021092371A2 discloses a series of novel lipid ligand structures.

[0006] There is still a need in this field to develop more ligands based on hydrophobic groups for more efficient extrahepatic delivery. Summary of the Invention

[0007] In one aspect, the present invention provides an oligonucleotide having at least 15 nucleotides sufficiently complementary to a target mRNA, the oligonucleotide comprising two identical or different hydrophobic groups located at the 5' and 3' ends of the oligonucleotide, respectively, each hydrophobic group being independently selected from compounds of formula (I): (I) in Indicates the position where it is linked to the remainder of the oligonucleotide; The groups are defined below.

[0008] In another aspect, the present invention provides a method for administering the aforementioned oligonucleotides to a subject via extrahepatic delivery.

[0009] In another aspect, the present invention provides a vector comprising a nucleotide sequence encoding the aforementioned oligonucleotide.

[0010] In another aspect, the present invention provides a cell containing the aforementioned oligonucleotides or the aforementioned carriers.

[0011] In another aspect, the present invention provides pharmaceutical compositions comprising the aforementioned oligonucleotides, the aforementioned carriers, or the aforementioned cells, and optionally pharmaceutically acceptable loads or excipients.

[0012] In another aspect, the present invention provides a kit comprising the aforementioned oligonucleotides, the aforementioned vectors, or the aforementioned cells. Invention Details

[0014] definition

[0015] Chemical definition

[0016] The definitions of specific functional groups and chemical terms are described in more detail below.

[0017] When listing a range of values, it is assumed that each value and the subranges within that range are included. For example, "C 1-6 Alkyl groups include C1, C2, C3, C4, C5, C6, and C6. 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.

[0018] “C 1-30 "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group having 1 to 30 carbon atoms. In some embodiments, C 5-25 Alkyl, C 10-20 Alkyl, C 1-20 Alkyl, C1-10 Alkyl and C 1-6 Alkyl groups are preferred. C 1-6 Examples of alkyl groups include: methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5), and n-hexyl (C6). The term "C" is used in conjunction with the preceding text. 1-6 "Alkyl" also includes heteroalkyl, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkyl group may be optionally substituted by one or more substituents, for example, substituted by 1 to 5 substituents, 1 to 3 substituents, or substituted by 1, 2, 3, 4, or 5 substituents. Common alkyl abbreviations include: Me(-CH3), Et(-CH2CH3), iPr(-CH(CH3)2), nPr(-CH2CH2CH3), n-Bu(-CH2CH2CH2CH3) or i-Bu(-CH2CH(CH3)2).

[0019] “C 2-30 "Alkenyl" refers to a straight-chain or branched hydrocarbon group having 2 to 30 carbon atoms and at least one carbon-carbon double bond. In some embodiments, C 10-25 alkenyl, C 2-10 alkenyl, C 2-6 alkenyl and C 2-4 Alkenyl groups are preferred; in some embodiments, C 8-28 Straight-chain alkenyl groups are preferred. C 2-6 Examples of alkenyl groups include: vinyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), pentenyl (C5), pentadienyl (C5), hexenyl (C6), and so on. The term "C" is used in conjunction with these groups. 2-6 "Alkenyl" also includes heteroalkenyl groups, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkenyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents, or by 1 substituent, for example, by 1, 2, 3, 4, or 5 substituents.

[0020] “C 2-30 "Alkyne" refers to a straight-chain or branched hydrocarbon group having 2 to 30 carbon atoms, at least one carbon-carbon triple bond, and optionally one or more carbon-carbon double bonds. In some embodiments, C 10-25 alkynyl group, C 2-10 alkynyl group, C 2-6alkynyl group and C 2-4 The alkynyl group is 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), pentyynyl (C5), hexynyl (C6), etc. The term "C" is used in conjunction with other alkynyl groups. 2-6 "Alkyne" also includes heteroyne, wherein one or more (e.g., 1, 2, 3 or 4) carbon atoms are replaced by heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). The alkynyl group may be optionally substituted by one or more substituents, for example, by 1 to 5 substituents, 1 to 3 substituents or 1 substituent.

[0021] “C 1-10 Alkylene, C 2-10 "Ideinyl" and "C" 2-10 "Iso-ynyl group" refers to the group with C removed. 1-10 Alkyl, C 2-10 alkenyl and C 2-10 The other hydrogen atom of the alkynyl group forms a divalent group, which can be substituted or unsubstituted. In some embodiments, C 2-8 Alkylene, C 3-7 Alkylene, C 4-6 Alkylene, C 1-4 Alkylene, C 2-4 Alkylene and C 1-3 Alkylenes are preferred. Unsubstituted alkylenes include, but are not limited to: methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), butylene (-CH2CH2CH2CH2-), pentylene (-CH2CH2CH2CH2CH2-), hexylene (-CH2CH2CH2CH2CH2CH2-), and so on. Exemplary substituted alkylenes, for example, those substituted with one or more alkyl (methyl) groups, include, but are not limited to: substituted methylene (-CH(CH3)-, -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, -CH2C(CH3) 2- ), substituted propylidenes (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), etc.

[0022] “C 0-10 "alkylene" refers to chemical bonds and the aforementioned "C" 1-10 Alkylene".

[0023] The term "imendene" in "lipids containing imendene" refers to the -CH=CH- group.

[0024] "Halogen" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0025] Therefore, "C" 1-20 "halogenated alkyl", "C" 1-6 "Halogenated alkyl" and "C" 1-4 "Halogenated alkyl" refers to the above "C" 1-20 Alkyl", C 1-6 "alkyl" and "C" 1-4 "alkyl" is substituted with one or more halogen groups. In some embodiments, C 1-4 Halogenated alkyl groups are particularly preferred, and C4 groups are more preferred. 1-2 Halogenated alkyl groups. Exemplary alkyl halogenated groups include, but are not limited to: -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. The alkyl halogenated group can be substituted at any available connection point, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0026] “C 1-30 "Alkoxy" refers to the group C as defined above. 1-30 The alkyl group is attached to the remainder of the molecule via an oxygen atom. In some embodiments, C 5-25 Alkoxy, C 10-20 Alkoxy, C 1-20 Alkoxy, C 1-10 Alkoxy, C 1-6 Alkoxy, C 1-4 Alkoxy and C 1-3 Alkoxy groups are preferred. Exemplary alkoxy groups include -OCH3, -OCH2CH3, and -OCH2CH2CH3.

[0027] The term "hydroxyl group" refers to the -OH group.

[0028] Term "C" 1-6 "alkylhydroxyl" refers to a C that has at least one hydroxyl group substituted. 1-6 Alkyl group. This group is formed by C... 1-6 The alkyl group is attached to the remainder of the molecule.

[0029] The term "amino" refers to the -NH2 group.

[0030] The term "carboxyl group" refers to the -COOH group.

[0031] The term "sulfonic acid group" refers to the -S(O)2OH group.

[0032] Term "C" 1-6 "Acyl" refers to -C(O)-C 1-6 Alkyl group. Therefore, the term "C" 1-6 "Acyloxy group" refers to a carbon atom bonded to the remaining part of the molecule via an oxygen atom. 1-6 Acyl group, i.e. -OC(O)-C 1-6 alkyl.

[0033] “C 3-12 "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 12 ring carbon atoms and zero heteroatoms. 3-7 "Cycloalkyl" refers to a non-aromatic cyclic hydrocarbon group having 3 to 7 ring carbon atoms and zero heteroatoms. In some embodiments, C 3-6 Cycloalkyl groups are particularly preferred, and C10 is more preferred. 4-6 cycloalkyl, more preferably C 5-6 Cycloalkyl groups. Cycloalkyl groups also include ring systems in which the aforementioned cycloalkyl ring is fused with one or more aryl or heteroaryl groups, wherein the bonding point is on the cycloalkyl ring, and in such cases, the number of carbons continues to represent the number of carbons in the cycloalkyl system. 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), cycloheptanetrienyl (C7), and so on.

[0034] “C 3-12 "Cycloalkylene" refers to the alkylene oxide that has had C removed. 3-12 The divalent group formed by the other hydrogen atom of the cycloalkyl group, and can be substituted or unsubstituted C. 3-12 Cycloalkylene. In some embodiments, C 3-7 Cycloalkylene compounds are preferred. In some embodiments, C 5-7 Cycloalkylene and C 5-6 Cycloalkylene compounds are preferred.

[0035] “C 6-14 "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic) 4n+2 aromatic ring system (e.g., having 6 or 10 shared π electrons arranged in a ring) having 6-14 ring carbon atoms and zero heteroatoms. In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C6 aryl"). 10"Aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). Aryl also includes a ring system in which the above-mentioned aryl ring is fused with one or more cycloalkyl or heterocyclic groups, and the connection point is on the aryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system.

[0036] “C 6-14 "Asyl" refers to the group that has been depleted of C. 6-14 The aryl group is a divalent group formed by the other hydrogen atom, and can be substituted or unsubstituted C. 6-14 Aryl groups. In some implementations, C 6-10 Aryl groups are preferred.

[0037] "5-12-membered heterocyclic group" refers to a group having a 5- to 12-membered non-aromatic ring system with a cyclic carbon atom and 1 to 6 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. "3-7-membered heterocyclic group" refers to a group having a 3- to 7-membered non-aromatic ring system with a cyclic carbon atom and 1 to 4 cyclic heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclic groups containing one or more nitrogen atoms, the linking point can be a carbon or nitrogen atom, provided the valence allows. In some embodiments, a 5-7-membered heterocyclic group is preferred, which is a 5- to 7-membered non-aromatic ring system with a cyclic carbon atom and 1 to 4 cyclic heteroatoms; a 5-6-membered heterocyclic group is preferred, which is a 5- to 6-membered non-aromatic ring system with a cyclic carbon atom and 1 to 3 cyclic heteroatoms. Heterocyclic groups also include ring systems in which the aforementioned heterocyclic ring is fused with one or more cycloalkyl groups, wherein the linking point is on the cycloalkyl ring, or ring systems in which the aforementioned heterocyclic ring is fused with one or more aryl or heteroaryl groups, wherein the linking point is on the heterocyclic ring; and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclic ring system. Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirropropyl, oxetane, and thiorenyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirrobutyl, oxetane, and thiorenyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolidinyl, and pyrrolidin-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to: dioxasulfuranyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to: piperazinyl, morpholinyl, disulfuranyl, and dioxalyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazinanyl. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to: azirheptanyl, oxasulfuranyl, and thioheptanyl. Exemplary 5-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclic groups) include, but are not limited to: dihydroindolyl, isodihydroindolyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, benzoxazolinone, etc. Exemplary 6-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 6,6-bicyclic heterocyclic groups) include, but are not limited to: tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc.

[0038] Heterocyclic groups also include spiroheterocyclic groups, which are groups in which two rings (e.g., heterocycles and carboalkyl groups) share a carbon atom, wherein at least one ring is a heterocyclic group as defined above. More specifically, the spiroheterocyclic group is a spirocycle formed by two 4-membered rings, two 5-membered rings, two 6-membered rings, one 4-membered ring and one 5-membered ring, one 4-membered ring and one 6-membered ring, or one 5-membered ring and one 6-membered ring, wherein at least one ring is a 4-6 membered heterocyclic group as defined above, preferably a 4-6 membered heterocyclic group containing one, two, or three O, N, or S heteroatoms, more preferably a 4-6 membered heterocyclic group containing one N heteroatom. Specific spiroheterocyclic groups include, but are not limited to: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

[0039] "5-14 membered heteroaryl" refers to a group comprising a 4n+2 aromatic ring system of a 5-14 membered monocyclic or bicyclic ring (e.g., having 6 or 10 shared π electrons arranged in a ring) having a ring carbon atom and 1-5 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl containing one or more nitrogen atoms, the bonding point can be a carbon or nitrogen atom, provided the valence allows. A heteroaryl bicyclic system may include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems in which the aforementioned heteroaryl ring is fused with one or more cycloalkyl or heterocyclic groups, and the bonding point is on the heteroaryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the heteroaryl ring system. In some embodiments, 5-10 membered heteroaryl is particularly preferred, which is a 4n+2 aromatic ring system of a 5-10 membered monocyclic or bicyclic ring having a ring carbon atom and 1-4 ring heteroatoms. In some embodiments, 5-6 membered heteroaryl groups are particularly preferred, which are 4n+2 aromatic ring systems of 5-6 membered monocyclic or bicyclic rings having a cyclic carbon atom and 1-4 cyclic heteroatoms. Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to: pyrrole, furanyl, and thiophene. 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 tetraazinyl. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azirmonoheptatrienyl, oxadiazinyl, and thioheptatrienyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazole, benzotriazolyl, benzothiophene, isobenzothiophene, benzofuranyl, benzoisofuranyl, benzoimidazolyl, benzoxazolyl, benzoisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, indazinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthidyl, pteridinyl, quinolinyl, isoquinolinyl, zolinyl, quinoxolinyl, phthalazinyl, and quinazolinyl.

[0040] "5-12-membered heteroaryl" refers to a divalent group formed by removing another hydrogen atom from a 5-12-membered heteroaryl group, and can be substituted or unsubstituted. In some embodiments, 5-10-membered heteroaryl is preferred. In some embodiments, 5-7-membered and 5-6-membered heteroaryl are preferred.

[0041] The carbonyl group, whether used alone or in combination with other terms (such as aminocarbonyl), is represented as -C(O)-.

[0042] "Oxyto" means =O.

[0043] The term "lipid" broadly refers to any chemical group that has an affinity for lipids. One way to characterize the hydrophobicity of a hydrophobic group is through the octanol-water partition coefficient logK. ow K ow This represents the ratio of the concentration of a chemical substance in the octanol phase to its concentration in the aqueous phase at equilibrium in a two-phase system. Typically, the logK of the hydrophobic portion... ow More than 1, more than 1.5, more than 2, more than 3, more than 4, more than 5, or more than 10.

[0044] In this invention, "lipid" can be a molecule with a straight-chain alkyl group as its backbone, which may optionally be replaced by one or more hydroxyl or carbonyl groups, i.e., a "straight-chain lipid". When the straight-chain alkyl group is replaced by one or more carbonyl groups, one or more carbon atoms of the straight-chain alkyl group exist in the form of -C(O)-. The total number of carbon atoms present in various forms in a lipid is the total number of carbon atoms contained in the lipid molecule. When a lipid is represented as "-C14-", it indicates that the lipid contains 14 carbon atoms. When a lipid is represented as "-C(O)-C11-", it indicates that the lipid contains a total of 12 carbon atoms, one of which exists in the form of -C(O)-, and the other 11 carbon atoms are represented as "C11", and so on. -C(O)-C13-, -C(O)-C14-, -C(O)-C15-, -C(O)-C16-, -C(O)-C17-, -C(O)-C18-, -C(O)-C19-, -C(O)-C20-, -C(O)-C21-, etc., represent lipids containing one -C(O)- and different numbers of carbon atoms. When a lipid is represented as "-CC(OH)-C14-", it indicates that the lipid contains a total of 16 carbon atoms, one of which is substituted with a hydroxyl group (-OH). When a lipid is represented as "-C(O)-C19:4-", it indicates that the lipid contains 20 carbon atoms, one of which is in the form of -C(O)-, and the other 19 carbons contain 4 double bonds, represented as "C19:4", and so on. Exemplary groups are... .

[0045] The term "hydrophobic group" refers to a lipid that optionally contains a terminal group at its end. When the lipid does not contain a terminal group (or the terminal group is hydrogen), the term "hydrophobic group" is synonymous with "lipid." The terminal group is as defined by the T group in this invention.

[0046] When any variable (such as R) appears more than once in the composition or structure of a compound, the definition of that variable is independent each time it appears. Therefore, for example, if a group is substituted by 0-2 Rs, that group can optionally be substituted by up to two Rs, where the definition of R is independent each time it appears. Furthermore, combinations of substituents and / or their variants are only permitted if the combination produces a stable compound.

[0047] When one of the variables is a single bond, it means that the two groups connected by that single bond are directly linked. For example, when L in ALB represents a single bond, the structure of ALB is actually AB.

[0048] When the linking group (i.e., the divalent group) listed does not specify its linking direction, the linking direction is arbitrary. For example, when the linking group L in ALB is -MW-, -MW- can be linked to variables A and B in the same direction according to the reading order from left to right, forming A-MW-B, or it can be linked to ring A and ring B in the opposite direction according to the reading order from left to right, forming A-WM-B. Preferably, in this document, when the divalent group -MW- is in the nucleotide chain, its left-to-right linking direction represents the linking direction from the nucleotide chain to the terminal group, for example, the linking direction from the nucleotide chain to the 3' end or the linking direction from the nucleotide chain to the 5' end. For example, in general formula (III), (III) When L1' is At that time, the connection direction from left to right corresponds to the connection direction from the Z end of the nucleotide chain to the 5' end (i.e., T'). structure; When L1 is At that time, the connection direction from left to right corresponds to the connection direction from the Z end to the 3' end (i.e., T) of the nucleotide chain, that is... structure.

[0049] Combinations of linking groups, substituents, and / or their variants are permitted only if a stable compound can be obtained. Unless otherwise specified, when a group has one or more linkable sites, any one or more sites of that group can be linked to other groups by chemical bonds. When the linking positions of chemical bonds are variable and there is an H atom at a linkable site, when a linkable site with an H atom is linked to a chemical bond, the number of H atoms at that site decreases accordingly as the number of linked chemical bonds increases, and the group becomes a group with the corresponding valence.

[0050] The alkyl, alkenyl, and ynyl groups defined in this article are optional substituted groups.

[0051] Exemplary substituents on carbon atoms 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 -NR bb 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, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, wherein each alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently bounded by 0, 1, 2, 3, 4, or 5 R groups. dd Group substitution; Or the two hydrogen-bearing groups on the carbon atom: =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 replace; R aaEach of them is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R aa Groups are combined to form heterocyclic or heteroaryl rings, 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; R bb Each is independently 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, heterocyclic, aryl and heteroaryl, or two R bb Groups are combined to form heterocyclic or heteroaryl rings, 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; R cc Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R cc Groups are combined to form heterocyclic or heteroaryl rings, 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; R dd Each 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)(ORee 2. Alkyl, haloalkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, wherein each alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl group is independently marked by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution, or two geminal radicals dd Substituents can combine to form =O or =S; R ee Each is independently selected from alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl is independently surrounded by 0, 1, 2, 3, 4, or 5 R groups. gg Group substitution; R ff Each is independently selected from hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl, or two R ff The 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. gg Group substitution; R gg Each of these 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 groups, -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C 1-6 Alkyl), -C(=O)(C 1-6 Alkyl group, -CO2H, -CO2(C 1-6 Alkyl), -OC (=O)(C 1-6 Alkyl), -OCO2(C 1-6 Alkyl groups, -C(=O)NH2, -C(=O)N(C 1-6Alkyl)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 groups, -NHC(=O)NH2, -C(=NH)O(C 1-6 Alkyl group), -OC (=NH)(C 1-6 Alkyl group), -OC (=NH)OC 1-6 Alkyl group, -C(=NH)N(C 1-6 Alkyl)2、-C(=NH)NH(C 1-6 Alkyl groups, -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2、-OC(NH)NH(C 1-6 Alkyl groups, -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 groups, -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 1-6 Alkyl), C(=S)NH2, -C(=O)S(C 1-6 Alkyl), -C(=S)SC 1-6 Alkyl, -SC(=S)SC 1-6 Alkyl group, -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-C6 alkenyl, C2-C6 ynyl, C3-C7 cycloalkyl, C6-C 10Aryl, C3-C7 heterocyclic, C5-C 10 heteroaryl; or two ethryl groups gg Substituents can combine to form =O or =S; where X - It is a counterion.

[0052] Exemplary substituents on the 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, heterocyclic, aryl, and heteroaryl, or two R atoms attached to a nitrogen atom. cc The 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.

[0053] Other definitions

[0054] The term "extrahepatic delivery" in this document refers to the delivery of oligonucleotides or double-stranded RNA molecules to tissues outside the liver, including but not limited to the eye, central nervous system, lung, muscle, kidney, heart, spleen, and pancreas. Those skilled in the art will understand that different administration methods are required to improve delivery efficiency depending on the target tissue. For example, delivery to the central nervous system may be via intrathecal injection, delivery to the eye may be via intraocular injection, delivery to muscle may be via intramuscular injection, and delivery to the lung may be via aerosol formulation or direct systemic administration, including but not limited to subcutaneous injection.

[0055] The term "extrahepatic expression" in this article refers to genes that are specifically or non-specifically expressed in extrahepatic tissues, including but not limited to the eyes, central nervous system, lungs, muscles, kidneys, heart, spleen, and pancreas.

[0056] In this invention, muscle tissue includes, but is not limited to, skeletal muscle, cardiac muscle, and smooth muscle. Skeletal muscle includes, but is not limited to, the quadriceps femoris, latissimus dorsi, erector spinae, rectus abdominis, and trapezius. Adipose tissue includes, but is not limited to, subcutaneous fat, gonadal fat, periabdominal white adipose tissue (pgWAT), inguinal white adipose tissue (iWAT), and brown adipose tissue (BAT).

[0057] The term "oligonucleotide" in this document refers to a nucleic acid molecule (RNA or DNA) having a length of less than 100, 200, 300, or 400 nucleotides. Oligonucleotides can be single-stranded or double-stranded. When an oligonucleotide is single-stranded, it is, for example, ASO. When an oligonucleotide is double-stranded, it is, for example, siRNA, miRNA, or shRNA. An oligonucleotide may optionally contain terminal modifications (as defined below) at the 5' and / or 3' ends of one or both strands.

[0058] 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 proteins-encoding genes). 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 called target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. Additionally, RNA other than mRNA (e.g., tRNA) and viral RNA can also be targeted.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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 continuous portion of the mRNA of interest. For example, a polynucleotide is at least partially complementary to PCSK9 mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding PCSK9. The terms “complementary,” “fully complementary,” “fully complementary,” and “substantially complementary” can be used relative to base pairing between the sense and antisense strands of the siRNA, or between the antisense strand of the siRNA reagent and the target sequence.

[0063] "Perfect complementarity" means that in order to maintain the overall double-stranded character of the molecule, the sense strand only needs to be complementary to the antisense strand to a certain extent. In other words, although perfect complementarity is usually required, in some cases, especially in the antisense strand, there may be one or more mismatches (relative to the target mRNA), such as 6, 5, 4, 3, 2 or 1, but the sense and antisense strands can still maintain the overall double-stranded character of the molecule.

[0064] "shRNA" refers to short hairpin RNA. shRNA consists of two short inverted repeat sequences. The shRNA cloned into the shRNA expression vector comprises two short inverted repeat sequences separated by a stem-loop sequence, forming a hairpin structure controlled by the polIII promoter. Subsequently, 5-6 T molecules are added as a transcription terminator for RNA polymerase III.

[0065] Nucleosides are compounds composed of purine or pyrimidine bases and ribose or deoxyribose, while nucleotides are compounds composed of purine or pyrimidine bases, ribose or deoxyribose, and phosphate. A "base" is the basic building block for the synthesis of nucleosides, nucleotides, and nucleic acids. Its constituent elements include nitrogen, hence it is also called a "nitrogenous base." In this article, unless otherwise specified, the capital letters A, U, T, G, and C represent the base composition of nucleotides, namely adenine, uracil, thymine, guanine, and cytosine, respectively.

[0066] The term "modification" of nucleotides as used herein includes, but is not limited to, methoxy modification, fluorination modification, thiophosphate linkage, or conventional protecting group protection. For example, a fluorinated nucleotide refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosome is replaced by fluorine, and a methoxy modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribosome is replaced by a methoxy group.

[0067] In this document, "modified nucleotides" include, but are not limited to, nucleotides modified with 2'-O-methyl, nucleotides modified with 2'-fluorine, nucleotides modified with 2'-deoxy-, inosine ribonucleotides, debased nucleotides, reverse abased deoxyribonucleotides, nucleotides containing a thiophosphate group, nucleotides modified with vinyl phosphate, locked nucleotides, nucleotides modified with 2'-amino, nucleotides modified with 2'-alkyl, morpholinonucleotides, aminophosphates, non-natural bases containing nucleotides, and terminal nucleotides linked to cholesterol derivatives or dodecanoic acid diecamide groups, deoxyribonucleotides, or those protected by conventional protecting groups. For example, a nucleotide modified with 2'-fluorine refers to a nucleotide in which the hydroxyl group at the 2' position of the ribosyl group is replaced by fluorine. A nucleotide modified with 2'-deoxy- refers to a nucleotide in which the 2'-hydroxyl group of the ribosyl group is replaced by a methoxy group.

[0068] As used herein, the term "terminal modification" refers to modifications made to the 5' and / or 3' ends of the positive strand of an siRNA molecule, such as coupling capping modifications or structures. Exemplary positive strand terminal modifications include, but are not limited to, inverted abase-free deoxyribonucleotides (IBs) and STMs.

[0069] Depending on its location / linking method within the siRNA, IB can include the following two structures (used for the 5' end and 3' end of the nucleic acid strand, respectively):

[0070] IB is well known in the art, see, for example, F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16 and PCT publications WO2016011123 and WO2019051402.

[0071] The structure of an unmodified STM is ; STM can be modified, for example Rs is selected from hydrogen, isopropyl or cyclohexyl; STM is well known in the field, see, for example, PCT Publication No. WO2024002006.

[0072] "Ligand moiety" refers to the chemical part conjugated to siRNA that can alter the distribution, targeting, or lifespan of the siRNA. In a preferred embodiment, such a ligand provides enhanced affinity for selected targets (e.g., molecules, cells or cell types), compartments (e.g., cell or organ compartments, tissues, organs, or regions of the body) compared to siRNAs, for example, those without such a ligand.

[0073] "Reactive phosphorus group" refers to a phosphorus-containing group contained in a nucleotide unit or nucleotide analog unit that can react with a hydroxyl or amino group contained in another molecule, particularly in another nucleotide unit or another nucleotide analog, via a nucleophilic attack reaction. Typically, such a reaction produces an ester-type nucleoside bond linking 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 phosphorous amides, H-phosphonates, alkyl-phosphonates, phosphate esters, or phosphate ester analogs, including but not limited to: native phosphate esters, thiophosphate esters, dithiophosphate esters, boron phosphate esters, boron thiophosphate esters, phosphonates, halogen-substituted phosphonates and phosphate esters, aminophosphate esters, phosphate diesters, phosphate triesters, thiophosphate diesters, thiophosphate triesters, diphosphate esters, and triphosphate esters, preferably -P(OCH2CH2CN)(N(iPr)2).

[0074] A "protecting group" is any atom or group of atoms added to a molecule to prevent existing groups in the molecule from undergoing undesirable chemical reactions. A protecting group can be an unstable chemical motif known in the art, used to protect reactive groups, such as hydroxyl, amino, and thiol groups, from unwanted or untimely reactions during chemical synthesis. Protecting groups are typically used selectively and / or orthogonally to protect sites during reactions at other reactive sites, and can then be removed to leave unprotected groups intact or usable for further reactions.

[0075] A non-limiting list of protecting groups includes benzyl; substituted benzyl; alkyl carbonyl and alkoxy carbonyl (e.g., tert-butoxycarbonyl (BOC), acetyl, or isobutyryl); arylalkyl carbonyl and arylalkoxy carbonyl (e.g., benzyloxycarbonyl); substituted methyl ether (e.g., methoxymethyl ether); substituted diethyl ether; substituted benzyl ether; tetrahydropyranyl ether; silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyloxymethyl, [2-(trimethylsilyl)ethoxy]methyl) alkyl or tert-butyldiphenylsilyl); esters (e.g., benzoates); carbonates (e.g., methoxymethyl carbonate); sulfonates (e.g., toluenesulfonate or methanesulfonate); noncyclic ketals (e.g., dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane, 1,3-dioxolane, and those described herein); noncyclic acetals; cyclic acetals (e.g., those described herein); noncyclic hemiacetals; cyclic hemiacetals; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiopentane); orthoesters (e.g., those described herein); and triarylmethyl groups (e.g., tert-butyldiphenylsilyl groups). For example, triphenylmethyl; monomethoxytriphenylmethyl (MMTr); 4,4′-dimethoxytriphenylmethyl (DMTr or DMT); 4,4′,4″-trimethoxytriphenylmethyl (TMTr); and those described herein). Preferred protecting groups are selected from acetyl (Ac), benzoyl (Bzl), benzyl (Bn), isobutyryl (iBu), phenylacetyl, benzyloxymethyl acetal (BOM), β-methoxyethoxymethyl ether (MEM), methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB), methyl thiomethyl ether, Neopentanoyl (Piv), tetrahydropyranyl (THP), triphenylmethyl (Trt), methoxytriphenylmethyl[(4-methoxyphenyl)diphenylmethyl] (MMT), dimethoxytriphenylmethyl, [bis-(4-methoxyphenyl)phenylmethyl (DMT), trimethylsilyl ether (TMS), tert-butyldimethylsilyl ether (TBDMS), tri-isopropylsilyloxymethyl ether (TOM), tri-isopropylsilyl ether (TIPS), methyl ether, ethoxyethyl ether (EE), N,N-dimethylformamidinium and 2-cyanoethyl (CE).

[0076] The protective base in this invention also includes, but is not limited to, those mentioned above. , , , , -DMT, -O-DMT, and -N3.

[0077] A "hydroxyl protecting group" is a group that prevents the hydroxyl group from undergoing chemical reactions and can be removed under specific conditions to restore the hydroxyl group. These mainly include silane-type protecting groups, acyl-type protecting groups, or ether-type protecting groups, with the following being preferred: 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.

[0078] 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.

[0079] 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.

[0080] The compounds of this invention may include one or more asymmetric centers and therefore may exist in a variety of stereoisomeric forms, such as enantiomers and / or diastereomers. For example, the compounds of this invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of mixtures of stereoisomers, including racemic mixtures and mixtures rich in one or more stereoisomers. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)- isomers, and their racemic mixtures and other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this invention. In this document, for compounds or connecting structures containing chiral centers, they may be in (S) absolute configuration, or in (R) absolute configuration, or a mixture of both, i.e., racemic forms. Isomers can be separated from mixtures by methods known to those skilled in the art, including chiral high-performance liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric synthesis.

[0081] The present invention also includes isotopically labeled compounds (isotopic variants) that are equivalent to those described in formula (I), but in which one or more atoms are replaced by atoms with atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, respectively, for example... 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. Other isotopes of the present invention containing the aforementioned isotopes and / or other atoms, their prodrugs, and pharmaceutically acceptable salts of said compounds or said prodrugs are all within the scope of this invention. Certain isotope-labeled compounds of the present invention, for example, those incorporating radioactive isotopes (e.g.,...) 3 H and 14 Those in category C) can be used for drug and / or substrate tissue distribution determination. Tritium, i.e. 3 H and carbon-14, i.e. 14 Carbon isotopes are particularly preferred because they are easy to prepare and detect. Subsequently, they are replaced by heavier isotopes, such as deuterium, i.e., 2H, because higher metabolic stability can provide therapeutic benefits, such as prolonged in vivo half-life or reduced dosage requirements, may be preferred in some cases. Isotope-labeled compounds of formula (I) of the present invention and their prodrugs can generally be prepared by using readily available isotope-labeled reagents instead of non-isotope-labeled reagents when performing the processes described below and / or the techniques disclosed in the examples and preparation examples.

[0082] Compounds of the present invention

[0083] This invention relates to an oligonucleotide having at least 15 nucleotides sufficiently complementary to a target mRNA, wherein the oligonucleotide comprises two identical or different hydrophobic groups located at the 5' and 3' ends of the oligonucleotide, respectively, and each hydrophobic group is independently a compound of formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. (I) in This indicates the position where it is attached to the remainder of the oligonucleotide; the other groups are defined below.

[0084] This invention relates to an oligonucleotide having at least 15 nucleotides sufficiently complementary to a target mRNA, the oligonucleotide comprising two identical or different hydrophobic groups located at the 5' and 3' ends of the oligonucleotide, respectively, and each hydrophobic group being independently a compound of formula II or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. (II) in This indicates the position where it is attached to the remainder of the oligonucleotide; the other groups are defined below.

[0085] This invention relates to an oligonucleotide, wherein the oligonucleotide is a compound of formula III or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: (III).

[0086] Where L2 and L2' are each independently -L a -L b -L c -L d -L e - The other groups are defined below.

[0087] This invention relates to compounds of formula V or their pharmaceutically acceptable salts, tautomers, or stereoisomers: (V) Each group is defined below.

[0088] Lipid R

[0089] In one embodiment, the lipid is a lipid having 10 to 30 carbon atoms; in one embodiment, the lipid contains 10 carbon atoms; in one embodiment, the lipid contains 11 carbon atoms; in one embodiment, the lipid contains 12 carbon atoms; in one embodiment, the lipid contains 13 carbon atoms; in one embodiment, the lipid contains 14 carbon atoms; in one embodiment, the lipid contains 15 carbon atoms; in one embodiment, the lipid contains 16 carbon atoms; in one embodiment, the lipid contains 17 carbon atoms; in one embodiment, the lipid contains 18 carbon atoms; in one embodiment, the lipid contains 19 carbon atoms. Carbon atoms; in one embodiment, the lipid contains 20 carbon atoms; in one embodiment, the lipid contains 21 carbon atoms; in one embodiment, the lipid contains 22 carbon atoms; in one embodiment, the lipid contains 23 carbon atoms; in one embodiment, the lipid contains 24 carbon atoms; in one embodiment, the lipid contains 25 carbon atoms; in one embodiment, the lipid contains 26 carbon atoms; in one embodiment, the lipid contains 27 carbon atoms; in one embodiment, the lipid contains 28 carbon atoms; in one embodiment, the lipid contains 29 carbon atoms; in one embodiment, the lipid contains 30 carbon atoms.

[0090] In one implementation, R is C 8-28 Straight-chain alkyl; in one embodiment, R is C 8-28 A straight-chain alkenyl group; in a further embodiment, R comprises one alkenyl group; in a further embodiment, R comprises four alkenyl groups; in a further embodiment, R comprises one hydroxyl substituent; in a further embodiment, R comprises two hydroxyl substituents; in a further embodiment, R comprises three hydroxyl substituents; in a further embodiment, one carbon atom of R is present in the form of a carbonyl group, also referred to herein as "carbonyl-substituted" or "oxo-substituted"; in a further embodiment, the carbon atoms at both ends of R are present in the form of a carbonyl group.

[0091] In one embodiment, R is optionally substituted by one, two, three, four, or five substituents independently selected from hydroxyl and oxo groups.

[0092] In one specific implementation, R is selected from -C(O)-C11-, -C(O)-C12-, -C(O)-C13-, -C(O)-C14-, -C(O)-C15-, -C(O)-C16-, -C(O)-C17-, -C(O)-C18-, -C(O)-C19-, -C(O)-C20-, -C(O)-C21-, -CC(OH)-C14-, -C(O)-C19:4-, -C(O)-C7-C=C-C8-, -CC(OH)-C14-, -C14-, and -C16-.

[0093] T

[0094] In one embodiment, T is H; in one embodiment, T is hydroxyl; in one embodiment, T is acetoxy; in one embodiment, T is carboxyl; in one embodiment, T is sulfonic acid; in one embodiment, T is tetrazolium (e.g.) In one implementation, T is C. 1-3 Alkyl group; in one embodiment, T is -OCH2CH2OH; in one embodiment, T is -OCH2CH(OH)CH2OH; in one embodiment, T is -OCH(CH2OH)2.

[0095] Hydrophobic group RT

[0096] In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is In one embodiment, the hydrophobic group is .

[0097] m and n

[0098] In one implementation, m and n can be any integers, provided that the lipid portion (i.e., R) contains a total of 10-30 carbon atoms, preferably 12, 14, 16, 18, 20 or 22 carbon atoms.

[0099] In one implementation, each m and n is independently selected from integers from 0 to 50, preferably from integers from 3 to 25, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25.

[0100] In one embodiment, R contains a total of 10 to 25 carbon atoms, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, preferably a total of 12, 14, 16, 18, 20 or 22 carbon atoms.

[0101] L1 and L1'

[0102] In one embodiment, L1 is absent; in one embodiment, L1 is a chemical bond; in one embodiment, L1 is -L x -L y -L z -

[0103] In one embodiment, L1' is a chemical bond; in another embodiment, L1 is... In one implementation, L1 is In one implementation, L1 is In one implementation, L1 is In one implementation, L1 is In the above implementation, each k is independently selected from 1, 2, 3, 4, 5, 6, 7, or 8.

[0104] In one specific implementation, L1 is In one specific implementation, L1 is In one specific implementation, L1 is In one specific implementation, L1 is ; In one embodiment, L1' is absent; in one embodiment, L1' is a chemical bond; in one embodiment, L1' is -L x -L y -L z -

[0105] In one embodiment, L1' is a chemical bond; in another embodiment, L1' is... In one implementation, L1' is In one implementation, L1' is In one implementation, L1' is In one implementation, L1' is In the above implementation, each k is independently selected from 1, 2, 3, 4, 5, 6, 7, or 8.

[0106] In one specific implementation, L1' is In one specific implementation, L1' is In one specific implementation, L1' is In one specific implementation, L1' is .

[0107] L x L y L z

[0108] In one implementation, L x For chemical bonds; in one embodiment, L x For -O-; in one implementation, L x For -S-; in one implementation, L x For -C(O)-; in one implementation, L x For -NR a -, for example -NH-; in one embodiment, L x -C(O)NR a -; In one implementation, L x For -NR a C(O)-.

[0109] In one implementation, L y C 1-10 Alkylene, the C 1-10 The alkylene group is optionally selected from -C by one, two, or three. 0-6 alkylene -OH, -C 0-6 Alkylene -NH2, -C 0-6 Alkylene-CN, C 1-4 Alkyl or C 1-4 Halogenated alkyl substitution.

[0110] In one specific implementation, L y for In one implementation, L y for In one implementation, L y for .

[0111] In one implementation, L z For chemical bonds; in one embodiment, L z For -O-; in one implementation, L z For -S-; in one implementation, L z For -SS-; in one implementation, L z For -C(O)-; in one implementation, L z For -NR a -; In one implementation, L z -C(O)NR a -; In one implementation, L z For -NR a C(O)-.

[0112] Each R a Independently selected from H and C 0-6 alkylene -OH, -C 0-6 Alkylene -NH2, -C 0-6 Alkylene-CN, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups.

[0113] L2 and L2'

[0114] In one implementation, L2 is -L a -L b -L c -L d -L e -; In one implementation, L2 is -L a -L b -L c -L d -L e -; In one implementation, L2 is -L b -L c -L d -L e -; In one implementation, L2 is -L c -L d -L e -; In one implementation, L2 is -L d -L e -; In one implementation, L2 is -L e -

[0115] In one implementation, L2' is -L a -Lb -L c -L d -L e -; In one implementation, L2' is -L a -L b -L c -L d -L e -; In one implementation, L2' is -L b -L c -L d -L e -; In one implementation, L2' is -L c -L d -L e -; In one implementation, L2' is -L d -L e -; In one implementation, L2' is -L e -

[0116] In one implementation, L2 is In one implementation, L2 is Where q is 1, 2, 3, 4, 5, 6, 7, 8, or 9; in one implementation, L2 is In one implementation, L2 is In one implementation, L2 is In one implementation, L2 is In one implementation, L2 is In one implementation, L2 is In one implementation, L2 is In one implementation, L2 is In one implementation, L2 is .

[0117] In one implementation, L2' is In one implementation, L2' is Where q is 1, 2, 3, 4, 5, 6, 7, 8, or 9; in one implementation, L2' is In one implementation, L2' is In one implementation, L2' is In one implementation, L2' is In one implementation, L2' is In one implementation, L2' is In one implementation, L2' is In one implementation, L2' is In one implementation, L2' is .

[0118] L a L b L c L d and L e

[0119] In one implementation, L a Selected from chemical bonds, , , or .

[0120] In one implementation, L a It does not exist; in one implementation, L a For chemical bonds; in one embodiment, L a for In one implementation, L a for In one implementation, L a for In one implementation, L a for In one implementation, L a for In one implementation, L a for In one implementation, L a for In one implementation, L a for Where q is an integer from 1 to 9.

[0121] In one implementation, L b Selected from chemical bonds, , , or .

[0122] In one implementation, L b It does not exist; in one implementation, L b For chemical bonds; in one embodiment, L b for In one implementation, L b for In one implementation, L b for In one implementation, L b for In one implementation, Lb for In one implementation, L b for In one implementation, L b for In one implementation, L b for Where q is an integer from 1 to 9.

[0123] In one implementation, L c Selected from chemical bonds, , , or .

[0124] In one implementation, L c It does not exist; in one implementation, L c For chemical bonds; in one embodiment, L c for In one implementation, L c for In one implementation, L c for In one implementation, L c for In one implementation, L c for In one implementation, L c for In one implementation, L c for In one implementation, L c for Where q is an integer from 1 to 9.

[0125] In one implementation, L d Selected from chemical bonds, , , or .

[0126] In one implementation, L d It does not exist; in one implementation, L d For chemical bonds; in one embodiment, L d for In one implementation, L d for In one implementation, L d for In one implementation, L d for In one implementation, L d for In one implementation, L d for In one implementation, L d for In one implementation, L d for Where q is an integer from 1 to 9.

[0127] In one implementation, L e Selected from chemical bonds, , , or .

[0128] In one implementation, L e It does not exist; in one implementation, L e For chemical bonds; in one embodiment, L e for In one implementation, L e for In one implementation, L e for In one implementation, L e for In one implementation, L e for In one implementation, L e for In one implementation, L e for In one implementation, L e for Where q is an integer from 1 to 9.

[0129] R1, R2, ring A, R b R c R d p and q

[0130] In one embodiment, R1 is -O-; in another embodiment, R1 is -S-; in yet another embodiment, R1 is -C(O)-; in a third embodiment, R1 is -NR. d -, for example -NH-; in one embodiment, R1 is -C(O)NR d -; In one implementation, R1 is -NR d C(O)-.

[0131] In one embodiment, R2 is a chemical bond; in one embodiment, R2 is -O-; in one embodiment, R2 is -S-; in one embodiment, R2 is -SS-; in one embodiment, R2 is -C(O)-; in one embodiment, R2 is -NR. d -, for example -NH-; in one embodiment, R2 is -C(O)NR d -、-NR d C(O)- or -S(O) m -NR d -; In one embodiment, ring A is a 3-12 membered cyclohexane; in another embodiment, ring A is a C 6-14 A aryl group; in one embodiment, ring A is a 5-14 member heteroaryl group; in another embodiment, ring A is a 5-14 member heterocyclic group.

[0132] In one implementation, R b For H; in one implementation, R b -C 0-6 alkylene-OH; in one embodiment, R b -C 0-6 Alkylene-NH2; in one embodiment, R b -C 0-6 Alkylene-CN; in one embodiment, R b -C 0-6 alkylene-C(O)OH; in one embodiment, R b C 1-6 Alkyl; in one embodiment, R b C 1-6 Halogenated alkyl groups.

[0133] In one implementation, R c For H; in one implementation, R c -C 0-6 alkylene-OH; in one embodiment, R c -C 0-6 Alkylene-NH2; in one embodiment, R c -C 0-6 Alkylene-CN; in one embodiment, R c -C 0-6 alkylene-C(O)OH; in one embodiment, R c C 1-6 Alkyl; in one embodiment, R c C 1-6 Halogenated alkyl groups.

[0134] In one implementation, R d For H; in one implementation, R d C 1-6 Alkyl; in one embodiment, R d C 1-6 Halogenated alkyl groups.

[0135] In one implementation, each p is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0136] In one implementation, each q is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0137] -L2-RT and -L2'-R'-T' are each independently selected from Table A: Table A

[0138] Z

[0139] In one embodiment, the 5' end of Z includes a terminal modification; in one embodiment, the 3' end of Z includes a terminal modification; in one embodiment, the 5' end and the 3' end of Z each include a terminal modification; in one embodiment, neither the 5' end nor the 3' end of Z includes a terminal modification.

[0140] In one embodiment, Z is siRNA; in another embodiment, Z is miRNA; in yet another embodiment, Z is shRNA.

[0141] Z'

[0142] In one embodiment, Z' is siRNA; in another embodiment, Z' is miRNA; in yet another embodiment, Z' is shRNA.

[0143] End modification

[0144] In one implementation, end modification is In one implementation, the end modification is In one implementation, the end modification is In one implementation, the end modification is In one implementation, the end modification is ;in Indicates the position where it is linked to the oligonucleotide. Indicates the position relative to the hydrophobic group; wherein R s It is hydrogen, isopropyl or cyclohexyl, and X is hydroxyl or mercapto.

[0145] In one implementation, P1 is In one implementation, P1 is In one implementation, P1 is In one implementation, P1 is In one implementation, P1 is In one embodiment, P1 is -DMT; in one embodiment, P1 is -O-DMT; in one embodiment, P1 is -N3.

[0146] M

[0147] In one implementation, M is In one implementation, M is R s It is hydrogen; in one embodiment, M is R s It is isopropyl; in one embodiment, M is R s It is cyclohexyl. In one implementation, M is... R s It is hydrogen; in one embodiment, M is R s It is isopropyl; in one embodiment, M is R s It is cyclohexyl. In one implementation, M is... X is a hydroxyl group; in one embodiment, M is... X is a thiol group; in one embodiment, M is... X is a hydroxyl group; in one embodiment, M is... X is a thiol group.

[0148] M'

[0149] In one implementation, M' is In one implementation, M' is R s It is hydrogen; in one embodiment, M' is R s It is isopropyl; in one embodiment, M' is... R s It is cyclohexyl. In one implementation, M' is... R sIt is hydrogen; in one embodiment, M' is R s It is isopropyl; in one embodiment, M' is... R s It is cyclohexyl. In one implementation, M' is... X is a hydroxyl group; in one embodiment, M' is... X is a thiol group; in one embodiment, M' is... X is a hydroxyl group; in one embodiment, M' is... X is a thiol group.

[0150] Any technical solution or any combination thereof in any of the above specific embodiments can be combined with any technical solution or any combination thereof in other specific embodiments. For example, any technical solution or any combination thereof of T can be combined with any technical solution or any combination thereof of L1, L2, R, L1', L2', R', T', M, M', Z, etc. This invention aims to include combinations of all these technical solutions; due to space limitations, they will not be listed one by one.

[0151] Specifically, the present invention relates to the following technical solutions.

[0152] In one embodiment, the present invention relates to oligonucleotides having at least 15 nucleotides sufficiently complementary to a target mRNA, the oligonucleotide comprising two identical or different hydrophobic groups located at the 5' and 3' ends of the oligonucleotide, respectively, each hydrophobic group being independently selected from compounds of formula (I) or their pharmaceutically acceptable salts, tautomers, or stereoisomers: (I) in Indicates the position where it is linked to the remainder of the oligonucleotide; R is a lipid, preferably a lipid having 10 to 30 carbon atoms; T is selected from hydrogen, hydroxyl, amino, carboxyl, sulfonic acid group (-S(O)2OH), C 1-6 Acetoxy, C 1-6 Alkyl, C 1-6 Alkyl or 5-10 heteroaryl groups, preferably, T is selected from amino, sulfonic acid (-S(O)2OH), C 1-6 Acetoxy, C 1-6 Alkyl, C 1-6 Alkoxy or 5-10 heteroaryl groups; The T is optionally composed of 1, 2, or 3 molecules selected from hydroxyl, amino, C 1-6 Alkyl hydroxyl, C 1-6 Alkyl or C 1-6Substituents of haloalkyl groups; Preferably, R is a lipid with 10 to 30 carbon atoms; T is selected from hydrogen, hydroxyl, acetoxy, carboxyl, sulfonic acid, tetrazolium, or optionally substituted C. 1-6 Alkoxy, preferably, the C 1-6 The alkoxy group is optionally surrounded by 1, 2, or 3 groups selected from hydroxyl, amino, C 1-6 Alkyl hydroxyl, C 1-6 Alkyl or C 1-6 Substitution of alkyl halogens.

[0153] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein the lipids comprise 10-30 carbon atoms, preferably 12, 14, 16, 18, 20 or 22 carbon atoms.

[0154] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein the lipid is a linear lipid optionally comprising 1-8 olefinic bonds, the lipid optionally being substituted with one or more hydroxyl or oxo groups; preferably, the lipid is optionally substituted with one, two, three, four or five substituents independently selected from hydroxyl and oxo groups.

[0155] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein the R is selected from C 8-28 Straight-chain alkyl or C 8-28 Straight-chain alkenyl, the C 10-28 Straight-chain alkyl and C 8-28 The straight-chain alkenyl group may optionally be substituted with one, two, three, four or five independent substituents selected from hydroxyl and oxo groups; Preferably, R is selected from C 10-28 Straight-chain alkyl groups or C groups containing 1-4 olefinic bonds 8-28 Straight-chain alkenyl, the C 10-28 Straight-chain alkyl groups or C groups containing 1-4 olefinic bonds 8-28 The straight-chain alkenyl group is optionally substituted by one, two, or three substituents independently selected from hydroxyl and oxo groups; More preferably, the lipid is selected from -C(O)-C11-, -C(O)-C12-, -C(O)-C13-, -C(O)-C14-, -C(O)-C15-, -C(O)-C16-, -C(O)-C17-, -C(O)-C18-, -C(O)-C19-, -C(O)-C20-, -C(O)-C21-, -CC(OH)-C14-, -C(O)-C19:4-, -C(O)-C7-C=C-C8-, -CC(OH)-C14-, -C14-, and -C16-.

[0156] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein the T is selected from sulfonic acid groups, tetrazolium groups (e.g., ... ) or optional replacement of C 1-3 Alkoxy, preferably, the C 1-3 Alkyl groups are optionally surrounded by one or two groups selected from hydroxyl and C. 1-3 Substituents of alkyl hydroxyl groups (e.g., -CH2OH).

[0157] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein the T is C 1-3 Alkoxy, the C 1-3 The alkoxy group is optionally substituted with one or two substituents selected from hydroxyl and -CH2OH; More preferably, the T is selected from -OH, -C(O)OH, -OCH3, -OCH2CH2OH, -OCH2CH(OH)CH2OH, -OCH(CH2OH)2, -S(O)2OH and -tetrazolyl, preferably -OCH3, -OCH2CH2OH, -OCH2CH(OH)CH2OH, -OCH(CH2OH)2, -S(O)2OH and .

[0158] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein -RT are each independently selected from: , , , , , , , , , , , , , or ; Where m and n can be any integers, provided that R contains a total of 10-30 carbon atoms; Preferably, m and n are independently selected from integers from 0 to 50, preferably from integers from 3 to 25, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25; R contains a total of 10 to 25 carbon atoms, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, preferably a total of 12, 14, 16, 18, 20 or 22 carbon atoms.

[0159] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein the oligonucleotides are selected from compounds of formula (III) or their pharmaceutically acceptable salts, tautomers, or stereoisomers: (III) in, The T' terminus corresponds to the 5' end of the oligonucleotide, and the T' terminus corresponds to the 3' end of the oligonucleotide. Z is an oligonucleotide; L1 and L1' are each independently selected from chemical bonds or -L x -L y -L z -; Among them, L x Selected from chemical bonds, -O-, -S-, -C(O)-, -NR a -、-C(O)NR a -or-NR a C(O)-; L y Selected from C 1-10 Alkylene, the C 1-10 The alkylene group is optionally selected from -C by one, two, or three. 0-6 alkylene -OH, -C 0-6 Alkylene -NH2, -C 0-6 Alkylene-CN, C 1-4 Alkyl or C 1-4 Halogenated alkyl substitution; L z Selected from chemical bonds, -O-, -S-, -SS-, -C(O)-, -NR a -、-C(O)NR a -or-NR a C(O)-; L x and L z At most one of them is a chemical bond; Each R a Independently selected from H and C 0-6 alkylene -OH, -C 0-6 Alkylene -NH2, -C 0-6 Alkylene-CN, C 1-6 Alkyl or C 1-6 Halogenated alkyl L2 and L2' are each independently -L a -L b -L c -L d -L e -, preferably selected from -L a -L b -L c -L d -L e -、-L b -L c -L d -L e -、-L c -L d -L e -、-L d -L e -、-L e -, more preferably -L c -L d -L e -、-L d -L e -or-L e -; Among them, L a L b L c L d and L e Each is independently selected from chemical bonds, , , or ; R1 is selected from -O-, -S-, -C(O)-, -NR d -、-C(O)NR d -or-NR d C(O)-; R2 is selected from chemical bonds, -O-, -S-, -SS-, -C(O)-, and -NR. d -、-C(O)NR d -、-NR d C(O)- or -S(O) m -NR d -; Cyclone A is selected from 3-12 membered cyclohexene alkyl groups, C 6-14 Arene, 5-14 membered heteroaryl or 5-14 membered heterocyclic, preferably 5-14 membered heterocyclic; Each R b and R c Independently selected from H, -C 0-6 alkylene -OH, -C 0-6 Alkylene -NH2, -C0-6 Alkylene-CN, -C 0-6 Alkylene-C(O)OH, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; Each R d Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; m is selected from 0, 1, or 2; Each p is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; q is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; The definitions of R' and T' are the same as those of R and T; R and T are as defined above; Preferably, L1 and L1' are each independently selected from chemical bonds, , , , or Each k is independently selected from 1, 2, 3, 4, 5, 6, 7, or 8; L2 and L2' are each independently -L a -L b -L c -L d -L e -, where L a L b L c L d and L e Each is independently selected from chemical bonds, , , , , , , , , , , -C(O)-5-12-membered heterocyclic group -NH- or ; Each p is independently selected from an integer between 0 and 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each q is independently selected from an integer between 0 and 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; The definitions of R' and T' are the same as those of R and T; R and T are as defined above; More preferably, L1 and L1' are each independently selected from chemical bonds, , , , or Each k is independently selected from 2, 3, 4, 5, 6, 7, or 8. For example, L1 and L1' are each independently selected from chemical bonds. , , , or ; Where L a L b L c L d and L e Each independently exists or is selected from chemical bonds. , , , , , , , , , , , , , or ; Each q is independently selected from an integer from 1 to 9, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9; R and T are defined as above.

[0160] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein L2 and L2' are each independently a chemical bond or selected from the group consisting of:

[0161] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein -L2-RT and -L2'-R'-T' are each independently selected from the groups listed in Table A above:

[0162] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein the oligonucleotides are selected from compounds of formula (III) or their pharmaceutically acceptable salts, tautomers, or stereoisomers: (III) in, The T' terminus corresponds to the 5' end of the oligonucleotide, and the T' terminus corresponds to the 3' end of the oligonucleotide; Z is an oligonucleotide; L1 and L1' are each independently selected from chemical bonds or -L x -L y -L z -; Among them, L x Selected from chemical bonds or -C(O)-; L y Selected from C 1-10 Alkylene, the C 1-10 The alkylene group is optionally selected from -C by one, two, or three. 0-6 alkylene-OH, C 1-4 Alkyl or C 1-4 Halogenated alkyl substitution; L z Selected from chemical bonds, -S-, -SS-, or -NH-; L x and L z At most one of them is a chemical bond; L2 and L2' are each independently -L d -L e -or-L e -, more preferably -L e -; Among them, L d and L e Each is independently selected from chemical bonds or ; R1 is selected from -C(O)-, -NH-, -C(O)NH- or -NHC(O)-, preferably selected from -C(O)- or -NH-; R2 is selected from chemical bonds, -C(O)-, -NH-, -C(O)NH- or -NHC(O)-, preferably selected from -C(O)- or -NH-; Each R b and R c Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; p is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; q is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R and R' are independently selected from C 10-28 Straight-chain alkyl groups or C groups containing 1-4 olefinic bonds 8-28 Straight-chain alkenyl groups, preferably C-terminated by one or two oxygen atoms. 10-20 Straight-chain alkyl; T and T' are independently selected from -OH, -C(O)OH, or C 1-3 Alkoxy; Preferably, L1 is selected from chemical bonds or ; L1' is selected from chemical bonds, , or ; Each k is independently selected from 2, 3, 4, 5, 6, 7, or 8; L2 and L2' are each independently -L d -L e -, where L d and L e Each is independently selected from chemical bonds, or ; Each p is independently selected from an integer between 0 and 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each q is independently selected from an integer between 0 and 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; -R'-T' and -RT are each independently selected from: , , , , , Preferred selection and ; Each n is independently selected from integers between 10 and 22; More preferably, L1 is a chemical bond or Preferably, chemical bonds; L1' is a chemical bond or Preferred ; L2 and L2' are each independently selected from chemical bonds. or Preferred ; q is 3, 4, or 5; -R'-T' and -RT are independent of each other. and ; Each n is independently selected from an integer between 10 and 20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 12, 13, 14, 15, 16, 17 or 18, more preferably 14, 15, 16, 17 or 18.

[0163] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein the 5' and / or 3' ends of Z contain end modifications, said end modifications preferably being modified or unmodified IB or STM.

[0164] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein Z is selected from compounds of formula IV: (IV) Where Z' represents the remaining portion of the oligonucleotide; M and M' are each independently selected from chemical bonds, , , , or ; in, Indicates the position connected to Z'; Indicates the position connected to T'-R'-L2'-L1'- or -L1-L2-RT; Ring B is selected from 3-7 membered heterocyclic groups or C 3-7 Cycloalkyl groups, preferably 5-7 membered heterocyclic groups, more preferably 6 membered heterocyclic groups; R s Selected from hydrogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups and C 3-7 cycloalkyl; X is selected from hydroxyl or mercapto groups; Y is selected from O or S; Preferably, Z' is the remaining portion of the oligonucleotide; M and M' are each independently selected from chemical bonds, , , , or Preferably selected from chemical bonds, , or ; in, Indicates the position connected to Z'; Indicates the position connected to T'-R'-L2'-L1'- or -L1-L2-RT; R s Selected from hydrogen, C1-6 Alkyl and C 3-6 Cycloalkyl, preferably selected from hydrogen, isopropyl or cyclohexyl; X is selected from hydroxyl or thiol groups.

[0165] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein at least one of M and M' is selected from... , or ,in Indicates the position connected to Z'; Indicates the position connected to T'-R'-L2'-L1'- or -L1-L2-RT; R s Selected from hydrogen, isopropyl or cyclohexyl.

[0166] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein at least one of M and M' is .

[0167] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein at least one of T and T' is a carboxyl group, for example, both T and T' are carboxyl groups.

[0168] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein T' is a carboxyl group and T is a hydroxyl group, or T' is a hydroxyl group and T is a carboxyl group.

[0169] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein T' is hydrogen and T is a hydroxyl group, or T' is hydrogen and T is a carboxyl group.

[0170] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein T' is a hydroxyl group and T is a hydroxyl group.

[0171] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein the oligonucleotides are selected from compounds of formula (III) or their pharmaceutically acceptable salts, tautomers, or stereoisomers: (III) Where Z is ; As defined in Table B, T'-R'-L2'-L1'-M'- and -M-L1-L2-RT are connected to Z' via sulfates or thiosulfates: Table B

[0172] In Table B, the structures of LL50-LL128 are as defined in Table A. "s" indicates that the two adjacent structures are connected by a phosphate ester or a thiophosphate ester. Other structures are shown in the following table:

[0173] In a further embodiment, the present invention relates to oligonucleotides as described above, wherein Z or Z' is a small interfering RNA (siRNA) or a short hairpin RNA (shRNA), preferably used to suppress genes expressed extrahepatically; Preferably, the gene expressed outside the liver is expressed in one or more tissues selected from the group consisting of: eye, central nervous system, lung, muscle, heart, kidney, fat, spleen and pancreas, wherein the muscle is preferably the quadriceps femoris or cardiac muscle, and the fat is preferably subcutaneous fat, gonadal fat, pgWAT, iWAT or BAT.

[0174] In a further embodiment, the present invention relates to an oligonucleotide as described above, wherein the oligonucleotide is siRNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand containing a sequence fully complementary to the sense strand and the target mRNA, and the two hydrophobic groups being located at the 5' and 3' ends of the sense strand, respectively.

[0175] In a further embodiment, the present invention relates to an oligonucleotide as described above, wherein the oligonucleotide is siRNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand containing a sequence fully complementary to the sense strand and the target mRNA, the two hydrophobic groups being located at the 5' and 3' ends of the antisense strand, respectively.

[0176] In one embodiment, the present invention relates to a method of administering oligonucleotides to a subject via extrahepatic delivery, the oligonucleotides being as defined in this application, the extrahepatic delivery comprising delivery to one or more tissues of the following group: eye, central nervous system, lung, muscle, kidney, fat, and spleen, the muscle preferably being the quadriceps femoris or heart, and the fat preferably being subcutaneous fat or gonadal fat.

[0177] In one embodiment, the present invention relates to a method of administering oligonucleotides to a subject via extrahepatic delivery, the method comprising delivering the oligonucleotides via systemic or local administration, preferred administration methods including any of the following: intravenous injection, subcutaneous injection, intrathecal injection, intraocular injection, intramuscular injection, and aerosol spray.

[0178] In one embodiment, the present invention relates to a cell containing oligonucleotides as described in this application.

[0179] In one embodiment, the present invention relates to a pharmaceutical composition comprising an oligonucleotide as described in this application, or a cell as described in this application, and optionally a pharmaceutically acceptable carrier or excipient.

[0180] In one embodiment, the present invention relates to a kit comprising oligonucleotides as described in this application, cells as described in this application, or pharmaceutical compositions as described in this application.

[0181] In one embodiment, the present invention relates to a compound of formula V or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: (V) L2, R, and T are defined as above. Where P1 is a protecting group, such as a carboxyl protecting group, preferably selected from... , , , , -DMTr, -O-DMTr, or -N3.

[0182] In one embodiment, the present invention relates to a compound of formula V or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein P1 is or .

[0183] In one embodiment, the present invention relates to a compound of formula V or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the compound is selected from the compounds listed in Table C below:

[0184] The present invention also provides a vector comprising a nucleotide sequence encoding the oligonucleotide described herein. The vector of the present invention is capable of amplifying or expressing the nucleotide encoding the oligonucleotide described herein linked thereto.

[0185] For example, oligonucleotides targeting a specific gene can be expressed from transcription units inserted into DNA or RNA vectors. Expression can be transient (lasting hours to weeks) or persistent (lasting weeks to months or longer), depending on the specific construct used and the target tissue or cell type. The coding nucleotides of the oligonucleotide can be introduced into linear constructs, circular plasmids, or viral vectors. The nucleotides of the oligonucleotide can be integrated into the cell genome for stable expression or expressed through stable extrachromosomal inheritance. Generally, oligonucleotide expression vectors are typically DNA plasmids or viral vectors.

[0186] Viral vector systems containing coding sequences of oligonucleotides include, but are not limited to: (a) adenovirus vectors; (b) retrovirus vectors; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) microRNA viral vectors; (i) poxvirus vectors; and (j) helper virus-dependent adenoviruses or enteroviruses.

[0187] The present invention also provides a cell containing the oligonucleotides or vectors described herein, wherein the oligonucleotides or vectors described herein are capable of transcription in the cell.

[0188] The present invention also provides a method for administering oligonucleotides to a subject via extrahepatic delivery, the oligonucleotides being as defined above, the extrahepatic delivery comprising delivery to one or more tissues from the group consisting of: eye, central nervous system, lung, muscle, heart, kidney, fat, spleen, and pancreas, the muscle preferably being the quadriceps femoris or cardiac muscle, and the fat preferably being subcutaneous fat, gonadal fat, pgWAT, iWAT, or BAT.

[0189] In one embodiment, the method includes delivering the oligonucleotide via systemic or local administration, the local administration being any of the following: intrathecal injection, intraocular injection, intramuscular injection, and aerosol spray.

[0190] This invention specifically relates to the following technical solutions: The compound and substituent numbers appearing in technical solutions A1-A16 below are only valid in technical solutions A1-A16. In case of any conflict between the numbers in other parts of this document and those numbers, the definitions in other parts of this document shall prevail.

[0191] Technical Solution A1. An oligonucleotide, wherein the oligonucleotide has at least 15 nucleotides that are sufficiently complementary to the target mRNA, and one or more sites of the oligonucleotide are coupled with a hydrophobic group, wherein the hydrophobic group optionally comprises 1-3 hydroxyl groups.

[0192] Technical Solution A2. The oligonucleotide of Technical Solution A1, wherein the oligonucleotide is a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand containing a sequence sufficiently complementary to the sense strand and the target mRNA, wherein one or more sites of the sense strand and / or the antisense strand are coupled with a hydrophobic group, the hydrophobic group optionally containing 1 to 3 hydroxyl groups.

[0193] Technical solution A3. An oligonucleotide of technical solution A1 or A2, wherein the oligonucleotide comprises one or more compounds of formula I, formula II or formula III, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: (I) (II) (III) in, L1 and L2 represent hydrogen, or the position where it is attached to an adjacent nucleotide; L3 represents the position where it is attached to an adjacent nucleotide. R s Selected from H, D, halogens, C 1-6 Alkyl, C 3-6 cycloalkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration; m is 0, 1, 2, 3, 4, 5, or 6; R is -XL-R1; X is selected from chemical bonds, -C(O)-, -C(O)-C 0-10 Alkylene, -C(O)-C 2-10 alkenyl or -C(O)-C 2-10 Ethyne group; L is selected from chemical bonds, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -NHC(O)-CH(OR1)CH2O-, -C(O)NH-CH(OR1)CH2O-, -OC(O)-CH(OR1)CH2O -, -C(O)O-CH(OR1)CH2O-, -NHC(O)-CH(R1)-, -C(O)NH-CH(R1)-, -OC(O)-CH(R1)-, -C(O )O-CH(R1)-, -CH(OR1)CH2O-, -O-CH(R1)CH2O-, -O-CH2CH(R1)O-, -O-CH(CH(OH)CH2OH)- , -O-CH(CH(NH2)CH2OH)-, -O-CH(CH2OH)CH(OH)-, -NH-CH(CH2OH)CH(OH)-, -O-CH2CH(OH)CH(OH)-, -O-CH2CH(NH2)CH(OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)-, -NHC(O)-CH2 -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-O-CH2CH(OH)CH(OH)-, or -NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-; R1 is independently a C1 that is optionally substituted with 1-3 hydroxyl groups. 1-30 Alkyl, C 2-30 alkenyl or C 2-30 Alkyne group, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon atoms that are not adjacent in the group can be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group can be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms can be connected to form a saturated or unsaturated ring; Where C 0-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 1-30 Alkyl, C 2-30 alkenyl and C 2-30 The hydrogen atom in the alkynyl group can optionally be surrounded by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more halogens, C atoms, etc. 1-6 Alkyl or C 1-6The alkyl halide is replaced, and optionally deuterated, up to and including complete deuteration.

[0194] Technical Solution A4. The oligonucleotide of Technical Solution A3, wherein the oligonucleotide comprises a compound of Formula Ia, Formula IIa or Formula IIIa, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: (Ia) (IIa) (IIIa) R is -XL-R1; X is selected from chemical bonds, -C(O)- or -C(O)-C. 3-6 Alkylene; L is selected from chemical bonds, -NHC(O)- or -C(O)NH-; R s Selected from hydrogen, isopropyl, or cyclohexyl; R1 is selected from C1 cells that are optionally substituted with 1-3 hydroxyl groups. 10-24 The alkyl group, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms can be replaced by heteroatoms selected from O, S, and N, or the -CH2CH2- group can be replaced by -OC(O)-, -C(O)O-, -NHC(O)-, or -C(O)NH-, and the hydrogen atom can optionally be replaced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups are substituted, and optionally deuterated, up to and including complete deuteration; Other groups are as defined in technical solution A3.

[0195] Technical solution A5. The oligonucleotide of technical solution A4, wherein R1 is a C-nucleotide optionally substituted with one or two hydroxyl groups. 14-22 Alkyl groups, preferably C14 groups optionally substituted with one hydroxyl group. 14-16 alkyl.

[0196] Technical solution A6. The oligonucleotide of technical solution A5, wherein R is selected from the following structures: , , , , , , , or .

[0197] Technical solution A7. The oligonucleotide of technical solution A6, wherein R is selected from the following structures: or .

[0198] Technical solution A8. An oligonucleotide of any one of technical solutions A3-A7, wherein L1 is hydrogen.

[0199] Technical solution A9. An oligonucleotide of any one of technical solutions A3-A7, wherein the compound of formula I, formula II, formula III, formula Ia, formula IIa or formula IIIa is located at the 5' end of the positive strand.

[0200] Technical solution A10. An oligonucleotide of any one of technical solutions A3-A7, wherein the compound of formula I, formula II, formula III, formula Ia, formula IIa or formula IIIa is selected from the following structures:

[0201] in, This indicates that the oligonucleotide is linked to the remaining portion via a phosphate ester group or a thiophosphate ester group.

[0202] Technical solution A11. An oligonucleotide of any one of technical solutions A1-A10, wherein the oligonucleotide is a small interfering RNA (siRNA) or a short hairpin RNA (shRNA), preferably used to suppress genes expressed in the central nervous system.

[0203] Technical solution A12. A cell containing oligonucleotides as described in any one of technical solutions A1-A11.

[0204] Technical Solution A13. A pharmaceutical composition comprising an oligonucleotide as described in any one of Technical Solutions A1-A11, or cells as described in Technical Solution A12, and optionally a pharmaceutically acceptable carrier or excipient.

[0205] Technical Solution A14. A kit comprising an oligonucleotide as described in any one of Technical Solutions A1-A11, cells as described in Technical Solution A12, or a pharmaceutical composition as described in Technical Solution A13.

[0206] Technical solution A15. A compound represented by formula I', II' or III', or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: (I') (II') (III') Wherein P1, P2, and P3 are each an independent protecting group, preferably each independently 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-trichloro Ethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (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), 4,4'-dimethoxytriphenylmethyl, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

[0207] R' is -XLR 11 ; R 11 Independently, C can be arbitrarily replaced by one or two -OAc or -ODMTr. 14-22 Alkyl groups, preferably C14 substituted with one -OAc or -ODMTr. 14-16 alkyl; Other groups are as defined in any of the foregoing technical solutions.

[0208] Technical solution A16. The compound of technical solution A15, wherein P1 is DMTr, and P2 and P3 are each independently -P(OCH2CH2CN)(N(iPr)2).

[0209] The compound and substituent numbers appearing in technical solutions B1-B23 below are only valid in technical solutions B1-B23. In case of any conflict between the numbers in other parts of this document and those numbers, the meanings in other parts of this document shall prevail.

[0210] Technical solution B1. An oligonucleotide, wherein the oligonucleotide has at least 15 nucleotides that are sufficiently complementary to the target mRNA, and one or more sites of the oligonucleotide are coupled with a hydrophobic group, wherein the hydrophobic group optionally comprises 1-3 hydroxyl groups.

[0211] Technical Solution B2. The oligonucleotide of Technical Solution B1, wherein the oligonucleotide is a double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand containing a sequence sufficiently complementary to the sense strand and the target mRNA, wherein one or more sites of the sense strand and / or the antisense strand are coupled with a hydrophobic group, the hydrophobic group optionally containing 1 to 3 hydroxyl groups.

[0212] Technical solution B3. An oligonucleotide of technical solution B1 or B2, wherein the oligonucleotide comprises one or more compounds of formula I, formula II or formula III, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: (I) (II) (III) in, L1 and L2 represent hydrogen, or the position where it is attached to an adjacent nucleotide; L3 represents the position where it is attached to an adjacent nucleotide. R s Selected from H, D, halogens, C 1-6 Alkyl, C 3-6 cycloalkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration; m is 0, 1, 2, 3, 4, 5, or 6; R is -XL-R1; X is selected from chemical bonds, -C(O)-, -C(O)-C 0-10 Alkylene, -C(O)-C 2-10 alkenyl or -C(O)-C 2-10 Ethyne group; L is selected from chemical bonds, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -NHC(O)-CH(OR1)CH2O-, -C(O)NH-CH(OR1)CH2O-, -OC(O)-CH(OR1)CH2O -, -C(O)O-CH(OR1)CH2O-, -NHC(O)-CH(R1)-, -C(O)NH-CH(R1)-, -OC(O)-CH(R1)-, -C(O )O-CH(R1)-, -CH(OR1)CH2O-, -O-CH(R1)CH2O-, -O-CH2CH(R1)O-, -O-CH(CH(OH)CH2OH)- , -O-CH(CH(NH2)CH2OH)-, -O-CH(CH2OH)CH(OH)-, -NH-CH(CH2OH)CH(OH)-, -O-CH2CH(OH)CH(OH)-, -O-CH2CH(NH2)CH(OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)-, -NHC(O)-CH2 -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-O-CH2CH(OH)CH(OH)-, or -NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-; R1 is independently a C1 that is optionally substituted with 1-3 hydroxyl groups. 1-30 Alkyl, C 2-30 alkenyl or C 2-30 Alkyne group, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon atoms that are not adjacent in the group can be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group can be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms can be connected to form a saturated or unsaturated ring; Where C 0-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 1-30 Alkyl, C 2-30 alkenyl and C 2-30 The hydrogen atom in the alkynyl group can optionally be surrounded by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more halogens, C atoms, etc. 1-6 Alkyl or C 1-6The alkyl halide is replaced, and optionally deuterated, up to and including complete deuteration.

[0213] Technical Solution B4. The oligonucleotide of Technical Solution B3, wherein the oligonucleotide comprises a compound of Formula Ia, Formula IIa or Formula IIIa, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof: (Ia) (IIa) (IIIa) R is -XL-R1; X is selected from chemical bonds, -C(O)- or -C(O)-C. 3-6 Alkylene; L is selected from chemical bonds, -NHC(O)- or -C(O)NH-; R s Selected from hydrogen, isopropyl, or cyclohexyl; R1 is selected from C1 cells that are optionally substituted with 1-3 hydroxyl groups. 10-24 The alkyl group, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 non-adjacent carbon atoms can be replaced by heteroatoms selected from O, S, and N, or the -CH2CH2- group can be replaced by -OC(O)-, -C(O)O-, -NHC(O)-, or -C(O)NH-, and the hydrogen atom can optionally be replaced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups are substituted, and optionally deuterated, up to and including complete deuteration; Other groups are as defined in technical solution B3.

[0214] Technical solution B5. The oligonucleotide of technical solution B4, wherein R1 is a C-nucleotide optionally substituted with one or two hydroxyl groups. 14-22 Alkyl groups, preferably C14 groups optionally substituted with one hydroxyl group. 14-16 alkyl.

[0215] Technical solution B6. The oligonucleotide of technical solution B5, wherein R is selected from the following structures: , , , , , , , or .

[0216] Technical solution B7. The oligonucleotide of technical solution B6, wherein R is selected from the following structures: or .

[0217] Technical solution B8. An oligonucleotide of any one of technical solutions B3-B7, wherein L1 is hydrogen.

[0218] Technical solution B9. An oligonucleotide of any one of technical solutions B3-B7, wherein the compound of formula I, formula II, formula III, formula Ia, formula IIa or formula IIIa is located at the 5' end of the positive strand.

[0219] Technical solution B10. An oligonucleotide of any one of technical solutions B3-B7, wherein the compound of formula I, formula II, formula III, formula Ia, formula IIa or formula IIIa is selected from the following structures:

[0220] in, This indicates that the oligonucleotide is linked to the remaining portion via a phosphate ester group or a thiophosphate ester group.

[0221] Technical solution B11. An oligonucleotide of any one of technical solutions B1-B10, wherein the oligonucleotide is a small interfering RNA (siRNA) or a short hairpin RNA (shRNA), preferably used to suppress genes expressed in the central nervous system.

[0222] Technical solution B12. An oligonucleotide of any one of technical solutions B3-B7, wherein the oligonucleotide comprises two delivery groups, each of which is independently a compound of formula I, II, III, Ia, IIa or IIIa, and the two delivery groups are preferably identical, more preferably located at the 5' end and 3' end of the positive strand, respectively.

[0223] Technical solution B13. The oligonucleotide of technical solution B12, wherein the delivery group is selected from the following structures, wherein This indicates that it is linked to the 3' carbon or corresponding position of the previous nucleotide or nucleotide analog via a phosphate ester group, a thiophosphate ester group, or other linking group. This indicates that the linker is attached to the 5' carbon or corresponding position of the next nucleotide or nucleotide analog via a phosphate ester group, a thiophosphate ester group, or other linking group; when the corresponding structure is located at the end of the nucleic acid chain. or Correspondingly, this indicates the connection to hydrogen, terminal modifications, terminal protecting groups, or other structures that can be used at the ends of nucleic acid chains: .

[0224] Technical solution B14. The oligonucleotide of any one of technical solutions B1-B13, wherein the 3' end of the positive strand is connected to the following structure via a phosphate ester group or a thiophosphate ester: .

[0225] Technical Solution B15. A method for administering double-stranded RNA to a subject via extrahepatic delivery, said double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, said antisense strand containing a sequence sufficiently complementary to said sense strand and target mRNA, wherein one or more sites of said sense strand and / or antisense strand are coupled with a delivery group, said delivery group being selected from compounds of formula I, II, III, Ia, IIa or IIIa or their pharmaceutically acceptable salts, tautomers or stereoisomers, the meaning of each substituent as defined in any one of Technical Solutions B1-B14.

[0226] Technical Solution B16. The method of Technical Solution B15, wherein the double-stranded RNA comprises two identical or different delivery groups, the delivery groups being selected from LL301, LL311, LL321, LL331, LL341, LL351, LL371, LL381, LL391 or LL401, and the structure of each compound is as defined in Technical Solution B13.

[0227] Technical solution B17. The method of technical solution B15 or B16, wherein the extrahepatic delivery includes delivery to one or more tissues in the following group: eye, central nervous system, lung, muscle and kidney, preferably delivery to the eye or central nervous system.

[0228] Technical solution B18. The method of any one of technical solutions B15-B17, the method comprising delivering the double-stranded RNA by systemic administration or local administration, the local administration including any one of the following: intrathecal injection, intraocular injection, intramuscular injection and aerosol spray.

[0229] Technical solution B19. A cell containing oligonucleotides as described in any one of technical solutions B1-B14.

[0230] Technical Solution B20. A pharmaceutical composition comprising an oligonucleotide as described in any one of Technical Solutions B1-B14, or cells as described in Technical Solution B19, and optionally a pharmaceutically acceptable carrier or excipient.

[0231] Technical Solution B21. A kit comprising an oligonucleotide as described in any one of Technical Solutions B1-B14, cells as described in Technical Solution B19, or a pharmaceutical composition as described in Technical Solution B20.

[0232] Technical solution B22. The compound represented by formula I', formula II' or formula III' or its pharmaceutically acceptable salt, tautomer or stereoisomer: (I') (II') (III') Wherein P1, P2, and P3 are each an independent protecting group, preferably each independently 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-trichloro Ethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (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), 4,4'-dimethoxytriphenylmethyl, -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

[0233] R' is -XLR 11 ; R 11 Independently, C can be arbitrarily replaced by one or two -OAc or -ODMTr. 14-22 Alkyl groups, preferably C14 substituted with one -OAc or -ODMTr. 14-16 alkyl; Other groups are as defined in any of the foregoing technical solutions.

[0234] Technical solution B23. The compound of technical solution B22, wherein P1 is DMTr, and P2 and P3 are each independently -P(OCH2CH2CN)(N(iPr)2) or -C(O)CH2CH2C(O)OH.

[0235] The compound and substituent numbers appearing in technical solutions C1-C14 below are only valid in technical solutions C1-C14. In case of any conflict between the numbers in other parts of this document and those numbers, the meanings in other parts of this document shall prevail.

[0236] Technical solution C1. A compound of formula (I), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: (I) in, L1 and L2 are independently selected from H, reactive phosphorus groups, hydroxyl protecting groups, or solid supports; R s Selected from H, D, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration; m = 0, 1, 2, 3, 4, 5 or 6; R is -C(O)-C 0-10 Alkylene-L-R1, -C(O)-C 2-10 alkenyl-L-R1 or -C(O)-C 2-10 Imyethynyl-L-R1; L represents a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -NHC(O)-CH(OR1)CH2O-, -C(O)NH-CH(OR1)CH2O-, -OC(O)-CH(OR1)CH2O- , -C(O)O-CH(OR1)CH2O-, -NHC(O)-CH(R1)-, -C(O)NH-CH(R1)-, -OC(O)-CH(R1)-, -C(O) O-CH(R1)-, -CH(OR1)CH2O-, -O-CH(R1)CH2O-, -O-CH2CH(R1)O-, -O-CH(CH(OH)CH2OH)- , -O-CH(CH(NH2)CH2OH)-, -O-CH(CH2OH)CH(OH)-, -NH-CH(CH2OH)CH(OH)-, -O-CH2CH(OH)CH(OH)-, -O-CH2CH(NH2)CH(OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)-, -NHC(O)-CH2 -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-O-CH2CH(OH)CH(OH)-, or -NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-; R1 is independently C 1-30 Alkyl, C 2-30 alkenyl or C 2-30 Alkyne group, wherein the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 carbon atoms that are not adjacent in the group can be replaced by heteroatoms selected from O, S and N, or the -CH2CH2- group can be replaced by -OC(O)-, -C(O)O-, -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms can be connected to form a saturated or unsaturated ring; Where C 0-10 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 1-30 Alkyl, C 2-30 alkenyl and C 2-30 The hydrogen atom in the alkynyl group can optionally be surrounded by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more halogens, C atoms, etc. 1-6 Alkyl or C 1-6 The alkyl halide is replaced, and optionally deuterated, up to and including complete deuteration.

[0237] Technical solution C2. A compound of formula (I) of technical solution C1, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R is -C(O)-C 0-10 Alkylene-L-R1, preferably -C(O)-L-R1, preferably -C(O)-C 2-8 Alkylene-L-R1, more preferably -C(O)-C 3-7 Alkylene-L-R1, more preferably -C(O)-C 4-6 Alkylene-L-R1, more preferably -C(O)-C 1-3 Alkylene-L-R1.

[0238] Technical solution C3. A compound of formula (I) of technical solution C1 or C2, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L is a chemical bond, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -O-CH(CH(OH)CH2OH)-, -O-CH(CH(NH2)CH2OH)-, -O-CH(CH2OH)CH(OH)-, -NH-CH(CH2O) H)CH(OH)-, -O-CH2CH(OH)CH(OH)-, -O-CH2CH(NH2)CH(OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)-, -NHC(O)-CH2-O-CH(CH (NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-NH-CH(CH2OH)CH(OH)-, -NHC(O)-CH2-O-CH2CH(OH)CH (OH)- or -NHC(O)-CH2-O-CH2CH(NH2)CH(OH)-, preferably chemical bonds, -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -SS-, -NHC(O)O-, -NHC(O)NH-, -OC(O)O-, -OC(O)NH-, -O-CH(CH(OH)CH2OH)-, -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)- or -NHC(O) -CH2-O-CH(CH(NH2)CH2OH)-, preferably chemical bond, -NHC(O)-, -SS-, -NHC(O)O-, -O-CH(CH(OH)CH2OH)-, -O-CH(CH(NH2)CH2OH)-, -NHC(O)-CH2-O-CH(CH(OH)CH2OH)- or -NHC(O)-CH2-O-CH(CH(NH2)CH2OH)-, more preferably chemical bond, -NHC(O)-, -SS- or -NHC(O)O-, more preferably -NHC(O)-.

[0239] Technical solution C4. A compound of formula (I) of technical solution C1 or C2, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein L is -NHC(O)-CH(OR1)CH2O-, -C(O)NH-CH(OR1)CH2O-, -OC(O)-CH(OR1)CH2O-, -C(O)O-CH(OR1)CH2O-, -NHC(O)-CH(R1)-, -C(O)NH-CH( R1)-, -OC(O)-CH(R1)-, -C(O)O-CH(R1)-, -CH(OR1)CH2O-, -O-CH(R1)CH2O-, -O-CH2CH(R1)O-, preferably -NHC(O)-CH(OR1)CH2O-, -NHC(O)-CH(R1)- or -CH(OR1)CH2O-, more preferably -NHC(O)-CH(OR1)CH2O-.

[0240] Technical solution C5. A compound of formula (I) from any one of technical solutions C1-C4, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein R1 is independently C 1-30 Alkyl or C 2-30 The alkenyl group, wherein the 1, 2, 3, 4, 5, 6, 7, or 8 non-adjacent carbon atoms in the group can be replaced by heteroatoms selected from O, S, and N, or the -CH2CH2- group can be replaced by -NHC(O)- or -C(O)NH-, or the substituents on one or more carbon atoms can be linked to form a saturated or unsaturated ring; preferably, R1 is independently C 5-25 Alkyl groups, C-type carbons containing 1, 2, 3, 4, 5, or 6 double bonds. 10-25 Alkenyl groups, wherein 1, 2, 3, 4, or 5 carbon atoms are replaced by N heteroatoms and / or 1, 2, or 3 -CH2CH2- groups are replaced by -C(O)NH- groups. 5-25 The C13 of the steroid ring consists of alkyl groups or one or more substituents on one or more carbon atoms. 5-25 Alkyl; preferably, R1 is selected from the following groups: C6 alkyl, C8 alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 21 alkyl, , , , , , , , or .

[0241] Technical solution C6. The compound of formula (I) of any one of claims C1-C5, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein one of L1 and L2 is a reactive phosphorus group, preferably a phosphorus amide, H-phosphonate, alkyl-phosphonate, phosphate ester or phosphate ester analog, such as natural phosphate ester, thiophosphate ester, dithiophosphate ester, borane phosphate ester, borane thiophosphate ester, phosphonate ester, halogen-substituted phosphonate ester and phosphate ester, aminophosphate ester, phosphate diester, phosphate triester, thiophosphate diester, thiophosphate triester, diphosphate ester or triphosphate ester, preferably -P(OCH2CH2CN)(N(iPr)2).

[0242] Technical solution C7. A compound of formula (I) of any one of technical solutions C1-C5, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein L1 and L2 are selected from protecting groups, preferably hydroxyl protecting groups, such as 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-butoxycarbonyl (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.

[0243] Technical solution C8. A compound of formula (I) from any one of technical solutions C1-C7, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, selected from the following general formulas: (II) (III) (IV) (IVa) (IVb) (V) (VI) (VIa) (VIb) (VII) (VIIa) (VIII) or (VIIIa), Each group is defined as described in technical solutions C1-C7.

[0244] Technical solution C9. A compound of any one of technical solutions C1-C8, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein the compound is selected from the following: , , , , , , , , , , , , , , , , , , , or .

[0245] Technical Solution C10. A method for administering double-stranded RNA to a subject via extrahepatic delivery, said double-stranded RNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, said antisense strand containing a sequence sufficiently complementary to said sense strand and target mRNA, wherein one or more sites of said sense strand and / or antisense strand are coupled with a delivery group, said delivery group being selected from compounds of formula I' or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: (I') in, It indicates the H or hydroxyl protecting group, or the position where it is linked to an adjacent nucleotide; L2 indicates H or a solid support, or the position where it is linked to an adjacent nucleotide; R s Selected from H, D, halogens, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups, optionally deuterated, up to and including complete deuteration; m = 0, 1, 2, 3, 4, 5 or 6; R is a hydrophobic group; Preferably, It indicates the H or hydroxyl protecting group, or the position where it is linked to an adjacent nucleotide; L2 indicates H or a solid support, or the position where it is linked to an adjacent nucleotide; R s m and R are as defined in any of the technical solutions C1-C5.

[0246] Technical solution C11. The method of technical solution C10, wherein the compound of formula (I') is selected from the following general formula compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof: (II') (III') (IV') (IVa') (IVb') (V') (VI') (VIa') (VIb') (VII') (VIIa') (VIII') or (VIIIa'), in, It indicates the position of H or the linking point to the adjacent nucleotide; L2 indicates H or the position where it is linked to an adjacent nucleotide; Other groups are as defined in technical solutions C1-C5.

[0247] Technical solution C12. The method of technical solution C10, wherein the compound of formula (I') is selected from the following compounds, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, wherein the compound is selected from the following: , , , , , , , , , , , , , , , , , , , or , in One represents H or indicates the position where it is linked to an adjacent nucleotide, and the other... The symbol H indicates the position where the nucleotide is attached to an adjacent nucleotide.

[0248] The method of any one of technical solutions C10-C12, wherein the extrahepatic delivery includes delivery to one or more tissues of the group below: eye, central nervous system, lung, muscle and kidney, preferably delivery to the eye or central nervous system.

[0249] Technical solution C14. The method of any one of technical solutions C10-C13, wherein the method comprises delivering the double-stranded RNA by systemic administration or local administration, wherein the local administration includes any one of the following: intrathecal injection, intraocular injection, intramuscular injection and aerosol spray.

[0250] The compound and substituent numbers appearing in the following technical solutions D1-D23 are only valid in technical solutions D1-D23. In case of any conflict between the numbers in other parts of this document and those numbers, the meanings in other parts of this document shall prevail.

[0251] D1. An oligonucleotide, wherein the oligonucleotide has at least 15 nucleotides that are sufficiently complementary to the target mRNA, the oligonucleotide comprising two identical or different hydrophobic groups located at the 5' and 3' ends of the oligonucleotide, respectively, and each hydrophobic group being independently a compound of Formula I or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: (I) in Indicates the position where it is linked to the remainder of the oligonucleotide; R is a lipid with 10 to 30 carbon atoms; T is selected from hydrogen, hydroxyl group, sulfonic acid group, tetrazolium group, or optionally substituted C. 1-6 Alkyl group.

[0252] D2. The oligonucleotide of technical solution D1, wherein the lipid contains 10-30 carbon atoms, preferably 12, 14, 16, 18, 20 or 22 carbon atoms.

[0253] D3. An oligonucleotide of technical solution D1 or D2, wherein the lipid is a linear lipid optionally containing 1-8 imenoyl groups, and the lipid may optionally be substituted with one or more hydroxyl or carbonyl groups.

[0254] D4. An oligonucleotide from any one of technical solutions D1-D3, wherein T is selected from hydrogen, hydroxyl, sulfonic acid group, tetrazolium group, C 1-3 Alkoxy, -OCH2CH2OH, -OCH2CH(OH)CH2OH or -OCH(CH2OH)2.

[0255] D5. An oligonucleotide of any one of technical solutions D1-D4, wherein each of the hydrophobic groups is independently a compound of formula II or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: (II) R1 and R3 are each independently -C(O)- or -CH2-; R2 is C 8-28 A straight-chain alkyl group, optionally containing one alkenyl group, optionally substituted with one or more hydroxyl groups.

[0256] D6. Oligonucleotides of any one of technical solutions D1-D5, wherein -RT are each independently selected from: , , , , , , , , , , , , or ; Where m and n can be any integers, provided that R contains a total of 10-30 carbon atoms, preferably 12, 14, 16, 18, 20 or 22 carbon atoms.

[0257] D7. An oligonucleotide of any one of technical solutions D1-D6, wherein the oligonucleotide is a compound of formula III or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof: (III) Where Z is an oligonucleotide; L1 and L1' are either absent or selected from chemical bonds. or ; L2 and L2' are each independently -L a -L b -L c -L d -L e -, where L a L b L c L d and L e Each independently exists or is selected from chemical bonds. , , , , , , or ; p is an integer between 0 and 10; q is an integer between 0 and 10; The definitions of R' and T' are the same as those of R and T, which are defined as in any of the technical solutions D1-D5.

[0258] D8. Oligonucleotides of technical solution D7, wherein L a L b L c L d and L e Each independently exists or is selected from chemical bonds. , , , , , , , , , , or ; q is an integer from 1 to 9.

[0259] D9. Oligonucleotides of technical solution D7 or D8, wherein L2 and L2' are each independently chemical bonds or selected from the following group:

[0260] D10. Oligonucleotides of any one of technical solutions D7-D9, wherein -L2-RT and -L2'-R'-T' are each independently selected from the following group:

[0261] D11. An oligonucleotide of any one of technical solutions D7-D10, wherein the 5' and / or 3' ends of Z contain a terminal modification, wherein the terminal modification is preferably a modified or unmodified IB or STM.

[0262] D12. An oligonucleotide of any one of technical solutions D7-D11, wherein Z is a compound of formula IV: (IV) Z' is an oligonucleotide, and M and M' are each independently selected from chemical bonds. , , , or .

[0263] in Indicates the position connected to Z'; Indicates the position relative to the hydrophobic group; R s Selected from hydrogen, isopropyl or cyclohexyl; X is selected from hydroxyl or mercapto.

[0264] D13. An oligonucleotide of any one of the technical solutions D7-D12, wherein Z or Z' is a small interfering RNA (siRNA) or a short hairpin RNA (shRNA), preferably used to suppress genes expressed outside the liver.

[0265] D14. The oligonucleotide of technical solution D13, wherein the gene expressed outside the liver is expressed in one or more tissues selected from the group consisting of: eye, central nervous system, lung, muscle, kidney, fat and spleen, wherein the muscle is preferably the quadriceps femoris or heart, and the fat is preferably subcutaneous fat or gonadal fat.

[0266] D15. An oligonucleotide of any one of technical solutions D7-D14, wherein the oligonucleotide is siRNA, the siRNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand containing a sequence fully complementary to the sense strand and the target mRNA, and the two hydrophobic groups being located at the 5' end and 3' end of the sense strand, respectively.

[0267] D16. A method for administering oligonucleotides to a subject via extrahepatic delivery, said oligonucleotides as defined in any one of technical solutions D1-D14, said extrahepatic delivery comprising delivery to one or more tissues of the following group: eye, central nervous system, lung, muscle, kidney, fat and spleen, said muscle preferably being the quadriceps femoris or heart, said fat preferably being subcutaneous fat or gonadal fat.

[0268] D17. The method of technical solution D16, the method comprising delivering the oligonucleotide by systemic administration or local administration, the local administration including any one of the following: intrathecal injection, intraocular injection, intramuscular injection and aerosol spray.

[0269] D18. Cells containing oligonucleotides as described in any one of technical solutions D1-D15.

[0270] D19. A pharmaceutical composition comprising an oligonucleotide as described in any one of technical solutions D1-D15, or cells as described in technical solution D18, and optionally a pharmaceutically acceptable carrier or excipient.

[0271] D20. A kit comprising an oligonucleotide as described in any one of technical solutions D1-D15, cells as described in technical solution D18, or a pharmaceutical composition as described in technical solution D19.

[0272] D21. The compound represented by Formula V or its pharmaceutically acceptable salt, tautomer, or stereoisomer: (V) Where L2, R, and T are as defined in any of the technical solutions 1-10, Where P1 is a protecting group, preferably selected from... , , , , -DMT, -O-DMT, or -N3.

[0273] D22. The compound of technical solution D21, wherein P1 is or .

[0274] D23. The compounds of technical solution D22 are selected from the following group:

[0275] Synthesis Examples

[0276] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0277] abbreviation

[0278] Example 1: Preparation of intermediate compounds

[0279] Example 1.1 Preparation of compound EE1

[0280] 1. Synthesis of Compound 2

[0281] In a MeOH (100 mL) solution of compound 1 (10 g, 67.949 mmol, synthesized according to the method in patent WO2023143571A1), TEA (20.63 g, 203.846 mmol) and CBZ-OSU (25.40 g, 101.923 mmol) were added sequentially. The resulting reaction solution was stirred overnight at room temperature. A large amount of water was added, and the mixture was extracted with DCM (100 mL), dried over anhydrous Na₂SO₄, and the organic solvent was removed by vacuum distillation. Purification by silica gel column chromatography yielded compound 2 (12.9 g, 45.857 mmol, 67.49%).

[0282] 2. Synthesis of Compound 3

[0283] Compound 2 (10 g, 17.13 mmol) was dissolved in DMF (100 mL). TBSCl (3.87 g, 25.70 mmol) and imidazole (2.33 g, 34.27 mmol) were added under ice bath conditions, and the reaction was carried out overnight at room temperature. The reaction was stopped by adding sufficient water. The mixture was extracted three times with water and ethyl acetate. The combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. The resulting compound was purified by silica gel column chromatography to give compound 3 (10.3 g, 86%).

[0284] 3. Synthesis of Compound 4

[0285] Compound 3 (10.3 g, 14.76 mmol) was dissolved in methanol (100 mL), and palladium on carbon (1 g) was added. The reaction was carried out overnight under a hydrogen atmosphere. Water (100 mL) was added, and the mixture was extracted with ethyl acetate (3 × 100 mL). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation to give crude product compound 4 (8 g, 96%).

[0286] 4. Synthesis of Compound 5

[0287] Compound 4 (5 g, 8.87 mmol) and 2-tetradecylethylene oxide (2.35 g, 9.76 mmol) were added to a mixed solvent of ethanol (100 mL) and water (50 mL), stirred vigorously, and heated at 50 °C for 48 hours. Water (100 mL) was added, and the mixture was extracted with ethyl acetate (3 × 100 mL). The combined organic phases were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. The mixture was purified by silica gel column chromatography to give compound 5 (6.1 g, 85%).

[0288] 5. Synthesis of Compound 6

[0289] Compound 5 (6.1 g, 7.58 mmol), triethylamine (2.3 g, 22.75 mmol), DMAP (93 mg, 0.76 mmol), and acetic anhydride (1.16 g, 11.38 mmol) were dissolved in dichloromethane (50 mL) and reacted overnight at room temperature. Water (100 mL) was added, and the mixture was extracted with ethyl acetate (3 × 100 mL). The combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation to give crude compound 6 (5.6 g, 87%).

[0290] 6. Synthesis of Compound 7

[0291] Compound 6 (5.6 g, 6.62 mmol) and TBAF (9.93 mL in 1 M THF, 9.93 mmol) were dissolved in tetrahydrofuran (60 mL) and reacted at room temperature for 3 hours. Water (100 mL) was added, and the mixture was extracted with ethyl acetate (3 × 100 mL). The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. The mixture was purified by silica gel column chromatography to give compound 7 (4.0 g, 82%).

[0292] 7. Synthesis of compound EE1

[0293] Compound 7 (1 g, 1.37 mmol) was dissolved in dichloromethane (10 mL). 4,5-Dicyanoimidazole (0.13 g, 1 mmol) and compound 8 (0.62 g, 2 mmol) were added sequentially under nitrogen protection in an ice bath. The reaction was carried out at room temperature for 3 hours. Water (50 mL) was added, and the mixture was extracted with dichloromethane (3 × 20 mL). The combined organic phases were washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and the organic solvent was removed by evaporation under reduced pressure. The resulting product was purified by silica gel column chromatography to obtain EE1 (0.9 g, 71%).

[0294] 1 H NMR (400 MHz, CDCl3) δ 7.44 (ddd, J = 7.6, 3.5, 2.1 Hz, 2H), 7.35-7.27 (m, 6H), 7.24-7.15 (m, 1H), 6.88-6.78 (m, 4H), 4.99 (tt, J = 7.1, 2.8Hz, 1H), 4.05-3.88 (m, 2H), 3.80 (s, 9H), 3.65-3.56 (m, 2H), 3.35-3.25 (m,1H), 3.14-2.98 (m, 1H), 2.70-2.23 (m, 8H), 2.03-1.93 (m, 4H), 1.57 (d, J =1.5 Hz, 3H), 1.31-1.23 (m, 22H), 1.19 (ddd, J = 9.1, 4.3, 2.3 Hz, 10H), 1.16 (s, 1H), 0.91-0.87 (m, 3H). 31 P NMR (162 MHz, CDCl3) δ 149.09, 149.04, 148.57,148.41.

[0295] Example 1.2 Preparation of compound EE2

[0296] 1. Preparation of compound 2c

[0297] In 25 oCompound 2a (8.0 g, 24.4 mmol, synthesized using the method described in patent WO2023143571A1) and compound 2b (1.43 mL, 13.7 mmol) were dissolved in MeOH (80.0 mL) under condition C, and acetic acid (3.79 mL, 36.7 mmol) was added. 25 o After stirring at C for 5 hours, sodium cyanoborohydride (4.54 g, 73.3 mmol) was added, and stirring continued for 12 hours. Dichloromethane (200 mL) was added to the reaction liquid, and the mixture was washed three times with saturated sodium bicarbonate solution (50 mL x 3) and saturated brine (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, PE / EtOAc = 1 / 0 to 20 / 1) to give compound 2c (6.8 g, yield 68.0%).

[0298] 1 H NMR (400 MHz, CDCl3) δ 7.27-7.38 (m, 10H), 4.44-4.71 (m, 4H), 3.91-4.03 (m, 2H), 3.56-3.70 (m, 4H), 2.56-2.65 (m, 2H), 2.46-2.50 (m, 2H), 2.11-2.22 (m, 1H), 1.69-1.83 (m, 4H), 1.61 (d, J = 12.0 Hz, 1H), 1.06-1.28 (m,5H).

[0299] 2. Preparation of compound 2d

[0300] In 25 o Compound 2c (6.8 g, 16.6 mmol) was added to THF (70.0 mL) at temperature C, followed by Pd / C 10% (1.0 g, 9.3 mmol) and Pd(OH)₂ (1.0 g, 7.12 mmol). At 50 °C... o The mixture was stirred for 12 hours at 15 psi under a hydrogen atmosphere. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, DCM / MeOH = 1 / 0 to 10 / 1) to give compound 2d (3.0 g, yield 78.7%).

[0301] 1H NMR (400 MHz, CD3OD) δ 3.71-3.88 (m, 4H), 3.54-3.63 (m, 2H), 2.58-2.62 (m, 2H), 2.46-2.50 (m, 2H), 2.15-2.26 (m, 1H), 1.77-1.91 (m, 4H), 1.63 (d, J = 12.0 Hz, 1H), 1.21-1.31 (m, 5H).

[0302] 3. Preparation of compound 2f

[0303] Compound 2d (2.45 g, 10.7 mmol) was dissolved in DMF (20.0 mL), and the temperature was lowered to 0. o After step C, add NaH (1.28 g, 32.0 mmol) and tetrabutylammonium iodide (0.39 g, 1.07 mmol), and mix the liquid at 0°C. o After stirring at C for 0.5 hours, compound 2e (3.76 g, 10.684 mmol) was added, and the reaction mixture was stirred for another 12 hours. A saturated aqueous solution of ammonium chloride (50 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (50.0 mL x 3). The organic phases were combined and concentrated under reduced pressure. The crude product compound was purified by column chromatography (silica gel, DCM / EtOAc = 1 / 0 to 1 / 1) to give compound 2f (650 mg, yield 13.4%).

[0304] 1 H NMR (400 MHz, CD3OD) δ 3.90-3.96 (m, 1H), 3.71-3.80 (m, 2H), 3.45-3.64 (m, 5H), 2.57-2.68 (m, 2H), 2.46-2.50 (m, 2H), 2.16-2.25 (m, 1H), 1.78-1.91 (m, 4H), 1.53-1.69 (m, 3H), 1.10-1.41 (m, 32H), 0.86-0.95 (m, 3H).

[0305] 4. Preparation of compound EE2

[0306] In 25 oCompound 2f (790 mg, 1.74 mmol) was dissolved in DCM (10.0 mL) at C, followed by the addition of DCI (154 mg, 1.31 mmol) and compound 2g (787 mg, 2.62 mmol). The solution was then incubated at 25 °C. o Stirring at C for 2 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (silica gel, PE (0.02% TEA) / EA = 1 / 0 to 10 / 1) to give compound EE2 (485 mg, yield 42.6%).

[0307] 1 H NMR (400 MHz, CDCl3) δ 3.77-3.94 (m, 5H), 3.51-3.75 (m, 5H), 3.38-3.48 (m, 2H), 2.59-2.69 (m, 4H), 2.38-2.55 (m, 2H), 2.15-2.26 (m, 1H), 1.70-1.82 (m, 4H), 1.61-1.65 (m, 1H), 1.52-1.56 (m, 1H), 1.26 (s, 26H), 1.19 (dd,J = 2.4, 6.4 Hz, 16H), 0.85-0.92 (m, 3H).

[0308] LCMS: m / z = 654.5 (M+H) + .

[0309] 1.3 Preparation of compounds EE3, EE5 and EE6

[0310] Compound EE3 can be prepared by replacing compound 2e with 1-iodotetradecane using the method of Example 1.2.

[0311]

[0312] 1H NMR (400 MHz, DMSO-d6) δ 3.82-3.68 (m, 4H), 3.70-3.60 (m, 2H), 3.60-3.51 (m, 2H), 3.45-3.41 (m, 2H), 3.38-3.34 (m, 2H), 2.75 (t, J = 6.0 Hz,2H), 2.44-2.37 (m, 1H), 2.36-2.28 (m, 1H), 2.17-2.11 (m, 1H), 1.74-1.66 (m,4H), 1.58-1.51 (m, 1H), 1.49-1.42 (m, 2H), 1.24 (s, 22H), 1.21-1.16 (m, 5H), 1.16-1.11 (m, 12H), 1.10-1.05 (m, 2H), 0.85 (t, J = 6.7 Hz, 3H).

[0313] LCMS: m / z = 626.61 (M+H) +

[0314] 1.4 Preparation of compound EE4

[0315] 1. Preparation of compound 4b

[0316] Nitrogen atmosphere -70 o Add 2 mL of anhydrous DCM solution containing 0.939 mL of DMSO (13.1 mmol) to 10.0 mL of anhydrous DCM containing 0.477 mL of oxalyl chloride (5.64 mmol), and incubate at -70°C. o The reaction was carried out at C for 30 minutes. Then, an anhydrous DCM (2 mL) solution of compound 4a (1.0 g, 1.88 mmol) and TEA (3.13 mL, 22.5 mmol, 12.0 eq) were added, and the mixture was reacted at -70°C for 30 minutes. o The reaction was carried out at C for 45 minutes. After the reaction was complete, saturated NaHCO3 (10.0 mL) aqueous solution was added to the reaction solution, and DCM (20.0 mL) was used. 2) Extract, combine the organic phases, and wash with saturated brine (20.0 mL). 2) Dry with anhydrous sodium sulfate. After filtration, concentrate the filtrate to obtain crude compound 4b (990 mg).

[0317] 2. Preparation of compound 4d

[0318] Under a nitrogen atmosphere, at -30 o At -30°C, n-BuLi (2.46 mL, 6.16 mmol) (2.50 M in n-hexane) was added to a 40.0 mL solution of compound 4c (1.12 g, 5.60 mmol) in THF. o The reaction was carried out at C for 30 minutes. Then, a THF solution (10.0 mL) of compound 4b (990 mg, 1.86 mmol) was added. o The reaction was carried out at C for 2 hours. A saturated ammonium chloride aqueous solution (60.0 mL) was added to the reaction solution, followed by EtOAc (20.0 mL). 2) Extraction, combined organic phases and washed with saturated brine (20.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give compound 4d (810 mg, 58.7%).

[0319] 1 H NMR (400 MHz, acetone- d 6 ) δ 7.49-7.19 (m, 9H), 6.89-6.85 (m, 4H), 4.60-4.56 (m, 1H), 4.12-3.98 (m, 1H), 3.78 (s, 6H), 3.55-3.51 (m, 1H), 3.30-3.25 (m, 1H), 3.20-3.09 (m, 1H), 2.80-2.59 (m, 3H), 2.50-2.40 (m, 1H), 2.21-2.14 (m, 3H), 1.70-1.74 (m, 4H), 1.59-1.57 (m, 1H), 1.51-1.37 (m, 4H), 1.34-1.11 (m, 24H), 0.87 (t, J = 6.8 Hz, 3H).

[0320] 3. Preparation of compound 4e

[0321] Pd(OH)₂ / C (1.0 g, 7.12 mmol) was added to anhydrous THF (100 mL), followed by compound 4d (810 mg, 1.09 mmol), and the mixture was heated in a hydrogen atmosphere at 50 psi for 25 minutes. oThe reaction was carried out at C for 24 hours. The reaction solution was filtered through diatomaceous earth, the filtrate was concentrated, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 10 / 1) to give compound 4e (560 mg, 64.6%).

[0322] 1 H NMR (400 MHz, acetone- d 6 ) δ 7.49-7.19 (m, 9H), 6.88-6.86 (m, 4H), 4.24-3.89 (m, 1H), 3.78-3.68 (m, 7H), 3.48-3.30 (m, 1H), 3.36-3.22 (m, 1H),3.17-3.10 (m, 1H), 2.69-2.57 (m, 3H), 2.44-2.37 (m, 1H), 2.24-2.16 (m, 1H),1.75-1.66 (m, 4H), 1.59-1.42 (m, 3H), 1.42-1.11 (m, 31H), 0.87 (t, J = 6.8Hz, 3H).

[0323] 4. Preparation of compound EE4

[0324] Compound 4e (560 mg, 0.755 mmol) was dissolved in DCM (5.60 mL) and then... o Compound 4f (0.360 mL, 1.13 mmol) and DCI (98.0 mg, 0.83 mmol) were added sequentially at temperature C. After addition, the solution was heated to 25 °C. o The reaction was carried out at C for 1.5 hours. Methyl tert-butyl ether (30.0 mL) was added to the reaction solution, followed by washing with saturated NaHCO3 aqueous solution (30.0 mL). 6) The organic phase was collected and washed with brine (30.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 1 to 15 / 1) to give compound EE4 (670 mg, 79.0%).

[0325] 1 H NMR (400 MHz, acetone- d 6) δ 7.49-7.19 (m, 9H), 6.90-6.86 (m, 4H), 4.12-4.02 (m, 1H), 3.92-3.79 (m, 1H), 3.79-3.78 (m, 6H), 3.78-3.17 (m, 6H),2.76-2.26 (m, 7H), 1.76-1.56 (m, 6H), 1.49-1.37 (3H), 1.37-1.05 (m, 43H),0.88 (t, J = 6.8 Hz, 3H).

[0326] 1.5 Preparation of other intermediate compounds

[0327] The preparation of other intermediate compounds can refer to the above embodiments, with the key compounds (specifically as follows) replaced and adaptive adjustments made, as is well known to those skilled in the art.

[0328] Compound EE5 can be prepared by replacing compound 2e with 4,4'-(((16-iodohexadecyl)oxy(phenyl)methylene)di(methoxybenzene) using the method of Example 1.2.

[0329] Compound EE6 can be prepared by replacing compound 2e with 4,4'-(((14-iodotetradecyl)oxy)(phenyl)methylene)di(methoxybenzene) using the method of Example 1.2.

[0330]

[0331] 1H NMR (400 MHz, Chloroform-d) δ 7.48 – 7.40 (m, 2H), 7.35 – 7.30 (m,4H), 7.30 – 7.26 (m, 2H), 7.23 – 7.17 (m, 1H), 6.85 – 6.79 (m, 4H), 3.87(dtdd, J = 16.7, 8.1, 3.7, 1.8 Hz, 4H), 3.79 (s, 6H), 3.72 – 3.37 (m, 7H), 3.02 (t, J = 6.6 Hz, 2H), 2.63 (ddt, J = 15.9, 7.6, 3.1 Hz, 4H), 2.53 – 2.39(m, 2H), 2.18 (d, J = 7.4 Hz, 1H), 2.01 (s, 1H), 1.76 (d, J = 8.0 Hz, 4H), 1.63 (d, J = 6.6 Hz, 2H), 1.59 – 1.50 (m, 4H), 1.34 – 1.21 (m, 27H), 1.18(dd, J = 6.8, 3.1 Hz, 15H).

[0332] 1 H NMR (400 MHz, Chloroform-d) δ 7.46 – 7.41 (m, 2H), 7.35 – 7.24 (m,6H), 7.22 – 7.16 (m, 1H), 6.85 – 6.78 (m, 4H), 3.95 – 3.77 (m, 11H), 3.75 –3.50 (m, 5H), 3.46– 3.38 (m, 2H), 3.02 (t, J = 6.6 Hz, 2H), 2.69 – 2.58 (m,4H), 2.54 – 2.38 (m, 2H), 2.23 – 2.14 (m, 1H), 1.82 – 1.73 (m, 4H), 1.66 –1.49 (m, 6H), 1.40 – 1.22 (m, 24H), 1.21 – 1.16 (m, 12H).

[0333] Compound EE7 can be prepared by replacing compound 4c with 4,4'-((hexadec-15-yne-1-oxy)(phenyl)methylene)di(methoxybenzene) using the method of Example 1.4.

[0334] Compound EE8 can be prepared by replacing compound 4c with 4,4'-(phenyl(tetradec-13-yne-1-oxy)methylene)di(methoxybenzene) using the method of Example 1.4.

[0335] Compound EE9 can be prepared by replacing compound 4c with tetradec-1-yne using the method of Example 1.4.

[0336]

[0337] Compound EE10 can be prepared by replacing 2-tetradecyl ethylene oxide with 16-iodohexadecyl acetate using the method of Example 1.1.

[0338]

[0339] 1 H NMR (400 MHz, Chloroform-d) δ 7.46 – 7.41 (m, 2H), 7.36 – 7.24 (m,6H), 7.23 – 7.17 (m, 1H), 6.88 – 6.78 (m, 4H), 4.09 – 3.95 (m, 3H), 3.94 – 3.64 (m, 11H), 3.63 – 3.52 (m, 2H), 3.33 – 3.25 (m, 1H), 3.14 (ddd, J = 21.2,9.1, 6.5 Hz, 1H), 2.65 – 2.12 (m, 8H), 2.04 (s, 3H), 1.64 – 1.58 (m, 2H),1.46 – 1.23 (m, 26H), 1.21 – 1.10 (m, 12H).

[0340] Compound EE11 can be prepared by replacing 2-tetradecyl ethylene oxide, ethanol and water with 16-acetoxyhexadecanoic acid, N,N-diisopropylethylamine, (2-oxime-cyanoethyl acetate)-N,N-dimethylmorpholinourea hexafluorophosphate and dichloromethane.

[0341]

[0342] 1H NMR (400 MHz, Chloroform-d) δ 7.44 – 7.36 (m, 2H), 7.33 – 7.25 (m,6H), 7.24 – 7.14 (m, 1H), 6.85 – 6.79 (m, 4H), 4.05 (t, J = 6.8 Hz, 2H), 3.94– 3.81 (m, 2H), 3.79 (d, J = 2.8 Hz, 6H), 3.77 – 3.70 (m, 2H), 3.68 – 3.44(m, 4H), 3.35 – 3.26 (m, 1H), 3.23 – 3.05 (m, 2H), 2.68 – 2.41 (m, 2H), 2.34– 2.09 (m, 2H), 2.04 (s, 3H), 2.00 (s, 3H), 1.64 – 1.51 (m, 4H), 1.36 – 1.22 (m, 22H), 1.22 – 1.10 (m, 12H).

[0343] Compound EE12 can be prepared by replacing 2-tetradecyl ethylene oxide, ethanol and water with 6-(16-acetoxyhexadecanoic acid), N,N-diisopropylethylamine, (2-oxime-cyanoethyl acetate)-N,N-dimethylmorpholinourea hexafluorophosphate and dichloromethane.

[0344]

[0345] 1H NMR (400 MHz, DMSO-d6) δ 7.74 – 7.60 (m, 1H), 7.40 – 7.34 (m, 2H), 7.30 (t, J = 7.4 Hz, 2H), 7.26 – 7.19 (m, 5H), 6.88 (d, J = 8.4 Hz, 4H), 3.96(t, J = 6.7 Hz, 2H), 3.73 (s, 6H), 3.72 – 3.61 (m, 5H), 3.60 – 3.45 (m, 5H), 3.20 – 3.09 (m, 1H), 3.04 – 2.93 (m, 3H), 2.74 (t, J = 5.9 Hz, 1H), 2.72 –2.66 (m, 1H), 2.30 – 2.19 (m, 2H), 2.04 – 1.99 (m, 2H), 1.98 (s, 3H), 1.57 –1.49 (m, 2H), 1.49 – 1.40 (m, 4H), 1.39 – 1.31 (m, 2H), 1.22 (s, 26H), 1.16 –1.01 (m, 12H).

[0346] 1.6 Preparation of compound EE13

[0347] 1. Preparation of compound 13-2

[0348] For the synthesis of compound 13-1, please refer to the synthesis method of intermediate Int-1 in PCT Publication No. WO2023143571A1.

[0349] To a solution of (9Z)-9-octadecenoic acid (100 mg, 0.145 mmol) in dichloromethane (1 mL), diisopropylethylamine (1.071 mL, 6.479 mmol), EDCI (496.78 mg, 2.591 mmol), and HOBt (0.29 g, 2.182 mmol) were added. After stirring the reaction mixture at room temperature for half an hour, compound 13-1 (970.10 mg, 2.160 mmol) was added, and the reaction mixture was stirred at room temperature for two hours. After the reaction was completed as monitored by LCMS, the reaction mixture was washed once with water. The aqueous phase was separated and extracted twice with dichloromethane. The combined organic phases were washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by rapid column chromatography (0-100% ethyl acetate / petroleum ether) to obtain compound 13-2 (1.1 g, 1.204 mmol, 55.76%).

[0350] 1 H NMR (400 MHz, DMSO-d6) δ 7.40 – 7.34 (m, 2H), 7.31 (t, J = 7.5 Hz, 2H), 7.27 – 7.19 (m, 5H), 6.88 (dd, J = 9.0, 2.2 Hz, 4H), 5.37 – 5.27 (m,2H), 4.76 (t, J = 5.5 Hz, 1H), 4.68 (t, J = 5.6 Hz, 1H), 3.96 – 3.89 (m, 1H), 3.88 – 3.82 (m, 1H), 3.74 (s, 6H), 3.61 – 3.46 (m, 3H), 3.45 – 3.34 (m, 2H),3.30 – 3.24 (m, 1H), 3.18 – 3.10 (m, 1H), 3.00 (d, J = 6.4 Hz, 2H), 2.88 (dd,J = 13.1, 8.3 Hz, 1H), 2.29 – 2.16 (m, 2H), 2.13 – 2.06 (m, 1H), 1.49 – 1.35 (m, 2H), 1.23 (s, 20H), 0.84 (t, J = 6.6 Hz, 3H).

[0351] 2. Preparation of compound EE13

[0352] Under a nitrogen atmosphere, CTPPA (0.63 g, 2.102 mmol) and 4,5-dicyanimidazolium (0.20 g, 1.682 mmol) were added to a dichloromethane (10 mL) solution of compound 13-2 (1 g, 1.402 mmol), and the mixture was stirred at room temperature for one hour. After the reaction was completed as monitored by LCMS, the reaction mixture was washed once with saturated sodium bicarbonate solution. The aqueous phase was separated and extracted twice with dichloromethane. The combined organic phases were washed once with saturated brine and dried over anhydrous sodium sulfate. The mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified by rapid column chromatography (0-24% ethyl acetate / petroleum ether + 1% triethylamine) to obtain a colorless syrup EE13 (0.97 g, 1.062 mmol, 75.78%).

[0353] 1 H NMR (400 MHz, DMSO-d6) δ 7.40 – 7.35 (m, 2H), 7.30 (t, J = 7.6 Hz,2H), 7.27 – 7.19 (m, 4H), 6.90 – 6.85 (m, 4H), 5.37 – 5.26 (m, 2H), 3.99 –3.84 (m, 1H), 3.73 (s, 6H), 3.72 – 3.61 (m, 4H), 3.60 – 3.45 (m, 6H), 3.43 –3.32 (m, 1H), 3.25 – 3.08 (m, 1H), 3.04 – 2.94 (m, 1H), 2.73 (t, J = 5.9 Hz,1H), 2.71 – 2.62 (m, 1H), 2.32 – 2.18 (m, 2H), 2.17 – 2.08 (m, 1H), 2.04 –1.91 (m, 4H), 1.48 – 1.34 (m, 2H), 1.23 (s, 20H), 1.15 – 1.05 (m, 12H), 0.84(t, J = 6.5 Hz, 3H).

[0354] 1.7 Preparation of compound EE14

[0355] To a 2 mL solution of compound 13-2 (113 mg, 0.158 mmol) in dichloromethane, diisopropylethylamine (0.157 mL, 0.950 mmol), succinic anhydride (95.10 mg, 0.950 mmol), and 4-dimethylaminopyridine (4.84 mg, 0.040 mmol) were added. The reaction mixture was stirred overnight at room temperature. After the reaction was completed by LCMS monitoring, the mixture was washed once with saturated sodium bicarbonate solution. The aqueous phase was separated and extracted twice with dichloromethane. The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by reversed-phase rapid column chromatography (0-60% acetonitrile / water) to obtain a pale yellow oil EE14 (41 mg, 0.050 mmol, 31.54%).

[0356] 1 H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 7.41 – 7.35 (m, 2H), 7.31(t, J = 7.6 Hz, 2H), 7.27 – 7.18 (m, 5H), 6.91 – 6.84 (m, 4H), 5.37 – 5.26(m, 2H), 4.15 (dd, J = 11.6, 6.6 Hz, 1H), 4.06 (dd, J = 11.5, 4.9 Hz, 1H), 4.00 – 3.87 (m, 4H), 3.73 (s, 6H), 3.66 – 3.58 (m, 2H), 3.57 – 3.49 (m, 1H),3.48 – 3.39 (m, 1H), 3.23 – 3.08 (m, 1H), 3.05 – 2.95 (m, 1H), 2.48 – 2.43(m, 1H), 2.31 – 2.17 (m, 2H), 2.16 – 2.06 (m, 1H), 2.02 – 1.93 (m, 4H), 1.49– 1.35 (m, 2H), 1.23 (s, 20H), 0.84 (t, J = 6.6 Hz, 3H).

[0357] 1.8 Preparation of compound EE15

[0358] Under a nitrogen atmosphere, COMU (172.92 mg, 0.404 mmol) and 2,3,5,6-tetrafluorophenol (67.05 mg, 0.404 mmol) were added sequentially to a solution of compound 15-1 (100 mg, 0.367 mmol) and Et3N (0.153 mL, 1.101 mmol) in N,N-dimethylformamide (5 mL), and the mixture was stirred at room temperature for two hours. The reaction solution was extracted with ethyl acetate and water. After separating the organic phase, the aqueous phase was extracted twice with ethyl acetate. The three organic phases were combined, washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (0-100% ethyl acetate / petroleum ether) to obtain EE15 (106 mg, 62.4%).

[0359] 1 H NMR (400 MHz, Chloroform- d ) δ 6.99 (tt, J = 9.9, 7.1 Hz, 1H), 3.64(td, J = 6.6, 2.6 Hz, 2H), 2.67 (t, J = 7.4 Hz, 2H), 1.78 (p, J = 7.4 Hz, 2H), 1.59 – 1.54 (m, 3H), 1.38 – 1.23 (m, 22H).

[0360] Compounds EE24, EE25, and EE26 were synthesized using the same method as compound EE15.

[0361] 1.9 Preparation of compound EE16

[0362] Under a nitrogen atmosphere, COMU (329 mg, 0.70 mmol) and 2,3,5,6-tetrafluorophenol (128 mg, 0.77 mmol) were added sequentially to an ultradry N,N-dimethylformamide (5 mL) solution of compound 16-1 (200 mg, 0.70 mmol) and Et3N (0.29 mL, 2.1 mmol), and the mixture was stirred at room temperature for two hours. The reaction solution was extracted with ethyl acetate and water. After separating the organic phase, the aqueous phase was extracted twice with ethyl acetate. The three organic phases were combined, washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel rapid column chromatography (elution: ethyl acetate / petroleum ether) to obtain EE16 (53 mg, 17.5%).

[0363] 1 H NMR (400 MHz, DMSO- d 6) δ 11.92 (s, 1H), 7.93 (tt, J = 10.9, 7.4 Hz, 1H), 2.76 (t, J = 7.2 Hz, 2H), 2.18 (t, J = 7.4 Hz, 2H), 1.67 (p, J = 7.2 Hz, 2H), 1.47 (p, J = 7.1 Hz, 2H), 1.40 – 1.32 (m, 2H), 1.30 – 1.20 (m, 20H).

[0364] 1.10 Preparation of compound EE17

[0365] 1. Preparation of compound 17-1

[0366] A methanol solution of compound 17-1 (500 mg, 1.5 mmol) and potassium hydroxide (252 mg, 4.5 mmol) was heated under reflux overnight. After cooling to room temperature, the methanol was concentrated under reduced pressure. The remaining residue was extracted with ethyl acetate (10 mL) and 1N HCl (25 mL), and the aqueous phase was extracted three times with ethyl acetate. The organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The residue was purified by rapid column chromatography (0-15% ethyl acetate / petroleum ether) to give compound 17-2 (260 mg, 60.7%).

[0367] LCMS: m / z = 287.1 (M+H) + .

[0368] 2. Preparation of compound EE17

[0369] Under a nitrogen atmosphere, COMU (164 mg, 0.38 mmol) and 2,3,5,6-tetrafluorophenol (64 mg, 0.38 mmol) were added sequentially to an ultradry N,N-dimethylformamide (5 mL) solution of compound 17-2 (100 mg, 0.35 mmol) and Et3N (0.146 mL, 1.05 mmol), and the mixture was stirred at room temperature for two hours. The reaction solution was extracted with ethyl acetate and water. After separating the organic phase, the aqueous phase was extracted twice with ethyl acetate. The three organic phases were combined, washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel rapid column chromatography (0-10% ethyl acetate / petroleum ether) to obtain compound EE17 (100 mg, 65.9%).

[0370] 1 H NMR (400 MHz, DMSO- d 6) δ 7.93 (tt, J = 10.9, 7.5 Hz, 1H), 3.28 (t, J = 6.6 Hz, 2H), 3.20 (s, 3H), 2.76 (t, J = 7.2 Hz, 2H), 1.67 (p, J = 7.2 Hz, 2H), 1.46 (p, J = 6.8 Hz, 2H), 1.39 – 1.18 (m, 22H).

[0371] Synthesize compound EE27 using the same method as compound EE17:

[0372] 1.11 Preparation of compound EE18

[0373] 1. Preparation of compound 1b

[0374] KOH (274 mg, 4.89 mmol) was added to a suspension of compound 1a (500 mg, 1.63 mmol) in ethylene glycol (5 mL), and the mixture was stirred overnight at 110 °C. Water (20 mL) and ethyl acetate (20 mL) were added to the reaction mixture, and the mixture was separated. The mixture was extracted three times with ethyl acetate (20 mL), and the combined organic phases were washed with saturated brine (20 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was subjected to column chromatography (eluent: petroleum ether and ethyl acetate) to give compound 1b (245 mg, 52% yield) as a white solid.

[0375] 2. Preparation of compound EE18

[0376] Compound 1c (130 mg, 0.78 mmol), triethylamine (0.3 mL, 2.13 mmol), and COMU (335 mg, 0.78 mmol) were added to a dichloromethane (2 mL) solution of compound 1b (245 mg, 0.71 mmol), and the mixture was stirred at room temperature for 12 hours. Water (10 mL) and ethyl acetate (10 mL) were added to the reaction mixture, and the mixture was separated. The mixture was extracted three times with ethyl acetate (10 mL), and the combined organic phases were washed with saturated brine (20 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was subjected to column chromatography (eluent: petroleum ether and ethyl acetate) to give a white solid compound EE18 (150 mg, 43% yield).

[0377] 1 H NMR (400 MHz, Chloroform-d) δ 6.98 (tt, J = 9.9, 7.0 Hz, 1H), 3.76– 3.71 (m, 2H), 3.55 – 3.51 (m, 2H), 3.47 (t, J = 6.7 Hz, 2H), 2.66 (t, J =7.4 Hz, 2H), 1.78 (p, J = 7.4 Hz, 2H), 1.59 (q, J = 7.0 Hz, 2H), 1.42 (td, J= 7.7, 7.0, 3.6 Hz, 2H), 1.32 – 1.24 (m, 24H).

[0378] Compounds EE28 and EE29 were synthesized using the same method as compound EE18:

[0379] 1.12 Preparation of compound EE19

[0380] 1. Preparation of compound 2b

[0381] KOH (231 mg, 4.13 mmol) was added to a glycerol (5 mL) suspension of compound 2a (500 mg, 1.63 mmol), and the mixture was stirred overnight at 110 °C. Water (20 mL) and ethyl acetate (20 mL) were added to the reaction mixture, and the mixture was separated. The mixture was extracted three times with ethyl acetate (20 mL), and the combined organic phases were washed with saturated brine (20 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was subjected to column chromatography (eluent: petroleum ether and ethyl acetate) to give compound 2b (156 mg, 30% yield) as a white solid.

[0382] 2. Preparation of compound EE19

[0383] Compound 2c (73 mg, 0.44 mmol), triethylamine (0.17 mL, 1.2 mmol), and COMU (188 mg, 0.44 mmol) were added to a DMF (2 mL) solution of compound 2b (150 mg, 0.40 mmol), and the mixture was stirred at room temperature for 12 hours. Water (10 mL) and ethyl acetate (10 mL) were added to the reaction mixture, and the mixture was separated. The mixture was extracted three times with ethyl acetate (10 mL), and the organic phases were combined. The mixture was washed with saturated brine (20 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was subjected to column chromatography (eluent: petroleum ether and ethyl acetate) to give compound EE19 (70 mg, 33% yield).

[0384] 1 H NMR (400 MHz, DMSO-d6) δ 7.93 (tt, J = 10.9, 7.4 Hz, 1H), 4.54 (d,J = 5.1 Hz, 1H), 4.42 (t, J = 5.6 Hz, 1H), 3.54 (h, J = 5.4 Hz, 1H), 3.37 –3.31 (m, 4H), 3.25 (td, J = 10.0, 5.7 Hz, 2H), 2.76 (t, J = 7.2 Hz, 2H), 1.67(p, J = 7.2 Hz, 2H), 1.46 (q, J = 6.7 Hz, 2H), 1.40 – 1.25 (m, 26H).

[0385] Compounds EE30 and EE31 were synthesized using the same method as compound EE19:

[0386] 1.13 Preparation of compound EE20

[0387] 1. Preparation of Compound 2

[0388] To a solution of compound 1 (2 g, 7.80 mmol) in DMF (20 mL), tetrafluorophenol (1.42 g, 8.58 mmol), COMU (3.67 g, 8.58 mmol), and TEA (2.37 g, 23.4 mmol) were added, and the mixture was stirred at 25 °C for 16 hours. Water (50 mL) and ethyl acetate (50 mL) were added to the reaction mixture, and the mixture was separated. The mixture was extracted three times with ethyl acetate (50 mL), and the organic phases were combined. The mixture was washed with saturated brine (100 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was subjected to column chromatography (eluent: petroleum ether and ethyl acetate) to give compound 2 (2.80 g, yield 88.7%).

[0389] 2. Preparation of Compound 3

[0390] To a mixed solution of compound 2 (300 mg, 0.74 mmol) in THF (2 mL) and H₂O (4 mL), 2a (180 mg, 0.82 mmol) and K₂CO₃ (205 mg, 1.48 mmol) were added, and the mixture was stirred at room temperature for 16 hours. Water (30 mL) was added to the reaction solution, and then 1 N hydrochloric acid was added to adjust the pH to 3. The mixture was filtered, and the filter cake was washed three times with water and dried to give a white solid compound 3 (280 mg, yield 82.1%).

[0391] 3. Preparation of compound EE20

[0392] To a DMF (2 mL) of compound 3 (60 mg, 0.13 mmol), tetrafluorophenol (23.8 mg, 0.14 mmol), COMU (61 mg, 0.14 mmol), and TEA (39.6 mg, 0.39 mmol) were added, and the mixture was stirred at 25 °C for 16 hours. Water (10 mL) and ethyl acetate (10 mL) were added to the reaction mixture, and the mixture was separated. The mixture was extracted three times with ethyl acetate (10 mL), and the organic phases were combined. The mixture was washed with saturated brine (20 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was subjected to column chromatography (eluent: petroleum ether and ethyl acetate) to give compound EE20 (40 mg, yield 50.4%).

[0393] 1 H NMR (400 MHz, Chloroform-d) δ 7.00 (tt, J = 9.9, 7.0 Hz, 1H), δ6.04 (t, J = 9.9, 7.0 Hz, 1H), δ 3.89 (t, J = 6.2 Hz, 2H), 3.65 (dddd, J =15.7, 5.9, 4.3, 2.5 Hz, 8H), 3.55 (t, J = 5.0 Hz, 2H), 3.44 (q, J = 5.2 Hz,2H), 2.95 (t, J = 6.2 Hz, 2H), 2.20 – 2.11 (m, 2H), 1.26 (dd, J = 11.0, 3.7Hz, 24H), 0.88 (t, J = 6.7 Hz, 3H).

[0394] 1.14 Preparation of compound EE20

[0395] 2. Preparation of compound 20-2

[0396] Triethylamine (16.58 g, 163.872 mmol) and ethyl trifluoroacetate (15.52 g, 109.248 mmol) were added to a methanol (100 mL) suspension of ((2S,6S)-morpholine-2,6-diyl)dimethylhydrochloride (10 g, 54.624 mmol). The mixture was stirred overnight at room temperature. After the reaction was monitored by LCMS, the solvent was concentrated to dryness under reduced pressure. The residue was purified by reversed-phase rapid column chromatography (0-20% aqueous ACN) to give compound 20-2 (11.1 g, 45.7 mmol, 83.60%). LCMS: m / z = 243.84 (M+H) +

[0397] 3. Preparation of compound 20-3

[0398] Under a nitrogen atmosphere and in an ice-water bath, a solution of DCM (50 mL) containing 6.27 g (18.513 mmol) of DTrCl was added dropwise to an ultradry pyridine solution (50 mL) of compound 20-2 (5 g, 20.570 mmol). The mixture was stirred overnight at room temperature. The reaction solution was diluted with dichloromethane, washed once with water, and the aqueous phase was separated and extracted twice with dichloromethane. The three organic phases were combined, washed once with saturated sodium bicarbonate solution and once with brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain the residue, and separated by rapid column chromatography (0-30%-60% EtOAc, in PE) to give compound 20-3 (2.7 g, 4.952 mmol, 24.08%).

[0399] 4. Preparation of compound EE20

[0400] Under a nitrogen atmosphere, bis(diisopropylamino)(2-cyanoethoxy)phosphine (1.82 g, 6.053 mmol) and 4,5-dicyanimidazole (0.62 g, 5.246 mmol) were added to an ultradry DCM (22 mL) solution of compound 20-3 (1.82 g, 6.053 mmol). The reaction solution was stirred at room temperature for one hour. The reaction was monitored by TLC until complete. The solution was washed with saturated sodium bicarbonate solution, and the aqueous phase was separated and extracted twice with dichloromethane. The three organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by rapid column chromatography (0-42% EtOAc in PE, eluent containing 1% Et3N) to obtain EE20 (2.8 g, 3.757 mmol, 93.10%). LCMS: m / z = 746.28 (M+H) + .

[0401] 1 H NMR (400 MHz, DMSO- d 6) δ 7.40 – 7.35 (m, 2H), 7.34 – 7.28 (m, 2H), 7.27 – 7.20 (m, 5H), 6.88 (dd, J = 8.9, 2.5 Hz, 4H), 4.06 (dq, J = 34.0, 4.1,3.3 Hz, 1H), 3.96 – 3.79 (m, 1H), 3.74 (s, 6H), 3.71 – 3.61 (m, 6H), 3.60 –3.39 (m, 4H), 3.24 – 2.88 (m, 2H), 2.78 – 2.63 (m, 2H), 1.18 – 1.01 (m, 12H).

[0402] 1.15 Preparation of Compound EE21

[0403] To a DCM (27 mL) solution of compound 20-3 (2.7 g, 4.967 mmol), DIEA (4.1 mL, 29.8 mmol), DMAP (0.12 g, 0.99 mmol) and succinic anhydride (2.98 g, 29.8 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was evaporated to dryness, and the residue was purified by rapid column chromatography (DCM:MeOH = 1:0 to 10:1) to obtain EE21 (1.6 g, 50.05%).

[0404] 1H NMR (400 MHz, DMSO- d 6) δ 11.92 (s, 1H), 7.38 (ddd, J = 8.3, 3.0,1.4 Hz, 2H), 7.31 (td, J = 7.6, 1.5 Hz, 2H), 7.27 – 7.21 (m, 5H), 6.89 (dt, J= 8.8, 1.3 Hz, 4H), 4.22 (td, J = 11.6, 6.3 Hz, 1H), 4.04 (dddd, J = 21.7,9.5, 6.0, 3.7 Hz, 2H), 3.86 (p, J = 5.9 Hz, 1H), 3.74 (s, 6H), 3.66 (ddd, J =17.2, 9.0, 3.8 Hz, 2H), 3.57 – 3.41 (m, 3H), 3.19 (dd, J = 9.9, 6.0 Hz, 1H), 3.11 – 2.94 (m, 2H), 2.45 (dtd, J = 7.4, 4.7, 4.2, 1.8 Hz, 2H). 19 F NMR (377MHz, DMSO- d 6) δ -68.10 (d, J = 44.1 Hz).

[0405] 1.16 Preparation of compound EE22

[0406] 1. Preparation of compound 22-2

[0407] To compound 22 - 1 (5 g, 38.12 mmol) and triethylamine (5.35 mL, 38.50 mmol) were added dropwise to a methanol (20 mL) solution, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was concentrated, and 1N HCl (30 mL) and ethyl acetate (100 mL) were added to the residue. The aqueous phase was extracted, the organic phases were combined, washed with saturated brine, dried over sodium sulfate, filtered, and concentrated to give compound 22-2 (8.4 g, yield: 97.0%).

[0408] 2. Preparation of compound 22-3

[0409] DIEA (2.2 mL, 13.35 mmol) and HATU (1.86 g, 4.89 mmol) were added to a DMF (20 mL) solution of compound 22-2 (2 g, 4.45 mmol). After stirring at room temperature for 30 minutes, compound 22-2a (2 g, 4.45 mmol, prepared using compound 20-1 as a starting material according to the preparation method of compound 20-3 in the above examples) was added. After stirring at room temperature for 18 hours, the mixture was diluted with water (20 mL), extracted three times with ethyl acetate (30 mL), washed with saturated sodium bicarbonate and brine, and subjected to rotary column chromatography (eluent: dichloromethane and methanol) to give compound 22-3 (2.6 g, yield: 88%).

[0410] 1 H NMR (400 MHz, DMSO- d 6) δ 9.37 (d, J = 6.2 Hz, 1H), 7.95 (s, 1H), 7.42 – 7.34 (m, 2H), 7.34 – 7.27 (m, 2H), 7.26 – 7.20 (m, 5H), 6.89 (dd, J =8.7, 2.4 Hz, 4H), 4.71 (dt, J = 32.8, 5.6 Hz, 1H), 3.96 – 3.80 (m, 1H), 3.74(s, 6H), 3.67 – 3.36 (m, 6H), 3.20 – 3.00 (m, 4H), 2.29 – 2.04 (m, 2H), 1.51– 1.33 (m, 4H), 1.24 – 1.12 (m, 2H).

[0411] 3. Preparation of compound EE22

[0412] To a solution of compound 22-3 (1.3 g, 1.97 mmol) in dichloromethane (10 mL), pyridine (0.80 mL, 9.87 mmol), compound 22-3a (0.77 g, 2.57 mmol), and DCI (0.28 g, 2.37 mmol) were added sequentially. The mixture was purged three times with nitrogen and reacted at 25 °C for 1 hour. The reaction solution was cooled to 0 °C, and saturated NaHCO3 aqueous solution (10 mL) was added. After stirring at 0 °C for 30 minutes, the mixture was separated and extracted with dichloromethane. The combined organic phases were washed with saturated sodium bicarbonate aqueous solution (20 mL) and saturated sodium chloride aqueous solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was subjected to silica gel column chromatography (eluent: petroleum ether and ethyl acetate, 1% triethylamine) to give compound EE22 (980 mg, yield: 58%). m / z: ES+ [M+H] + 859.47.

[0413] 1 H NMR (400 MHz, DMSO-d6) δ 9.36 (s, 1H), 7.38 (q, J = 3.2 Hz, 2H), 7.31 (t, J = 7.6 Hz, 2H), 7.27 – 7.19 (m, 5H), 6.88 (d, J = 8.4 Hz, 4H), 3.99– 3.85 (m, 1H), 3.79– 3.62 (m, 11H), 3.61 – 3.33 (m, 6H), 3.14 (q, J = 7.0Hz, 3H), 3.01 (d, J = 6.0 Hz, 1H), 2.76 – 2.72 (m, 1H), 2.71 – 2.67 (m, 1H), 2.31 – 2.05 (m, 2H), 1.44 (dq, J = 13.6, 7.0, 5.7 Hz, 4H), 1.24 (q, J = 5.3Hz, 2H), 1.11 (dtd, J = 12.5, 6.7, 3.3 Hz, 12H).

[0414] 1.17 Preparation of compound EE23

[0415] 1、 Preparation of compound 2

[0416] At 0 °C, MTTl (25.90 g, 83.86 mmol) was added to a pyridine (50 mL) solution of compound 1 (10 g, 76.23 mmol). The mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (eluents: dichloromethane and methanol) to give compound 2 (17 g, yield: 55.3%). m / z: ES+ [M+H]+ 403.20

[0417] 2. Preparation of compound 3

[0418] To a 10 mL solution of compound 2 (1.0 g, 2.48 mmol) in acetonitrile, N,N-diisopropylethylamine (0.52 mL, 2.97 mmol) and TSTU (0.9 g, 2.97 mmol) were added, and the mixture was stirred at room temperature for two hours. The reaction mixture was concentrated, and the residue was dissolved in dichloromethane, washed with saturated brine, dried over sodium sulfate, filtered, and concentrated to give crude compound 3 (1.2 g, yield: 96.7%), which was used directly in the next step. m / z: ES+ [M+H]+ 501.34

[0419] 3. Preparation of compound 4

[0420] A solution of compound 3 (1.2 g, 2.40 mmol) in DMSO (5 mL) was added to a TEA solution (7.2 mL, 7.2 mmol) containing compound 3a (0.42 g, 2.88 mmol), and the mixture was stirred overnight at 50 °C. Reversed-phase column chromatography (eluents: water and acetonitrile) yielded compound 4 (720 mg, yield: 56.4%). m / z: ES-[MH]- 531.47

[0421] 4. Preparation of compound 5

[0422] To a pyridine (7 mL) solution of compound 4 (720 mg, 1.35 mmol), DMTrCl (458 mg, 1.35 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (eluents: petroleum ether and ethyl acetate) to give compound 5 (550 mg, yield: 48.7%). m / z: ES+ [M+H] + 835.58

[0423] 5. Preparation of compound EE23

[0424] To a solution of compound 5 (7.0 g, 8.38 mmol) in dichloromethane (70 mL), pyridine (3.39 mL, 41.91 mmol), compound 5a (3.03 g, 10.06 mmol), and DCI (1.19 g, 10.06 mmol) were added sequentially. The mixture was purged with nitrogen three times and reacted at 25 °C for 1 hour. The reaction solution was cooled to 0 °C, and a saturated aqueous solution of NaHCO3 (70 mL) was added. After stirring at 0 °C for 30 minutes, the mixture was separated, and extracted three times with dichloromethane (70 mL). The combined organic phases were washed with a saturated aqueous solution of sodium bicarbonate (50 mL) and a saturated aqueous solution of sodium chloride (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was subjected to silica gel column chromatography (eluent: petroleum ether and ethyl acetate, 1% triethylamine) to give compound EE23 (6.0 g, yield: 69%). m / z: ES+ [M+H] + 1036. 48.

[0425] 1 H NMR (400 MHz, Chloroform- d ) δ 7.49 – 7.33 (m, 8H), 7.33 – 7.22 (m, 10H), 7.17 (q, J = 8.6, 7.3 Hz, 3H), 6.80 (dt, J = 8.6, 3.5 Hz, 6H), 4.08 –3.80 (m, 3H), 3.79 – 3.69 (m, 12H), 3.68 – 3.42 (m, 5H), 3.35 – 3.04 (m, 3H),2.62 – 2.45 (m, 2H), 2.32 – 2.07 (m, 4H), 1.50 (dh, J = 22.6, 7.3 Hz, 5H),1.39 – 1.23 (m, 2H), 1.15 (ddt, J = 13.6, 10.1, 4.4 Hz, 12H).

[0426] 1.18 Preparation of compound EE32

[0427] 1、 Preparation of compound 32-2

[0428] Under a nitrogen atmosphere, Ac₂O (17.2 mL, 183 mmol) was added to an ultradry dichloromethane (50 mL) solution of compound 32-1 (5 g, 18.3 mmol) and pyridine (29.7 mL, 367 mmol), and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was extracted with ethyl acetate and water. After separating the organic phase, the aqueous phase was extracted twice with ethyl acetate. The three organic phases were combined, washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (0-60% ethyl acetate / petroleum ether) to obtain compound 32-2 (5 g, 86.6%).

[0429] 2、 Preparation of compound 32-3

[0430] To an ultradry solution of compound 32-2 (5 g, 15.9 mmol) in N,N-dimethylformamide (50 mL), 2,3,5,6-tetrafluorophenol (2.90 g, 17.5 mmol), triethylamine (6.63 mL, 47.7 mmol), and COMU (7.49 g, 17.5 mmol) were added sequentially, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, the reaction solution was extracted with ethyl acetate and water. The organic phase was separated, and the aqueous phase was extracted twice with ethyl acetate. The three organic phases were combined, washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by reversed-phase column chromatography (0-100% water / acetonitrile) to obtain compound 32-3 (4.5 g, 61.2%). LCMS: m / z = 480.3 (M+NH4). +

[0431] 3、 Preparation of compound 32-4

[0432] To a tetrahydrofuran (15 mL) solution of compound 32-3 (4.5 g, 9.73 mmol), an aqueous solution (30 mL) of 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propionic acid (2.26 g, 10.2 mmol) and potassium carbonate (2.69 g, 19.5 mmol) were added sequentially, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, the reaction solution was diluted with water and the pH was adjusted to 3-4, resulting in the precipitation of a solid. The solid was filtered to obtain compound 32-4 (4.2 g, 83.4%). LCMS: m / z = 516.4 (MH) -

[0433] 4、 Preparation of compound EE32

[0434] To an ultradry solution of compound 32-4 (3.1 g, 5.99 mmol) in N,N-dimethylformamide (30 mL), 2,3,5,6-tetrafluorophenol (1.09 g, 6.59 mmol), triethylamine (2.49 mL, 17.9 mmol), and COMU (2.82 g, 6.59 mmol) were added sequentially, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, the reaction solution was extracted with ethyl acetate and water. The organic phase was separated, and the aqueous phase was extracted twice with ethyl acetate. The three organic phases were combined, washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by reversed-phase column chromatography (0-100% water / acetonitrile) to obtain compound EE32 (2.2 g, 55.2%). LCMS: m / z = 666 (M+H) + .

[0435] 1 H NMR (400 MHz, Chloroform-d) δ 7.01 (tt, J = 9.9, 7.0 Hz, 1H), 6.07(s, 1H), 4.05 (t, J = 6.8 Hz, 2H), 3.89 (t, J = 6.2 Hz, 2H), 3.71 – 3.64 (m,8H), 3.64 – 3.61 (m, 4H), 3.55 (dd, J = 5.6, 4.5 Hz, 2H), 3.48 – 3.39 (m,2H), 2.96 (t, J = 6.2 Hz, 2H), 2.19 – 2.12 (m, 2H), 2.04 (s, 3H), 1.62 (p, J= 6.9 Hz, 2H), 1.31 – 1.23 (m, 20H).

[0436] 1.19 Preparation of compound EE33

[0437] 1、 Preparation of compound 33-2

[0438] KOH (7.53 g, 134 mmol) was added to a suspension of compound 33-1 (15 g, 44.7 mmol) in ethylene glycol (50 mL), and the mixture was stirred overnight at 75 °C. Water (100 mL) and ethyl acetate (200 mL) were added to the reaction mixture, and the mixture was separated and extracted three times with ethyl acetate (200 mL). The combined organic phases were washed with saturated brine (200 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated. The residue was subjected to column chromatography (eluting with petroleum ether and ethyl acetate) to give compound 33-2 (9 g, 63.6%). LCMS: m / z = 315.4 (MH) -

[0439] 2. Preparation of compound 33-3

[0440] Under a nitrogen atmosphere, Ac₂O (26.7 mL, 284 mmol) was added to an ultradry dichloromethane (90 mL) solution of compound 33-2 (9 g, 28.4 mmol) and pyridine (46 mL, 569 mmol), and the mixture was stirred at room temperature for two hours. After the reaction was complete, the reaction solution was extracted with ethyl acetate and water. The organic phase was separated, and the aqueous phase was extracted twice with ethyl acetate. The three organic phases were combined, washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (0-60% ethyl acetate / petroleum ether) to obtain compound 33-3 (8.8 g, 86.3%). LCMS: m / z = 357.4 (MH) -

[0441] 3、 Preparation of compound 33-4

[0442] To an ultradry solution of compound 33-3 (8.8 g, 24.5 mmol) in N,N-dimethylformamide (90 mL), 2,3,5,6-tetrafluorophenol (4.48 g, 27.0 mmol), triethylamine (10.2 mL, 73.6 mmol), and COMU (11.6 g, 27.0 mmol) were added sequentially, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, the reaction solution was extracted with ethyl acetate and water. The organic phase was separated, and the aqueous phase was extracted twice with ethyl acetate. The three organic phases were combined, washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by reversed-phase column chromatography (0-100% water / acetonitrile) to obtain 33-4 (6.5 g, 52.3%). LCMS: m / z = 524.3 (M+NH4).+

[0443] 4. Preparation of compound 33-5

[0444] To a tetrahydrofuran (10 mL) solution of compound 33-4 (3.5 g, 6.91 mmol), an aqueous solution (20 mL) of 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propionic acid (1.61 g, 7.26 mmol) and potassium carbonate (1.91 g, 13.8 mmol) were added sequentially, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, the reaction solution was diluted with water and the pH was adjusted to 3-4, resulting in the precipitation of a solid. The solid was filtered to obtain compound 33-5 (3.59 g, 92.5%). LCMS: m / z = 560.4 (MH) -

[0445] 5. Preparation of compound EE33

[0446] To an ultradry solution of compound 33-5 (3.59 g, 6.39 mmol) in N,N-dimethylformamide (35 mL), 2,3,5,6-tetrafluorophenol (1.17 g, 7.03 mmol), triethylamine (2.67 mL, 19.2 mmol), and COMU (3.01 g, 7.03 mmol) were added sequentially, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, the reaction solution was extracted with ethyl acetate and water. The organic phase was separated, and the aqueous phase was extracted twice with ethyl acetate. The three organic phases were combined, washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by reversed-phase column chromatography (0-100% water / acetonitrile) to obtain compound EE33 (2.4 g, 52.9%). LCMS: m / z = 710 (M+H) + .

[0447] 1H NMR (400 MHz, Chloroform-d) δ 7.00 (tt, J = 9.9, 7.0 Hz, 1H), 6.01(s, 1H), 4.25 – 4.19 (m, 2H), 3.89 (t, J = 6.2 Hz, 2H), 3.71 – 3.59 (m, 10H), 3.55 (dd, J = 5.6, 4.5 Hz, 2H), 3.46 (td, J = 6.1, 5.5, 2.4 Hz, 6H), 2.95 (t,J = 6.2 Hz, 2H), 2.19 – 2.13 (m, 2H), 2.08 (s, 3H), 1.59 (dq, J = 13.8, 7.1Hz, 4H), 1.36 – 1.20 (m, 20H).

[0448] 1.20 Preparation of compound EE34

[0449] 1、 Preparation of compound 34-2

[0450] Under a nitrogen atmosphere, COMU (7.5 g, 17.5 mmol) and 2,3,5,6-tetrafluorophenol (2.9 g, 0.175 mmol) were added sequentially to a DMF (60 mL) solution of hexadecyl dicarboxylic acid monobenzyl ester (6.0 g, 15.9 mmol) and Et3N (6.6 mL, 47.8 mmol). After the addition was complete, the mixture was stirred at room temperature for two hours. After the reaction was complete, water was added to quench the reaction. The mixture was extracted three times with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (0-100% ethyl acetate / petroleum ether) to give compound 34-2 (6.2 g, 74.2%).

[0451] 2. Preparation of compound 34-3

[0452] Compound 34-2a (3.54 g, 11.437 mmol) was dissolved in water (50 mL) and THF (25 mL), potassium carbonate (3.16 g, 22.9 mmol) was added, followed by compound 34-2 (6 g, 11.4 mmol). The reaction was carried out overnight at room temperature. After the reaction was complete, the pH was adjusted to ~3, and the mixture was concentrated under reduced pressure to remove THF. The mixture was extracted three times with EA, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was pulped with EA / PE and filtered to obtain compound 34-3 (5.6 g, 8.4 mmol, 73.31%).

[0453] 1 H NMR (400 MHz, DMSO-d6) δ 12.15 (s, 1H), 7.80 (t, J = 5.7 Hz, 1H), 7.40 – 7.30 (m, 5H), 5.08 (s, 2H), 3.59 (t, J = 6.4 Hz, 2H), 3.48 (d, J = 2.6Hz, 16H), 3.38 (t, J = 5.9 Hz, 2H), 3.17 (q, J = 5.8 Hz, 2H), 2.48 (s, 2H), 2.44 (t, J = 6.4 Hz, 2H), 2.34 (t, J = 7.3 Hz, 2H), 2.04 (t, J = 7.4 Hz, 2H),1.50 (dq, J = 27.5, 7.1 Hz, 4H), 1.22 (s, 21H).

[0454] 3. Preparation of compound 34-4

[0455] Under a nitrogen atmosphere, COMU (4.0 g, 9.223 mmol) and 2,3,5,6-tetrafluorophenol (1.5 g, 9.2 mmol) were added sequentially to a DMF (60 mL) solution of compound 34-3 (5.6 g, 8.4 mmol) and Et3N (3.5 mL, 25.2 mmol). After the addition was complete, the mixture was stirred at room temperature for two hours. After the reaction was complete, water was added to quench the reaction. The mixture was extracted three times with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the residue. The residue was then slurried with PE / EA to give compound 34-4 (4.5 g, 65.8%).

[0456] 1 H NMR (400 MHz, DMSO- d6) δ 8.00 – 7.88 (m, 1H), 7.79 (t, J = 5.6 Hz,1H), 7.43 – 7.26 (m, 5H), 5.08 (s, 2H), 3.77 (t, J = 5.9 Hz, 2H), 3.57 – 3.47(m, 16H), 3.38 (t, J = 5.9 Hz, 2H), 3.17 (q, J = 5.8 Hz, 2H), 3.02 (t, J =5.9 Hz, 2H), 2.33 (t, J = 7.3 Hz, 2H), 2.04 (t, J = 7.4 Hz, 2H), 1.50 (dq, J = 27.7, 7.0 Hz, 4H), 1.22 (s, 22H).

[0457] 4. Preparation of compound EE34

[0458] Compound 34-4 (3.5 g, 4.290 mmol) was dissolved in ultradry THF (40 mL), and palladium on carbon (0.46 g, 4.290 mmol) was added. The reaction was carried out overnight under a hydrogen atmosphere. After the starting material disappeared as detected by TLC, the mixture was filtered through diatomaceous earth, concentrated to obtain a crude product, pulped in EA / PE, and filtered to obtain compound EE34 (2.3 g, 3.169 mmol, 73.87%).

[0459] 1H NMR (400 MHz, DMSO-d6) δ 11.96 (s, 1H), 7.94 (tt, J = 10.9, 7.4Hz, 1H), 7.79 (t, J = 5.6 Hz, 1H), 3.77 (t, J = 5.9 Hz, 2H), 3.60 – 3.44 (m,16H), 3.38 (t, J = 5.9 Hz, 2H), 3.17 (q, J = 5.8 Hz, 2H), 3.02 (t, J = 5.9Hz, 2H), 2.18 (t, J = 7.4 Hz, 2H), 2.04 (t, J = 7.4 Hz, 2H), 1.47 (q, J = 7.1Hz, 4H), 1.23 (s, 20H). 19 F NMR (377 MHz, DMSO-d6) δ -139.31, -139.35, -153.29, -153.33.

[0460] 1.21 Preparation of compound EE35

[0461] 1. Preparation of compound 35-2

[0462] DIPEA (4.6 mL, 26.6 mmol) and HATU (5.3 g, 13.9 mmol) were added to a DMF (50 mL) solution of hexadecanoic acid monobenzyl ester (5 g, 13.3 mmol), and the mixture was stirred at room temperature for 1 hour. Then, 3-[(8-amino-3,6-dioxaoct-1-yl)oxy]propanoic acid (4.41 g, 19.918 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction was quenched with water (20 mL), extracted with EA (20 mL x 3), the organic phase was washed with saturated sodium chloride solution (20 mL x 2), dried over anhydrous sodium sulfate, filtered, concentrated, and subjected to reversed-phase column chromatography (water:acetonitrile = 1:0 to 0:1) to give compound 35-2 (7.1 g, 92.2%).

[0463] 2、 Preparation of compound 35-3

[0464] To a DMF (50 mL) solution of compound 35-2 (5.3 g, 9.1 mmol), COMU (4.3 g, 10.1 mmol), TEA (3.8 mL, 27.4 mmol), and 2,3,5,6-tetrafluorophenol (1.8 g, 10.1 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was quenched with water, extracted three times with ethyl acetate, dried over sodium sulfate, filtered, concentrated, and the residue was subjected to silica gel column chromatography (PE:EA = 1:0 to 2:3) to give compound 35-3 (3.4 g, 50.7%).

[0465] 3、 Preparation of compound EE35

[0466] Palladium on carbon (10% Pd, containing 40-60% water) (0.5 g, 4.7 mmol) was added to a tetrahydrofuran (30 mL) solution of compound 35-3 (3.4 g, 4.7 mmol), and the mixture was stirred at room temperature for 16 hours under a hydrogen atmosphere. The reaction solution was filtered and concentrated to give compound EE35.

[0467] 1 H NMR (500 MHz, DMSO-d6) δ 11.95 (s, 1H), 7.94 (tt, J = 10.9, 7.4Hz, 1H), 7.79 (t, J = 5.7 Hz, 1H), 3.77 (t, J = 5.9 Hz, 2H), 3.53 (dddd, J =21.4, 13.2, 5.3, 2.5 Hz, 8H), 3.38 (t, J = 5.9 Hz, 2H), 3.17 (q, J = 5.9 Hz,2H), 3.02 (t, J = 5.9 Hz, 2H), 2.18 (t, J = 7.4 Hz, 2H), 2.03 (t, J = 7.4 Hz, 2H), 1.46 (q, J = 7.4 Hz, 4H), 1.23 (d, J = 6.4 Hz, 20H).

[0468] 1.22 Preparation of compound EE36

[0469] Synthesize compound EE36 using the same method as compound EE34:

[0470] 1H NMR (400 MHz, DMSO-d6) δ 11.96 (s, 1H), 7.95 (tt, J = 11.0, 7.4Hz, 1H), 7.81 (t, J = 5.7 Hz, 1H), 3.77 (t, J = 5.9 Hz, 2H), 3.57 – 3.45 (m,32H), 3.38 (t, J = 5.9 Hz, 2H), 3.17 (q, J = 5.8 Hz, 2H), 3.02 (t, J = 5.9Hz, 2H), 2.18 (t, J = 7.4 Hz, 2H), 2.04 (t, J = 7.4 Hz, 2H), 1.46 (q, J = 7.0Hz, 4H), 1.23 (s, 20H).

[0471] 1.23 Preparation of compound EE37

[0472] Synthesize compound EE37 using the same method as compound EE34:

[0473] 1 H NMR (500 MHz, DMSO- d 6) δ 11.96 (s, 1H), 7.94 (tt, J = 10.9, 7.4 Hz, 1H), 7.79 (t, J = 5.7 Hz, 1H), 3.77 (t, J = 6.0 Hz, 2H), 3.57 – 3.46 (m, 8H), 3.38 (t, J = 5.9 Hz, 2H), 3.17 (q, J = 5.9 Hz, 2H), 3.02 (t, J = 5.9 Hz, 2H), 2.18 (t, J = 7.4 Hz, 2H), 2.03 (t, J = 7.4 Hz, 2H), 1.46 (q, J = 8.2, 7.7 Hz, 4H), 1.22 (s, 24H).

[0474] 1.24 Preparation of compound EE38

[0475] 1. Synthesis of Compound 2

[0476] Compound 1 (5 g, 11.3 mmol) was dissolved in dry DCM (50 mL), and DIPEA (4.36 g, 33.8 mmol) and TSTU (3.7 g, 12.4 mmol) were added. The reaction solution was stirred at room temperature for 1 hour. After thin-layer chromatography showed complete conversion of the starting material to the intermediate, compound 1a (1.63 g, 12.401 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the solution was concentrated under reduced pressure and purified by reverse-phase C18 column chromatography (ACN:H2O, 0-40%-50%) to give compound 2 (6.1 g, 97.2%).

[0477] 2、 Synthesis of Compound 3

[0478] Compound 2 (4.8 g, 8.6 mmol) was dissolved in dry DMF (50 mL), and DIPEA (3.3 g, 25.9 mmol) and HATU (3.6 g, 9.5 mmol) were added. After reacting at room temperature for 0.5 hours, compound 2a (4.3 g, 9.5 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, water was added. The mixture was extracted three times with ethyl acetate (20 mL), and the organic phases were combined, dried, filtered, and concentrated. The solution was purified by silica gel column chromatography to give compound 3 (8.0 g, 94.0%).

[0479] 3、 Synthesis of compound 3a

[0480] Compound 3 (8 g, 8.101 mmol) was dissolved in DCM (60 mL), and piperidine (12 mL) was added. The mixture was stirred at room temperature for 2 hours. Thin-layer chromatography showed that the reaction was complete, and water was added. The mixture was extracted three times with DCM, the organic layers were combined, washed with saturated sodium chloride, dried over anhydrous Na₂SO₄, filtered, and concentrated to dryness. C16 reversed-phase column chromatography was used to purify compound 3a (4.7 g, 75.8%).

[0481] 4、 Preparation of compound 5

[0482] Acetic anhydride (3.925 mL, 41.60 mmol) was added to a solution of compound 4 (5 g, 16.64 mmol) in DCM (30 mL) and pyridine (20 mL), and the mixture was stirred at room temperature for 1 hour. The reaction solution was concentrated to give compound 5 (5.3 g, 93.0%).

[0483] 5. Preparation of Compound 6

[0484] To a DCM (50 mL) solution of compound 5 (5.3 g, 15.47 mmol), tetrafluorophenol (2.83 g, 17.02 mmol), COMU (7.29 mg, 17.02 mmol), and TEA (4.70 g, 46.42 mmol) were added, and the mixture was stirred at 25 °C for 2 hours. Water (50 mL) was added to the reaction mixture, and the mixture was separated. The mixture was extracted three times with dichloromethane (50 mL), and the combined organic phases were washed with saturated brine (50 mL), dried over sodium sulfate, filtered, and the concentrated filtrate was subjected to column chromatography (eluent: petroleum ether and ethyl acetate) to give compound 6 (5.0 g, 65.9%). m / z: ES + [M+H] + 490.31

[0485] 6. Preparation of Compound 7

[0486] To a solution of compound 3a (1.5 g, 1.96 mmol) in tetrahydrofuran (15 mL), diisopropylethylamine (1.03 mL, 5.87 mmol) and compound 6 (1.15 g, 2.35 mmol) were added, and the mixture was stirred at room temperature for one hour. The reaction solution was concentrated and subjected to reversed-phase column chromatography (eluents: water and methanol) to give compound 7 (2.1 g, 98%). m / z: ES+[M+H]+ 1090.71

[0487] 7. Preparation of compound EE38

[0488] At 0 °C, DIEA (1.9 mL, 11.55 mmol), DMAP (0.05 g, 0.38 mmol), and compound 7a (1.16 g, 11.555 mmol) were added to a DCM (20 mL) solution of compound 7 (2.1 g, 1.93 mmol). After stirring at room temperature for 2 hours, the solution was concentrated, and the residue was purified by reverse-phase C18 column chromatography (eluent: water and methanol) to give compound EE38 (1.58 g, yield: 68.9%). m / z: ES-[MH] - 1188.74.

[0489] 1 H NMR (400 MHz, Chloroform- d ) δ 7.45 – 7.35 (m, 2H), 7.33 – 7.26 (m,5H), 7.25 – 7.15 (m, 2H), 6.98 – 6.74 (m, 5H), 6.39 (d, J = 18.4 Hz, 1H),4.29 – 4.10 (m, 1H), 4.05 (t, J = 6.8 Hz, 3H), 3.88 (s, 2H), 3.78 (d, J = 1.8Hz, 6H), 3.75 – 3.69 (m, 2H), 3.64 – 3.58 (m, 7H), 3.54 (q, J = 5.1 Hz, 3H),3.47 – 3.38 (m, 3H), 3.34 – 2.88 (m, 7H), 2.60 – 2.39 (m, 6H), 2.36 – 2.12(m, 4H), 2.04 (s, 3H), 1.61 (q, J = 6.2, 5.0 Hz, 4H), 1.46 (dd, J = 15.8, 8.7Hz, 2H), 1.37 – 1.19 (m, 30H).

[0490] 1.25 Preparation of compound EE39

[0491] Synthesize compound EE39 using the same method as compound EE38:

[0492] 1H NMR (400 MHz, Chloroform-d) δ 7.33 – 7.24 (m, 7H), 7.19 – 7.14 (m,3H), 6.86 – 6.79 (m, 4H), 6.29 (d, J = 7.6 Hz, 1H), 4.35 (dd, J = 11.2, 7.5Hz, 1H), 4.17 – 4.00 (m, 2H), 3.86 (td, J = 6.1, 3.5 Hz, 1H), 3.80 (s, 6H), 3.77 – 3.66 (m, 6H), 3.63 (d, J = 2.9 Hz, 8H), 3.55 (d, J = 5.3 Hz, 2H), 3.44(q, J = 5.3 Hz, 2H), 3.31 – 3.21 (m, 2H), 3.15 (q, J = 7.4 Hz, 2H), 2.64 (d,J = 5.8 Hz, 2H), 2.48 (t, J = 5.5 Hz, 2H), 2.35 (p, J = 7.6 Hz, 2H), 2.18(dd, J = 8.9, 6.4 Hz, 2H), 1.62 (h, J = 7.4 Hz, 4H), 1.48 – 1.44 (m, 4H), 1.30 – 1.21 (m, 30H), 0.88 (t, J = 6.7 Hz, 3H).

[0493] 1.26 Preparation of compound EE40

[0494] Synthesize compound EE40 using the same method as compound EE38:

[0495] 1H NMR (400 MHz, Chloroform-d) δ 7.37 – 7.35 (t, J = 9.0 Hz, 3H), 7.28 – 7.10 (m, 5H), 6.82 (td, J = 5.4, 2.5 Hz, 5H), 5.44– 5.28 (m, 8H), 4.25(ddd, J = 34.4, 11.4, 7.2 Hz, 3H), 4.17 – 3.98 (m, 1H), 3.79 (d, J = 3.0 Hz, 6H), 3.73 (t, J = 5.9 Hz, 2H), 3.62 (d, J = 5.2 Hz, 9H), 3.55 (t, J = 5.2 Hz, 2H), 3.45 (dq, J = 15.9, 5.9, 5.2 Hz, 4H), 3.23 (p, J = 7.7, 7.2 Hz, 2H), 3.16 – 3.07 (m, 1H), 2.90 (q, J = 7.3 Hz, 1H), 2.82 (dq, J = 9.9, 4.3, 3.0Hz, 6H), 2.67 – 2.51 (m, 3H), 2.45 (q, J = 6.2 Hz, 2H), 2.28 (d, J = 10.0 Hz,1H), 2.19 (p, J = 7.9, 7.5 Hz, 2H), 2.07 (ddd, J = 25.7, 12.2, 5.8 Hz, 4H), 2.04 (s, 3H), 1.71 (p, J = 7.5 Hz, 2H), 1.55 (ddq, J = 44.3, 15.0, 7.6, 7.1Hz, 3H), 1.41 – 1.23 (m, 16H)

[0496] 1.27 Preparation of compound EE41

[0497] Synthesize compound EE41 using the same method as compound EE38:

[0498] 1H NMR (400 MHz, Chloroform-d) δ 7.45 – 7.37 (m, 2H), 7.34 – 7.24 (m,6H), 6.89 – 6.78 (m, 5H), 4.42 (dd, J = 10.9, 8.8 Hz, 1H), 4.24 – 4.18 (m,2H), 4.11 – 3.99 (m, 2H), 3.89 (ddt, J = 17.8, 8.3, 3.9 Hz, 3H), 3.79 (d, J =2.8 Hz, 6H), 3.73 (td, J = 5.9, 2.6 Hz, 2H), 3.63 (d, J = 4.5 Hz, 12H), 3.58– 3.51 (m, 4H), 3.46 (hept, J = 5.4 Hz, 8H), 3.29 – 3.05 (m, 4H), 2.68 – 2.53(m, 3H), 2.46 (q, J = 5.5 Hz, 2H), 2.37 – 2.22 (m, 1H), 2.17 (t, J = 7.7 Hz, 2H), 2.08 (s, 2H), 1.68 – 1.46 (m, 9H), 1.26 (d, J = 14.1 Hz, 22H).

[0499] 1.28 Preparation of compound EE42

[0500] Synthesize compound EE42 using the same method as compound EE38:

[0501] 1 H NMR (400 MHz, DMSO- d 6) δ 12.23 (s, 1H), 7.85 – 7.70 (m, 2H), 7.38(dd, J = 8.1, 3.6 Hz, 2H), 7.31 (t, J = 7.4 Hz, 2H), 7.27 – 7.20 (m, 5H), 6.89 (dd, J = 8.5, 1.9 Hz, 4H), 4.16 (dd, J= 11.6, 6.6 Hz, 1H), 4.11 – 4.07(m, 2H), 3.99 – 3.86 (m, 2H), 3.83 – 3.76 (m, 1H), 3.74 (s, 6H), 3.62 – 3.54(m, 4H), 3.54 – 3.51 (m, 2H), 3.50 – 3.45 (m, 8H), 3.37 (td, J = 6.3, 2.8 Hz, 6H), 3.17 (q, J = 5.8 Hz, 2H), 3.00 (p, J = 6.7 Hz, 4H), 2.48 – 2.43 (m, 2H), 2.31 – 2.18 (m, 4H), 2.03 (t, J = 7.4 Hz, 2H), 2.00 (s, 3H), 1.51 – 1.40 (m,6H), 1.40 – 1.31 (m, 4H), 1.22 (d, J = 2.2 Hz, 28H).

[0502] 1.29 Preparation of compound EE43

[0503] Synthesize compound EE43 using the same method as compound EE38:

[0504] 1H NMR (400 MHz, Chloroform-d) δ 7.46 – 7.38 (m, 2H), 7.37 – 7.24 (m,7H), 7.21 (q, J = 8.4, 7.3 Hz, 1H), 6.82 (dd, J = 4.4, Hz), 6 (s,1H), 4.37 (dd, J = 11.0, 8.5 Hz, 1H), 4.15 – 3.83 (m, 6H), 3.79 (d, J = 3.2Hz, 6H), 3.73 (t, J = 5.8 Hz, 2H.8), J = 3.6 – 3.40 (m,7H), 3.34 – 3.03 (m, 6H), 2.92 (q, J = 7.3 Hz, 1H), 2.57 (dt, J = 9.9, 6.7Hz, 2H), 2.46 (q, J = 5.0 Hz, 2. 4.1). 16.0, 8.4 Hz, 2H), 2.17(t, J = 7.7 Hz, 2H), 2.04 (s, 3H), 1.61 (p, J = 6.8 Hz, 6H), 1.54 – 1.45 (m,2H), 1.327 (H), 1.32H)., m 1.1.

[0505] 1.30 EE44's house

[0506] 1. Preparation of compound 44-2

[0507] To a DMF (40 mL) solution of compound 44-1 (2.23 g, 9.795 mmol), DIEA (4.415 mL, 26.713 mmol) and HATU (3.72 g, 9.795 mmol) were added. After reacting at room temperature for half an hour, 6-[(trifluoroacetyl)amino]hexanoic acid (4 g, 8.904 mmol) was added, and the reaction mixture was allowed to react overnight at room temperature. After the reaction was monitored by LCMS and TLC to indicate completion, ethyl acetate and water were added to the reaction mixture. The organic phase was separated, and the aqueous phase was extracted twice with ethyl acetate. The three organic phases were combined, washed once with saturated brine, dried over anhydrous sodium sulfate, and filtered. The residue was purified by rapid column chromatography (0-90% EA in PE) to give compound 44-2 (4.73 g, 80.67%). LCMS: m / z = 659.36 (M+H) + .

[0508] 2. Preparation of compound 44-3

[0509] Compound 44-2 (6.59 g, 10.011 mmol) was dissolved in 7M NH3 / MeOH (30 mL) and concentrated ammonia (30 mL), sealed in a tube, and incubated at 78°C. o The mixture was heated overnight at C. The reaction was monitored for completeness by TLC and LCMS. The solvent was concentrated to dryness under reduced pressure, and the residue was purified by reversed-phase rapid column chromatography (0-50% ACN aqueous solution) to give compound 44-3 (5.5 g, 97.71%). LCMS: m / z = 563.24 (M+H) + .

[0510] 3. Preparation of compound 44-4

[0511] Triethylamine (1.55 g, 15.294 mmol) and compound EE20 (4.65 g, 7.647 mmol) were added to an ultradry dichloromethane (10 mL) solution of compound 44-3 (4.3 g, 7.647 mmol). The reaction mixture was stirred at room temperature for half an hour. The reaction was monitored for completeness by LCMS and TLC. The solvent was concentrated to dryness under reduced pressure, and the residue was purified by reversed-phase column chromatography (0-100% ACN aqueous solution) to obtain compound 44-4 (8.6 g, 112.05%). LCMS: m / z = 1021.79 (M+NH4). + .

[0512] 4. Preparation of compound EE44

[0513] To a solution of compound 44-4 (8.6 g, 8.569 mmol) in dichloromethane (80 mL), DIEA (6.63 g, 51.412 mmol), DMAP (0.31 g, 2.571 mmol), and succinic anhydride (5.14 g, 51.412 mmol) were added, and the reaction mixture was stirred at room temperature for 1 hour. After the reaction was complete as monitored by TLC and LCMS, the solvent was concentrated to dryness under reduced pressure. The residue was purified by reversed-phase rapid column chromatography (0-100% MeOH aqueous solution) to obtain EE44 (8.4 g, 7.611 mmol, 88.82%). LCMS: m / z = 1104.76 (M+H) + .

[0514] 1 H NMR (400 MHz, DMSO- d6) δ 7.82 – 7.33 (m, 2H), 7.41 – 7.35 (m, 2H), 7.31 (t, J = 7.5 Hz, 2H), 7.27 – 7.19 (m, 4H), 6.89 (dd, J = 8.8, 1.8 Hz, 4H), 4.16 (dd, J = 11.6, 6.7 Hz, 1H), 4.06 (dd, J = 11.6, 4.9 Hz, 1H), 3.94 (dd, J = 11.8, 4.8 Hz, 2H), 3.79 (dt, J = 11.2, 6.0 Hz, 1H), 3.74 (s, 6H),3.63 – 3.52 (m, 4H), 3.52 – 3.44 (m, 8H), 3.42 – 3.35 (m, 6H), 3.17 (q, J =5.8 Hz, 2H), 3.05 – 2.95 (m, 4H), 2.48 – 2.43 (m, 2H), 2.28 (td, J = 6.7, 4.7Hz, 2H), 2.04 (t, J = 7.4 Hz, 2H), 1.51 – 1.40 (m, 4H), 1.40 – 1.30 (m, 1H), 1.23 (d, J = 2.9 Hz, 28H), 1.00 (d, J = 6.6 Hz, 1H), 0.88 – 0.82 (m, 3H).

[0515] 1.31 Preparation of compound EE45

[0516] Compound EE45 was synthesized using the same method as compound EE44. The synthesis method for DL0343 can be found, for example, in PCT Publication No. WO2024148329.

[0517] 1H NMR (400 MHz, Chloroform-d) δ 7.40 – 7.37 (t, J = 9.0 Hz, 3H),7.35 – 7.16 (m, 5H), 6.82 (td, J = 5.4, 2.5 Hz, 5H), 5.47 – 5.27 (m, 8H),4.25 (ddd, J = 34.4, 11.4, 7.2 Hz, 1H), 4.17 – 3.98 (m, 1H), 3.79 (d, J = 3.0Hz, 6H), 3.73 (t, J = 5.9 Hz, 2H), 3.62 (d, J = 5.2 Hz, 9H), 3.55 (t, J = 5.2Hz, 2H), 3.45 (dq, J = 15.9, 5.9, 5.2 Hz, 4H), 3.23 (p, J = 7.7, 7.2 Hz, 2H), 3.16 – 3.07 (m, 1H), 2.90 (q, J = 7.3 Hz, 1H), 2.82 (dq, J = 9.9, 4.3, 3.0Hz, 6H), 2.67 – 2.51 (m, 3H), 2.45 (q, J = 6.2 Hz, 2H), 2.28 (d, J = 10.0 Hz,1H), 2.19 (p, J = 7.9, 7.5 Hz, 2H), 2.07 (ddd, J = 25.7, 12.2, 5.8 Hz, 4H), 1.71 (p, J = 7.5 Hz, 2H), 1.55 (ddq, J = 44.3, 15.0, 7.6, 7.1 Hz, 3H), 1.41 –1.23 (m, 16H), 0.89 (t, J = 6.7 Hz, 3H).

[0518] 1.32 Preparation of compound EE46

[0519] Compound EE46 was synthesized using the same method as compound EE44. The synthetic method for 46-1 can be found, for example, in PCT Publication No. WO2024148329.

[0520] 1H NMR (400 MHz, DMSO-d6) δ 8.26 – 7.93 (m, 1H), 7.79 (q, J = 5.6 Hz,1H), 7.37 (dd, J = 7.9, 3.3 Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 7.24 (dd, J =8.8, 4.9 Hz, 5H), 6.88 (d, J = 8.4 Hz, 4H), 4.13 (dd, J = 12.4, 7.4 Hz, 1H), 4.08 – 4.00 (m, 1H), 3.93 – 3.86 (m, 1H), 3.78 (t, J = 5.1 Hz, 1H), 3.73 (s,6H), 3.65 – 3.48 (m, 6H), 3.15 (q, J = 5.8 Hz, 2H), 3.01 (dd, J = 12.5, 6.3Hz, 4H), 2.43 (q, J = 7.7 Hz, 2H), 2.34 – 2.18 (m, 6H), 2.06 – 2.00 (m, 2H), 1.98 (s, 3H), 1.58 – 1.50 (m, 2H), 1.49 – 1.34 (m, 6H), 1.32 – 1.14 (m, 32H).

[0521] According to the preparation method of the representative embodiments described above, all compounds shown in the specification can be easily prepared using commercially available raw material compounds and in combination with common knowledge in the art.

[0522] Intermediate compounds containing a tetrafluorophenol protecting group can also be conjugated to nucleic acid chains using the following strategies, which are also included within the scope of this invention.

[0523] 1. General suffix combination

[0524] 2. Symmetrical molecular post-combination (can also be done by sequentially combining single-chain molecules, followed by purification and annealing)

[0525] 3. Asymmetric suffix combination

[0526] 4. Multi-step suffix combination

[0527] Example 2: Synthesis of siRNA

[0528] The siRNA of the present invention was prepared using the solid-phase phosphoramide method well known in the art. Specific methods can be found, for example, in PCT publications WO2016081444 and WO2019105419, and are briefly described below.

[0529] 1.1 Synthesis of the Justice Chain (SS Chain)

[0530] The oligonucleotide was synthesized using a solid-phase phosphoramide method, employing a blank CPG solid support as the starting cycle. Nucleoside monomers (including the monomer compounds of this invention) were sequentially linked from the 3'-5' direction according to the nucleotide arrangement of the positive strand. Each linkage of a nucleoside monomer involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis scale was 5 μmol / L of oligonucleotides. The synthesis conditions are as follows: The nucleoside monomers were provided in a 0.05 mol / L acetonitrile solution, and the reaction conditions were identical for each step, i.e., a temperature of 25°C. o C. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution; the coupling reaction was activated twice using a 0.25 mol / L 5-ethylthiotetrazole (ETT)-acetonitrile solution; capping was performed twice using a 10% acetic anhydride-acetonitrile and pyridine / N-methylimidazolium / acetonitrile solution (10:14:76, v / v / v); oxidation was performed twice using a 0.05 mol / L iodine solution in tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v); thiolation was performed twice using a 0.2 mol / L phenylacetyl disulfide (PADS) solution in acetonitrile / 3-methylpyridine (1 / 1, v / v).

[0531] 1.2 Synthesis of the antisense chain (AS chain)

[0532] The solid-phase phosphoramide synthesis method utilizes a blank CPG solid-phase support as the starting cycle, and nucleotide monomers (including the monomer compounds of this invention) are sequentially linked from the 3'-5' direction according to the antisense strand nucleotide arrangement sequence. Each linkage of a nucleotide monomer involves four steps: deprotection, coupling, capping, oxidation, or thiolation. The synthesis conditions for 5 μmol oligonucleotides of the antisense strand are the same as those for the sense strand.

[0533] 1.3 Purification and Annealing of Oligonucleotides

[0534] 1.3.1 Ammonolysis

[0535] Add the synthesized solid support (sense or antisense chain) to a 5 mL centrifuge tube, then add 3% diethylamine / ammonia (v / v) or a mixed solution of ammonia and methylamine (50%-50%, v / v). Continue for 35 minutes. o C (or 55) oC) React in a constant temperature water bath for 16 hours (or 8 hours), filter, wash the solid support three times with ethanol / water, 1 mL each time, centrifuge and concentrate the filtrate, and then purify the crude product.

[0536] 1.3.2 Purification

[0537] Purification and desalting methods are well known to those skilled in the art. For example, a column packed with strong anion exchange material can be used for elution purification with a sodium chloride-sodium hydroxide system, the product collected and tubed, and desalting can be performed using a gel-packed purification column with pure water as the elution system.

[0538] 1.3.3 Annealing

[0539] According to the instructions, mix the sense chain (SS chain) and the antisense chain (AS chain) at a molar ratio of 1 / 1.05, and heat in a water bath to 70-95°C. o C, keep for 3-5 minutes, cool naturally to room temperature, and freeze-dry the system to obtain the product.

[0540] Test Implementation Example Sequence Information

[0541] The meanings of the abbreviations in this article are as follows: A, U, G, and C represent natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide, and cytosine ribonucleotide, respectively.

[0542] The 'd' indicates that the nucleotide adjacent to its right is a deoxyribonucleotide. For example, dA, dT, dG, and dC represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and cytosine deoxyribonucleotide, respectively.

[0543] i represents inosine ribonucleotide.

[0544] The 'm' indicates that the nucleotide adjacent to it on the left is a nucleotide modified with 2'-OCH3. For example, Am, Um, Gm, and Cm represent A, U, G, and C modified with 2'-OCH3, respectively.

[0545] The 'f' indicates that the nucleotide adjacent to it on the left is a 2'-F modified nucleotide. For example, Af, Uf, Gf, and Cf represent 2'-F modified A, U, G, and C, respectively.

[0546] “s” indicates that the two adjacent nucleotides, delivery vectors or other structures are linked by thiophosphate.

[0547] VP indicates that the nucleotide adjacent to its right is a vinyl phosphate modified nucleotide.

[0548] U(od) indicates that the hydrogen atom at the 2' position of the hydroxyl group of the natural uracil ribonucleotide has been replaced by a straight C22 aliphatic chain.

[0549] NHC6 and C6NH, when near the 5' end, are... This indicates that when it is closer to the 3' end, it is represented as .

[0550] C6S indicates .

[0551] MO1 indicates .

[0552] MO2 indicates .

[0553] -DCBO-DL0269 indicates .

[0554] -C6-NH-DL0312 indicates the following structure: , Starting with commercially available intermediates (structures shown below, available from, for example, PharmaBlock catalog number PBU1072), those skilled in the art can readily synthesize -C6-NH-DL0312.

[0555]

[0556] The structures of LL30-LL41 and LL117-LL120 are shown in the table below, where, This indicates that the oligonucleotide is linked to the remaining portion via a phosphate ester group or a thiophosphate ester group. Other structures beginning with LL are shown in this specification.

[0557] The preparation methods of the corresponding intermediate compounds of LL117-LL120 can be found in, for example, PCT Publication No. WO2024148329.

[0558]

[0559] Example 3: In vivo activity assay

[0560] Experimental methods

[0561] Male SD rats (8-10 weeks old) were randomly divided into two groups: a solvent control group (aCSF) with 3 rats per group and a compound group with 5 rats per group. The compound was prepared using sterile artificial cerebrospinal fluid at a concentration of 30 mg / mL. After anesthesia, the corresponding compound was administered via intrathecal injection (L4-L6 level) at a dose of 0.9 mg / rat (30 µL / rat).

[0562] On day 14 after drug administration, the animals were euthanized by CO2, and the following tissues were rapidly isolated and removed: brainstem, hippocampus, and frontal cortex. After the samples were removed, they were placed in 5-10 times the volume of RNAlater and incubated overnight at 2-8°C, and then transferred to -80°C for storage for subsequent detection of rSOD1 mRNA.

[0563] RNA was extracted from tissues using a nucleic acid extractor (Hangzhou Aosheng, Auto-pure96) following the instructions of the high-throughput tissue RNA extraction kit (Shanghai Fushen Biotechnology, FSF0035-TS). Reverse transcription was performed using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Quantitative PCR (ABI, QuantStudio3) was conducted using a 20 μL TaqMan™ Fast Advanced Master Mix (ABI, 4444965) for detection. Primer information is as follows: Table 1: Primer sequences

[0564] The residual inhibition rate is calculated using the following formula: Calculate 2 -△△Ct The value is then converted into a percentage to obtain the residual inhibition rate.

[0565] △△Ct=[(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)].

[0566] The target gene was rSOD1, the internal control was rGAPDH, and the control group was injected with artificial cerebrospinal fluid (aCSF).

[0567] Experimental results

[0568] Table 2. Inhibitory activity of the compounds of the present invention against SOD1 mRNA in different brain regions of SD rats (presented by residual inhibition rate).

[0569] Table 3. Inhibitory activity of the compounds of the present invention against SOD1 mRNA in different brain regions of SD rats (presented by residual inhibition rate).

[0570] Table 4. Inhibitory activity of the compounds of the present invention against SOD1 mRNA in different brain regions of SD rats (presented by residual inhibition rate).

[0571] Table 5. Inhibitory activity of the compounds of the present invention against SOD1 mRNA in different brain regions of SD rats (presented as residual inhibition rate).

[0572] Example 4: Peripheral drug delivery bioactivity test

[0573] Experimental methods

[0574] C57BL / 6 (male, 6-8 weeks old) mice were randomly divided into two groups: a solvent control group (n=3 per group) and a compound group (n=3 per group). The compound was prepared using physiological saline at a concentration of 5 mg / mL. The appropriate compound was administered subcutaneously at a dose of 5 mg / kg unless otherwise specified.

[0575] On day 14 or 28 post-drug administration, animals were euthanized with CO2, and the following tissues were rapidly isolated and removed: subcutaneous fat, gonadal fat, quadriceps femoris muscle, heart, liver, kidney, lung, spleen, and brain. After sample removal, the samples were divided into two portions, flash-frozen in liquid nitrogen, and then transferred to -80°C for storage for subsequent detection of mSOD1-mRNA levels.

[0576] Tissue RNA extraction was performed using a nucleic acid extractor (Hangzhou Aosheng, Auto-pure96) following the instructions of the High-Throughput Tissue RNA Extraction Kit (Shanghai Fushen Biotechnology, FSF0035-TS). (Special note: For adipose tissue: Transfer the adipose tissue to a 2ml centrifuge tube pre-filled with zirconium beads, add 1ml Trizol (Takara, 9108), and homogenize thoroughly using a tissue homogenizer (-5℃, 60Hz, 15min). After homogenization, centrifuge at high speed (12000 rpm) for 3min, carefully discard the upper oil phase with a pipette tip, and then centrifuge again at high speed (12000 rpm) for 3min. Use 350µl of tissue lysis supernatant for RNA extraction. For non-adipose tissue, follow the kit instructions.) Reverse transcription was performed using the PrimeScript™ II 1st Strand cDNASynthesis Kit (Takara, 6210B); TaqMan™ Fast Advanced Master Mix was also used. A 20 μL volume of (ABI, 4444965) was used for real-time PCR (ABI, QuantStudio3). The primers used are as follows: Table 6. Primer Sequences

[0577] The residual inhibition rate is calculated using the following formula: Calculate 2 -△△Ct The value is then converted into a percentage to obtain the residual inhibition rate.

[0578] △△Ct=[(Ct experimental group target gene-Ct experimental group internal reference)-(Ct control group target gene-Ct control group internal reference)].

[0579] The target gene was either mSOD1 or mAdipoq, selected according to the target of the compound. The internal control was mGAPDH, and the control group was injected with physiological saline. The experimental results are shown in the table below.

[0580] Table 7. Inhibitory effects of the compounds of the present invention on SOD1 in multiple tissues, results shown as residual inhibition rate (%)

[0581] Table 8. Inhibitory effects of the compounds of the present invention on SOD1 in multiple tissues.

[0582] pgWAT: Peri-abdominal white adipose tissue; iWAT: Inguinal white adipose tissue

[0583] / indicates no detection

[0584] Table 9. Inhibitory effects of the compounds of the present invention on SOD1 in multiple tissues.

[0585] pgWAT: Peri-abdominal white adipose tissue; iWAT: Inguinal white adipose tissue

[0586] Table 10. Inhibitory effects of the compounds of the present invention on SOD1 in multiple tissues.

[0587] pgWAT: Peri-abdominal white adipose tissue; iWAT: Inguinal white adipose tissue

[0588] Table 11. Inhibitory effects of the compounds of the present invention on Adipoq in multiple tissues (dose 2 mg / kg)

[0589] pgWAT: Peri-abdominal white adipose tissue; iWAT: Inguinal white adipose tissue; BAT: Brown adipose tissue

[0590] Example 5: Peripheral drug delivery bioactivity assay – protein level detection

[0591] C57BL / 6 (female, 6-8 weeks old) mice were randomly divided into two groups: a solvent control group (n=4 per group) and a compound group (n=4 per group). The compound was prepared using physiological saline at a concentration of 5 mg / mL. The corresponding compound was administered subcutaneously at a dose of 2 mg / kg.

[0592] Blood / samples were collected from the orbital venous plexus of mice before administration (referred to as day 0) and on days 14, 21 and 28 after administration (the sampling time and method were adjusted according to different experimental needs). Serum was collected at each time point to detect MouseAdiponectin protein.

[0593] ELISA assay: Detect serum Mouse Adiponectin protein (R&D, SMRP300) according to the ELISA kit instruction manual.

[0594] Table 12. Inhibitory activity of the compounds of the present invention against Mouse Adiponectin protein.

[0595] Table 13. Inhibitory activity of the compounds of the present invention against Mouse Adiponectin protein

[0596] Table 14. Inhibitory activity of the compounds of the present invention against Mouse Adiponectin protein

[0597] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. An oligonucleotide, wherein the oligonucleotide has at least 15 nucleotides sufficiently complementary to a target mRNA, the oligonucleotide comprising two identical or different hydrophobic groups located at the 5' and 3' ends of the oligonucleotide, respectively, each hydrophobic group being independently selected from compounds of formula (I) or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof: (I) in Indicates the position where it is linked to the remainder of the oligonucleotide; R is a lipid, preferably a lipid having 10 to 30 carbon atoms; T is selected from hydrogen, hydroxyl, amino, carboxyl, sulfonic acid group (-S(O)2OH), C 1-6 Acetoxy, C 1-6 Alkyl, C 1-6 Alkyl or 5-10 heteroaryl groups, preferably, T is selected from amino, sulfonic acid (-S(O)2OH), C 1-6 Acetoxy, C 1-6 Alkyl, C 1-6 Alkoxy or 5-10 heteroaryl groups; The T is optionally composed of 1, 2, or 3 molecules selected from hydroxyl, amino, C 1-6 Alkyl hydroxyl, C 1-6 Alkyl or C 1-6 Substituents of haloalkyl groups; Preferably, R is a lipid with 10 to 30 carbon atoms; T is selected from hydrogen, hydroxyl, acetoxy, carboxyl, sulfonic acid, tetrazolium, or optionally substituted C. 1-6 Alkoxy, preferably, the C 1-6 The alkoxy group is optionally surrounded by 1, 2, or 3 groups selected from hydroxyl, amino, C 1-6 Alkyl hydroxyl, C 1-6 Alkyl or C 1-6 Substituents of haloalkyl groups.

2. The oligonucleotide of claim 1, wherein the lipid comprises 10-30 carbon atoms, preferably 12, 14, 16, 18, 20 or 22 carbon atoms.

3. The oligonucleotide of claim 1 or 2, wherein the lipid is a linear lipid optionally comprising 1-8 olefinic bonds, the lipid optionally being substituted with one or more hydroxyl or oxo groups; preferably, the lipid is optionally substituted with one, two, three, four or five substituents independently selected from hydroxyl and oxo groups.

4. The oligonucleotide of any one of claims 1-3, wherein, The R is selected from C. 8-28 Straight-chain alkyl or C 8-28 Straight-chain alkenyl, the C 10-28 straight-chain alkyl and C 8-28 The straight-chain alkenyl group may optionally be substituted with one, two, three, four or five independent substituents selected from hydroxyl and oxo groups; Preferably, R is selected from C 10-28 Straight-chain alkyl groups or C groups containing 1-4 olefinic bonds 8-28 Straight-chain alkenyl, the C 10-28 Straight-chain alkyl groups or C groups containing 1-4 olefinic bonds 8-28 The straight-chain alkenyl group is optionally substituted by one, two, or three substituents independently selected from hydroxyl and oxo groups; More preferably, R is selected from -C(O)-C11-, -C(O)-C12-, -C(O)-C13-, -C(O)-C14-, -C(O)-C15-, -C(O)-C16-, -C(O)-C17-, -C(O)-C18-, -C(O)-C19-, -C(O)-C20-, -C(O)-C21-, -C(OH)-C14-, -C(O)-C19:4-, -C(O)-C7-C=C-C8-, -CC(OH)-C14-, -C14- and -C16-.

5. The oligonucleotide of any one of claims 1-4, wherein, The T is selected from sulfonic acid group, tetrazolium group (e.g., ...). ) or optional replacement of C 1-3 Alkoxy, preferably, the C 1-3 Alkyl groups are optionally surrounded by one or two groups selected from hydroxyl and C. 1-3 Substituents of alkyl hydroxyl groups (e.g., -CH2OH).

6. The oligonucleotide of any one of claims 1-5, wherein, The T is C 1-3 Alkoxy, the C 1-3 The alkoxy group is optionally substituted with one or two substituents selected from hydroxyl and -CH2OH; More preferably, the T is selected from -OH, -C(O)OH, -OCH3, -OCH2CH2OH, -OCH2CH(OH)CH2OH, -OCH(CH2OH)2, -S(O)2OH and -tetrazole, preferably -OCH3, -OCH2CH2OH, -OCH2CH(OH)CH2OH, -OCH(CH2OH)2, -S(O)2OH and .

7. The oligonucleotide of any one of claims 1-6, wherein -RT are each independently selected from: , , , , , , , , , , , , , or ; Where m and n can be any integers, provided that R contains a total of 10-30 carbon atoms; Preferably, Each m and n is independently selected from integers from 0 to 50, preferably from integers from 3 to 25, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25; R contains a total of 10 to 25 carbon atoms, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 carbon atoms, preferably 12, 14, 16, 18, 20 or 22 carbon atoms.

8. The oligonucleotide of any one of claims 1-7, wherein the oligonucleotide is selected from compounds of formula (III) or their pharmaceutically acceptable salts, tautomers or stereoisomers: (III) in, The T' terminus corresponds to the 5' end of the oligonucleotide, and the T' terminus corresponds to the 3' end of the oligonucleotide. Z is an oligonucleotide; L1 and L1' are each independently selected from chemical bonds or -L x -L y -L z -; Among them, L x Selected from chemical bonds, -O-, -S-, -C(O)-, -NR a -、-C(O)NR a -or-NR a C(O)-; L y Selected from C 1-10 Alkylene, the C 1-10 The alkylene group is optionally selected from -C by one, two, or three. 0-6 alkylene -OH, -C 0-6 Alkylene -NH2, -C 0-6 Alkylene-CN, C 1-4 Alkyl or C 1-4 Halogenated alkyl substitution; L z Selected from chemical bonds, -O-, -S-, -SS-, -C(O)-, -NR a -、-C(O)NR a -or-NR a C(O)-; L x and L z At most one of them is a chemical bond; Each R a Independently selected from H and C 0-6 alkylene -OH, -C 0-6 Alkylene -NH2, -C 0-6 Alkylene-CN, C 1-6 Alkyl or C 1-6 Halogenated alkyl L2 and L2' are each independently -L a -L b -L c -L d -L e -, preferably selected from -L a -L b -L c -L d -L e -、-L b -L c -L d -L e -、-L c -L d -L e -、-L d -L e -and-L e -, more preferably -L c -L d -L e -、-L d -L e -or-L e -; Among them, L a L b L c L d and L e Each is independently selected from chemical bonds, , , or ; R1 is selected from -O-, -S-, -C(O)-, -NR d -、-C(O)NR d -or-NR d C(O)-; R2 is selected from chemical bonds, -O-, -S-, -SS-, -C(O)-, and -NR. d -、-C(O)NR d -、-NR d C(O)- or -S(O) m -NR d -; Cyclone A is selected from 3-12 membered cyclohexene alkyl groups, C 6-14 Arene, 5-14 membered heteroaryl or 5-14 membered heterocyclic, preferably 5-14 membered heterocyclic; Each R b and R c Independently selected from H, -C 0-6 alkylene -OH, -C 0-6 Alkylene -NH2, -C 0-6 Alkylene-CN, -C 0-6 Alkylene-C(O)OH, C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; Each R d Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; m is selected from 0, 1, or 2; Each p is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each q is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; The definitions of R' and T' are the same as those of R and T; R and T are as defined in any one of claims 1-7; Preferably, L1 and L1' are each independently selected from chemical bonds, , , , or Each k is independently selected from 1, 2, 3, 4, 5, 6, 7, or 8; L2 and L2' are each independently -L a -L b -L c -L d -L e -, where L a L b L c L d and L e Each is independently selected from chemical bonds, , , , , , , , , , , -C(O)-5-12-membered heterocyclic group -NH- or ; Each p is independently selected from an integer between 0 and 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each q is independently selected from an integer between 0 and 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; The definitions of R' and T' are the same as those of R and T; R and T are defined as in any one of claims 1-7, preferably as in claim 7; More preferably, L1 and L1' are each independently selected from chemical bonds, , , , or Each k is independently selected from 2, 3, 4, 5, 6, 7, or 8. For example, L1 and L1' are each independently selected from chemical bonds. , , , or ; Where L a L b L c L d and L e Each independently exists or is selected from chemical bonds. , , , , , , , , , , , , , or ; Each q is independently selected from an integer from 1 to 9, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9; R and T are as defined in claim 7.

9. The oligonucleotide of claim 8, wherein L2 and L2' are each independently a chemical bond or selected from the group consisting of: 。 10. The oligonucleotide of any one of claims 8-9, wherein -L2-RT and -L2'-R'-T' are each independently selected from the groups listed in Table A of the specification.

11. The oligonucleotide of any one of claims 1-7, wherein the oligonucleotide is selected from compounds of formula (III) or their pharmaceutically acceptable salts, tautomers or stereoisomers: (III) in, The T' terminus corresponds to the 5' end of the oligonucleotide, and the T' terminus corresponds to the 3' end of the oligonucleotide. Z is an oligonucleotide; L1 and L1' are each independently selected from chemical bonds or -L x -L y -L z -; Among them, L x Selected from chemical bonds or -C(O)-; L y Selected from C 1-10 Alkylene, the C 1-10 The alkylene group is optionally selected from -C by one, two, or three. 0-6 alkylene-OH, C 1-4 Alkyl or C 1-4 Halogenated alkyl substitution; L z Selected from chemical bonds, -S-, -SS-, or -NH-; L x and L z At most one of them is a chemical bond; L2 and L2' are each independently -L d -L e -or-L e -, more preferably -L e -; Among them, L d and L e Each is independently selected from chemical bonds or ; R1 is selected from -C(O)-, -NH-, -C(O)NH- or -NHC(O)-, preferably selected from -C(O)- or -NH-; R2 is selected from chemical bonds, -C(O)-, -NH-, -C(O)NH- or -NHC(O)-, preferably selected from -C(O)- or -NH-; Each R b and R c Independently selected from H and C 1-6 Alkyl or C 1-6 Halogenated alkyl groups; p is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; q is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; R and R' are independently selected from C 10-28 Straight-chain alkyl groups or C groups containing 1-4 olefinic bonds 8-28 Straight-chain alkenyl groups, preferably C-terminated by one or two oxygen atoms. 10-20 Straight-chain alkyl; T and T' are independently selected from -OH, -C(O)OH, or C 1-3 Alkoxy; Preferably, L1 is selected from chemical bonds or ; L1' is selected from chemical bonds, , or ; Each k is independently selected from 2, 3, 4, 5, 6, 7, or 8; L2 and L2' are each independently -L d -L e -, where L d and L e Each is independently selected from chemical bonds, or ; Each p is independently selected from an integer between 0 and 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Each q is independently selected from an integer between 0 and 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; -R'-T' and -RT are each independently selected from: , , , , and Preferred selection and ; Each n is independently selected from integers between 10 and 22; More preferably, L1 is a chemical bond or Preferably, chemical bonds; L1' is a chemical bond or Preferred ; L2 and L2' are each independently selected from chemical bonds. or Preferred ; q is 3, 4, or 5; -R'-T' and -RT are independent of each other. and ; Each n is independently selected from an integer between 10 and 20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably 12, 13, 14, 15, 16, 17 or 18, more preferably 14, 15, 16, 17 or 18.

12. The oligonucleotide of any one of claims 8-11, wherein the 5' and / or 3' ends of Z contain terminal modifications, preferably modified or unmodified IB or STM.

13. The oligonucleotide of any one of claims 8-12, wherein Z is selected from compounds of formula IV: (IV) in, Z' represents the remaining portion of the oligonucleotide; M and M' are each independently selected from chemical bonds, , , , or ; in, Indicates the position connected to Z'; Indicates the position connected to T'-R'-L2'-L1'- or -L1-L2-RT; Ring B is selected from 3-7 membered heterocyclic groups or C 3-7 Cycloalkyl groups, preferably 5-7 membered heterocyclic groups, more preferably 6 membered heterocyclic groups; R s Selected from hydrogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups and C 3-7 cycloalkyl; X is selected from hydroxyl or mercapto groups; Y is selected from O or S; Preferably, Z' is the remaining portion of the oligonucleotide; M and M' are each independently selected from chemical bonds, , , , or Preferably selected from chemical bonds, , or ; in, Indicates the position connected to Z'; Indicates the position connected to T'-R'-L2'-L1'- or -L1-L2-RT; R s Selected from hydrogen, C 1-6 Alkyl and C 3-6 Cycloalkyl, preferably selected from hydrogen, isopropyl or cyclohexyl; X is selected from hydroxyl or thiol groups.

14. The oligonucleotide of claim 13, wherein, At least one of M and M' is selected from , or ,in Indicates the position connected to Z'; Indicates the position connected to T'-R'-L2'-L1'- or -L1-L2-RT; R s Selected from hydrogen, isopropyl or cyclohexyl.

15. The oligonucleotide of claim 14, wherein at least one of M and M' is .

16. The oligonucleotide of any one of claims 8-15, wherein, At least one of T and T' is a carboxyl group; for example, both T and T' are carboxyl groups.

17. The oligonucleotide of any one of claims 8-16, wherein, T' is a carboxyl group and T is a hydroxyl group, or T' is a hydroxyl group and T is a carboxyl group.

18. The oligonucleotide of any one of claims 8-15, wherein, T' is hydrogen, T is hydroxyl, or T' is hydrogen, T is carboxyl.

19. The oligonucleotide of any one of claims 8-15, wherein, T' is a hydroxyl group, and T is a hydroxyl group.

20. The oligonucleotide of any one of claims 8-19, wherein, The oligonucleotide is selected from compounds of formula (III) or their pharmaceutically acceptable salts, tautomers or stereoisomers: (III) Where Z is ; T'-R'-L2'-L1'-M'- and -M-L1-L2-RT are as defined in Table B of the specification, and are connected to Z' via sulfate or thiosulfate. In Table B, the structures of LL50-LL128 are as defined in claim 10, where "s" indicates that the two adjacent structures are connected by a phosphate ester or a thiophosphate ester. Other structures are defined in the following table: 。 21. The oligonucleotide of any one of claims 8-20, wherein Z or Z' is a small interfering RNA (siRNA) or a short hairpin RNA (shRNA), preferably used to suppress genes expressed outside the liver; Preferably, the gene expressed outside the liver is expressed in one or more tissues selected from the group consisting of: eye, central nervous system, lung, muscle, heart, kidney, fat, spleen and pancreas, wherein the muscle is preferably the quadriceps femoris or cardiac muscle, and the fat is preferably subcutaneous fat, gonadal fat, pgWAT, iWAT or BAT.

22. The oligonucleotide of any one of claims 8-21, wherein the oligonucleotide is siRNA, the siRNA having a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the antisense strand comprising a sequence sufficiently complementary to the sense strand and the target mRNA, the two hydrophobic groups being located at the 5' and 3' ends of the sense strand, respectively.

23. A method of administering an oligonucleotide to a subject via extrahepatic delivery, the oligonucleotide being as defined in any one of claims 1-22, the extrahepatic delivery comprising delivery to one or more tissues of the group consisting of: eye, central nervous system, lung, muscle, kidney, fat, and spleen, the muscle preferably being the quadriceps femoris or heart, and the fat preferably being subcutaneous fat or gonadal fat.

24. The method of claim 23, wherein the method comprises delivering the oligonucleotide by systemic or local administration, preferably by any of the following: intravenous injection, subcutaneous injection, intrathecal injection, intraocular injection, intramuscular injection, and aerosol spray.

25. A cell containing an oligonucleotide as described in any one of claims 1-22.

26. A pharmaceutical composition comprising an oligonucleotide as described in any one of claims 1-22, or a cell as described in claim 25, and optionally a pharmaceutically acceptable carrier or excipient.

27. A kit comprising an oligonucleotide as described in any one of claims 1-22, a cell as described in claim 25, or a pharmaceutical composition as described in claim 26.

28. The compound represented by formula V or its pharmaceutically acceptable salt, tautomer, or stereoisomer: (V) Wherein L2, R and T are as defined in any one of claims 1-11 and 16-19 Where P1 is a protecting group, such as a carboxyl protecting group, preferably selected from... , , , , -DMTr, -O-DMTr, or -N3.

29. The compound of claim 28, wherein P1 is or .

30. The compound of claim 29, wherein, The compounds are selected from those listed in Table C of the specification.

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