Lipid-modified nucleic acid compound, and preparation method and use therefor

Abstract

The present invention relates to a lipid-modified nucleic acid compound, and a preparation method and use therefor, and specifically relates to a conjugate as represented by formula I or a pharmaceutically acceptable salt thereof.

Description

A lipid-modified nucleic acid compound, preparation method and use thereof Technical Field

[0001] The present disclosure relates to the field of medicine, and in particular to a lipid-modified nucleic acid compound, a preparation method thereof, and uses thereof. Background Art

[0002] RNA interference (RNAi) is an effective method for silencing gene expression and holds broad application prospects. While RNAi drugs offer advantages such as a rich pipeline of candidate targets, a simple and efficient development process, and strong specificity, the number of RNAi drugs ultimately authorized by the FDA for clinical treatment remains very limited. These drugs face significant limitations, including poor stability, short half-life, high immunogenicity, weak targeting, and limited cytoplasmic penetration. Therefore, in addition to improving the stability and immunogenicity of RNAi drugs through chemical modification, they also require efficient and safe in vivo delivery systems to enhance membrane permeability.

[0003] RNAi drug delivery technologies primarily include liposome nanoparticles (LNPs), liver-targeted delivery mediated by N-acetylgalactosamine (GalNAC) modification, and antibody-mediated targeted delivery. GalNAC-conjugated RNA drugs can achieve efficient and specific liver delivery, but effective delivery methods for extrahepatic cells lacking ASGPR expression remain lacking. Therefore, the development of efficient in vivo delivery methods is needed to enable RNA drugs to be effective in extrahepatic tissues.

[0004] Summary of the Invention

[0005] The present disclosure provides a conjugate represented by formula I or a pharmaceutically acceptable salt thereof,

[0006] Wherein, A is a nucleic acid;

[0007] L 1 , L 2 , L 3 and L 4 Each is independently a bond, -O-, -S-, -NH-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, unsubstituted or replaced by R 3 Substituted C 1-10 alkylene;

[0008] R 3 is -OH;

[0009] L 5 -L 5A -L 5B -L 5C -L 5D-;

[0010] L 6 -L 6A -L 6B -L 6C- L 6D -;

[0011] L 5A , L 5B , L 5C , L 5D , L 6A , L 6B , L 6C and L 6D Each is independently a bond, -O-, -NH-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -(CH2) m1 -, unsubstituted or replaced by R 4 substituted 3-10 membered heteroaryl, unsubstituted or replaced by R 9 substituted 6-14 membered aryl groups;

[0012] R 4 and R 9 Each is independently -OH, -NH2, -CN, halogen, C 1-4 Alkyl, C 1-4 Alkoxy or C 1-4 alkyl halide;

[0013] R 1 and R 2 Each independently is an unsubstituted saturated or unsaturated C 1-25 Hydrocarbon, or one or more R 10 Substituted saturated or unsaturated C 1-25 hydrocarbon group;

[0014] Each R 10 are independently halogen or 3-6 membered cycloalkyl;

[0015] Alternatively, when a carbon atom is substituted with two R 10 When two R 10 Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl group;

[0016] m1 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0017] n is 1, 2, 3, 4, 5 or 6.

[0018] In some embodiments, only the following substituents or groups are defined, and the definitions of substituents or groups not involved are the same as those described in any other embodiment (hereinafter referred to as "in some embodiments").

[0019] A is nucleic acid;

[0020] L 1 , L 2 , L 3 and L 4 Each independently represents a bond, -NH-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, unsubstituted or replaced by R 3 Substituted C 1-10 alkyl;

[0021] R 3 is -OH;

[0022] L 5 -L 5A -L 5B -L 5C -L 5D -;

[0023] L 6 -L 6A -L 6B -L 6C- L 6D -;

[0024] L 5A , L 5B , L 5C , L 5D , L 6A , L 6B , L 6C and L 6D are each independently a bond, -O-, -NH-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -(CH2) m1 -, unsubstituted 3-10 membered heteroaryl, unsubstituted 6-14 membered aryl;

[0025] R 1 and R 2 Each independently is an unsubstituted saturated C 1-25 Hydrocarbon, unsubstituted unsaturated C 3-25 Hydrocarbon, one or more R 10 Substituted saturated C 1-25 hydrocarbon group;

[0026] Each R 10 are independently halogen or 3-6 membered cycloalkyl;

[0027] Alternatively, when a carbon atom is substituted with two R 10 When two R 10 Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl group;

[0028] m1 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0029] n is 1, 2, 3, 4, 5 or 6.

[0030] In some embodiments, L 1 , L 2 , L 3 and L 4 In the C 1-10 The alkyl group is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl or C 10 alkyl.

[0031] In some embodiments, L 1 , L 2 , L 3 and L 4 In the C 1-10 Alkyl is C 4-8 alkyl.

[0032] In some embodiments, L 1 , L 2 , L 3 and L 4 In the C 1-10 Alkyl is C 1-4 alkyl.

[0033] In some embodiments, L 1 , L 2 , L 3 and L 4 In the C 1-10 Alkyl is a straight chain or branched C 1-10 alkyl.

[0034] In some embodiments, L 1 , L 2 , L 3 and L 4 Each independently represents a bond, -NH-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, unsubstituted or replaced by R 3 Substituted C 1-10 alkyl.

[0035] In some embodiments, -L 1 -L 2 -L 3 -L 4 -for-by R 3 Substituted C 1-10 Alkyl-C(=O)-, - is R 3 Substituted C 1-10In some embodiments, the 3' end of the nucleic acid chain of the nucleic acid is connected to -OL 1 -connect.

[0036] In some embodiments, -L 1 -L 2 -L 3 -L 4 -for-by R 3 Substituted C 4-8 Alkyl-C(=O)-, - is R 3 Substituted C 4-8 In some embodiments, the 3' end of the nucleic acid chain of the nucleic acid is connected to -OL 1 -connect.

[0037] In some embodiments, -L 1 -L 2 -L 3 -L 4 -for-by R 3 Substituted C5 alkyl -C(=O)-, - is R 3 Substituted C6 alkyl -C(=O)-, - is R 3 Substituted C7 alkyl -C(=O)-, - is R 3 Substituted C8 alkyl-C(=O)-, - is R 3 Substituted C5 alkyl-, - by R 3 Substituted C6 alkyl-, - by R 3 Substituted C7 alkyl-, - by R 3 In some embodiments, the 3' end of the nucleic acid chain of the nucleic acid is connected to -OL 1 -connect.

[0038] In some embodiments, L 1 , L 2 , L 3 and L 4 Each is independently a bond, -NH-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -(CH2) m2 -、-(CH2) m3 -CH(CH2OH)-(CH2) m4 -, m2, m3 and m4 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the 3' end of the nucleic acid chain of the nucleic acid is connected to -OL 1 -connect.

[0039] In some embodiments, -L 1 -L 2 -L 3 -L4 - is -(CH2) m3 -CH(CH2OH)-(CH2) m4 -C(=O)-, -(CH2) m3 -CH(CH2OH)-(CH2) m4 -, m3 and m4 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the 3' end of the nucleic acid chain of the nucleic acid is connected to -OL 1 -connect.

[0040] In some embodiments, -L 1 -L 2 -L 3 -L 4 -for In some embodiments, the 3' end of the nucleic acid strand of the nucleic acid is conjugated to -OL 1 -connect.

[0041] In some embodiments, R 3 The number of is 1, 2 or 3.

[0042] In some embodiments, -L 1 -L 2 -L 3 -L 4 - is - unsubstituted C 1-10 Alkyl-C(=O)-, -unsubstituted C 1-10 In some embodiments, the 5' end of the nucleic acid chain of the nucleic acid is connected to -OL 1 -connect.

[0043] In some embodiments, -L 1 -L 2 -L 3 -L 4 - is - unsubstituted C 4-8 Alkyl-C(=O)-, -unsubstituted C 4-8 In some embodiments, the 5' end of the nucleic acid chain of the nucleic acid is connected to -OL 1 -connect.

[0044] In some embodiments, -L 1 -L 2 -L 3 -L 4- is -unsubstituted C5 alkyl-C(=O)-, -unsubstituted C6 alkyl-C(=O)-, -unsubstituted C7 alkyl-C(=O)-, -unsubstituted C8 alkyl-C(=O)-, -unsubstituted C5 alkyl-, -unsubstituted C6 alkyl-, -unsubstituted C7 alkyl-, -unsubstituted C8 alkyl-. In some embodiments, the 5' end of the nucleic acid chain of the nucleic acid is connected to -OL 1 -connect.

[0045] In some embodiments, -L 1 -L 2 -L 3 -L 4 - is -(CH2) m2 -C(=O)-, -(CH2) m2 -, m2 is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the 5' end of the nucleic acid chain of the nucleic acid is connected to -OL 1 -connect.

[0046] In some embodiments, -L 1 -L 2 -L 3 -L 4 -for In some embodiments, the 5' end of the nucleic acid strand of the nucleic acid is conjugated to -OL 1 -connect.

[0047] In some embodiments, L 5A , L 5B , L 5C , L 5D , L 6A , L 6B , L 6C and L 6D In the embodiment, the 3-10 membered heteroaryl group is a 3-10 membered heteroaryl group having 1, 2 or 3 heteroatoms selected from N, O and S.

[0048] In some embodiments, L 5A , L 5B , L 5C , L 5D , L 6A , L 6B , L 6C and L 6D In the embodiment, the 3-10 membered heteroaryl group is a 3-8 membered heteroaryl group having 1, 2 or 3 heteroatoms selected from N, O and S.

[0049] In some embodiments, L 5A , L 5B, L 5C , L 5D , L 6A , L 6B , L 6C and L 6D In the embodiment, the 3-10 membered heteroaryl group is a 3-6 membered heteroaryl group having 1, 2 or 3 heteroatoms selected from N, O and S.

[0050] In some embodiments, L 5A , L 5B , L 5C , L 5D , L 6A , L 6B , L 6C and L 6D In the embodiment, the 3-10 membered heteroaryl group is a 5-6 membered heteroaryl group having 1, 2 or 3 heteroatoms selected from N and O.

[0051] In some embodiments, L 5A , L 5B , L 5C , L 5D , L 6A , L 6B , L 6C and L 6D In the embodiment, the 3-10 membered heteroaryl group is a 5-6 membered heteroaryl group in which the heteroatom is N and the number of heteroatoms is 1, 2 or 3.

[0052] In some embodiments, L 5A , L 5B , L 5C , L 5D , L 6A , L 6B , L 6C and L 6D wherein the 3-10 membered heteroaryl group is a triazole group (e.g. ).

[0053] In some embodiments, L 5A , L 5B , L 5C , L 5D , L 6A , L 6B , L 6C and L 6D wherein the 6-14 membered aryl group is a 6-10 membered aryl group.

[0054] In some embodiments, L 5A , L 5B , L 5C , L 5D , L 6A , L6B , L 6C and L 6D wherein the 6-14 membered aryl group is a phenyl group (e.g. ).

[0055] In some embodiments, L 5A , L 5B , L 5C , L 5D , L 6A , L 6B , L 6C and L 6D are each independently a bond, -O-, -NH-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -(CH2) m1 -, unsubstituted or replaced by R 4 substituted 3-10 membered heteroaryl, unsubstituted or replaced by R 9 substituted 6-14 membered aryl, said R 4 、R 9 The definitions and selection ranges of m1 and m2 are as described in any embodiment of the present disclosure.

[0056] In some embodiments, L 5A , L 5B , L 5C , L 5D , L 6A , L 6B , L 6C and L 6D are each independently a bond, -O-, -NH-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -(CH2) m1 -, unsubstituted 3-10 membered heteroaryl, unsubstituted 6-14 membered aryl, the definition and selection range of m1 are as described in any scheme in the context of the present disclosure.

[0057] In some embodiments, -L 5A -L 5B -L 5C -L 5D - is a bond, -(CH2) m1 -NHC(=O)-, -C(=O)-, -(CH2) m1 -unsubstituted 3-10 membered heteroaryl-, -(CH2) m1 -unsubstituted 6-14 membered aryl-, -(CH2) m1 -O-unsubstituted 6-14 membered aryl-, the definition and selection range of m1 are as described in any scheme in the context of the present disclosure.

[0058] In some embodiments, -L 5A -L5B -L 5C -L 5D - is a bond, -(CH2) m1 -NHC(=O)-, -C(=O)-, -(CH2) m1 -unsubstituted 5-6 membered heteroaryl-, -(CH2) m1 -Unsubstituted phenyl-, -(CH2) m1 -O-unsubstituted phenyl-, the definition and selection range of m1 are as described in any scheme in the context of the present disclosure.

[0059] In some embodiments, -L 5A -L 5B -L 5C -L 5D - is the key,

[0060] In some embodiments, -L 6A -L 6B -L 6C -L 6D - is a bond, -(CH2) m1 -NHC(=O)-, -C(=O)-, -(CH2) m1 -unsubstituted 3-10 membered heteroaryl-, -(CH2) m1 -unsubstituted 6-14 membered aryl-, -(CH2) m1 -O-unsubstituted 6-14 membered aryl-, the definition and selection range of m1 are as described in any scheme in the context of the present disclosure.

[0061] In some embodiments, -L 6A -L 6B -L 6C- L 6D - is a bond, -(CH2) m1 -NHC(=O)-, -C(=O)-, -(CH2) m1 -unsubstituted 5-6 membered heteroaryl-, -(CH2) m1 -Unsubstituted phenyl-, -(CH2) m1 -O-unsubstituted phenyl-, the definition and selection range of m1 are as described in any scheme in the context of the present disclosure.

[0062] In some embodiments, -L 6A -L 6B -L 6C- L 6D - is the key,

[0063] In some embodiments, L 5 and L 6 same.

[0064] In some embodiments, L 5 and L 6 different.

[0065] In some embodiments, R 4 and R 9 wherein the halogen is fluorine, chlorine or bromine.

[0066] In some embodiments, R 4 and R 9 In the C 1-4 The alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.

[0067] In some embodiments, R 4 and R 9 In the C 1-4 Alkoxy is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy or tert-butoxy.

[0068] In some embodiments, R 4 and R 9 In the C 1-4 Haloalkyl groups include, but are not limited to, -CF3, -CHF2 or CH2F.

[0069] In some embodiments, R 4 and R 9 The number of is independently 1, 2 or 3.

[0070] In some embodiments, R 10 In some embodiments, R 10 wherein the halogen is fluorine.

[0071] In some embodiments, R 10 In some embodiments, the 3-6 membered cycloalkyl group is independently cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, the 3-6 membered cycloalkyl group is cyclopropyl or cyclobutyl. In some embodiments, the 3-6 membered cycloalkyl group is cyclopropyl.

[0072] In some embodiments, R 10 The number of is 1, 2 or 3. In some embodiments, R 10 The number is 1 or 2.

[0073] In some embodiments, R 1 and R 2 wherein the saturated or unsaturated C1-25 The hydrocarbon group is saturated C 1-25 Hydrocarbon, or unsaturated C containing 1-10 carbon-carbon double bonds 3-25 Hydrocarbon group.

[0074] In some embodiments, the saturated C 1-25 The hydrocarbon group is saturated C 6-25 In some embodiments, R 1 and R 2 In the saturated C 1-25 The hydrocarbon group is C 8-21 Hydrocarbyl (e.g. C8 hydrocarbyl, C9 hydrocarbyl, C 10 Hydrocarbon, C 11 Hydrocarbon, C 12 Hydrocarbon, C 13 Hydrocarbon, C 14 Hydrocarbon, C 15 Hydrocarbon, C 16 Hydrocarbon, C 17 Hydrocarbon, C 18 Hydrocarbon, C 19 Hydrocarbon, C 20 Hydrocarbon or C 21 hydrocarbon group).

[0075] In some embodiments, the unsaturated C 3-25 The hydrocarbon group is an unsaturated C 6-25 In some embodiments, R 1 and R 2 wherein the unsaturated C 3-25 The hydrocarbon group is an unsaturated C 8-21 Hydrocarbyl (e.g. C8 hydrocarbyl, C9 hydrocarbyl, C 10 Hydrocarbon, C 11 Hydrocarbon, C 12 Hydrocarbon, C 13 Hydrocarbon, C 14 Hydrocarbon, C 15 Hydrocarbon, C 16 Hydrocarbon, C 17 Hydrocarbon, C 18 Hydrocarbon, C 19 Hydrocarbon, C 20 Hydrocarbon or C 21 In some embodiments, R 1 and R 2 wherein the unsaturated C 3-25 The hydrocarbon group is an unsaturated C 8-21 Hydrocarbyl (e.g. C8 hydrocarbyl, C9 hydrocarbyl, C 10 Hydrocarbon, C11 Hydrocarbon, C 12 Hydrocarbon, C 13 Hydrocarbon, C 14 Hydrocarbon, C 15 Hydrocarbon, C 16 Hydrocarbon, C 17 Hydrocarbon, C 18 Hydrocarbon, C 19 Hydrocarbon, C 20 Hydrocarbon or C 21 hydrocarbon group).

[0076] In some embodiments, R 1 and R 2 In the C 1-25 The hydrocarbon group is a straight chain or branched C 1-25 Hydrocarbon group.

[0077] In some embodiments, the linear C 1-25 The hydrocarbon group is a straight chain C 3-25 In some embodiments, the straight chain C 1-25 The hydrocarbon group is a straight chain C 6-25 In some embodiments, the straight chain C 1-25 The hydrocarbon group is a straight chain C 8-21 Hydrocarbyl (e.g. C8 hydrocarbyl, C9 hydrocarbyl, C 10 Hydrocarbon, C 11 Hydrocarbon, C 12 Hydrocarbon, C 13 Hydrocarbon, C 14 Hydrocarbon, C 15 Hydrocarbon, C 16 Hydrocarbon, C 17 Hydrocarbon, C 18 Hydrocarbon, C 19 Hydrocarbon, C 20 Hydrocarbon or C 21 hydrocarbon group).

[0078] In some embodiments, the branched chain C 1-25 The hydrocarbon group is a branched C 3-25 In some embodiments, the branched C 1-25 The hydrocarbon group is a branched C 6-25 In some embodiments, the branched C 1-25 The hydrocarbon group is a branched C 8-21 Hydrocarbyl (e.g. C8 hydrocarbyl, C9 hydrocarbyl, C 10 Hydrocarbon, C 11 Hydrocarbon, C 12 Hydrocarbon, C 13 Hydrocarbon, C 14 Hydrocarbon, C 15 Hydrocarbon, C 16 Hydrocarbon, C 17Hydrocarbon, C 18 Hydrocarbon, C 19 Hydrocarbon, C 20 Hydrocarbon or C 21 hydrocarbon group).

[0079] In some embodiments, R 1 and R 2 Each independently is an unsubstituted saturated C 1-25 Hydrocarbon, unsubstituted unsaturated C 3-25 Hydrocarbon, one or more R 10 Substituted saturated C 1-25 Hydrocarbon group.

[0080] In some embodiments, R 1 and R 2 are independently unsubstituted saturated C 6-25 Hydrocarbon, unsubstituted unsaturated C 6-25 Hydrocarbon, one or more R 10 Substituted saturated C 6-25 Hydrocarbon group.

[0081] In some embodiments, R 1 and R 2 are independently unsubstituted saturated C 8-21 Hydrocarbyl (e.g. C8 hydrocarbyl, C9 hydrocarbyl, C 10 Hydrocarbon, C 11 Hydrocarbon, C 12 Hydrocarbon, C 13 Hydrocarbon, C 14 Hydrocarbon, C 15 Hydrocarbon, C 16 Hydrocarbon, C 17 Hydrocarbon, C 18 Hydrocarbon, C 19 Hydrocarbon, C 20 Hydrocarbon or C 21 hydrocarbon group), unsubstituted unsaturated C 8-21 Hydrocarbyl (e.g. C8 hydrocarbyl, C9 hydrocarbyl, C 10 Hydrocarbon, C 11 Hydrocarbon, C 12 Hydrocarbon, C 13 Hydrocarbon, C 14 Hydrocarbon, C 15 Hydrocarbon, C 16 Hydrocarbon, C 17 Hydrocarbon, C 18 Hydrocarbon, C 19 Hydrocarbon, C 20 Hydrocarbon or C 21 Hydrocarbon), one or more R 10 Substituted saturated C8-21 Hydrocarbyl (e.g. C8 hydrocarbyl, C9 hydrocarbyl, C 10 Hydrocarbon, C 11 Hydrocarbon, C 12 Hydrocarbon, C 13 Hydrocarbon, C 14 Hydrocarbon, C 15 Hydrocarbon, C 16 Hydrocarbon, C 17 Hydrocarbon, C 18 Hydrocarbon, C 19 Hydrocarbon, C 20 Hydrocarbon or C 21 hydrocarbon group).

[0082] In some embodiments, R 1 and R 2 are independently unsubstituted saturated C 8-21 Hydrocarbyl (e.g. C8 hydrocarbyl, C9 hydrocarbyl, C 10 Hydrocarbon, C 11 Hydrocarbon, C 12 Hydrocarbon, C 13 Hydrocarbon, C 14 Hydrocarbon, C 15 Hydrocarbon, C 16 Hydrocarbon, C 17 Hydrocarbon, C 18 Hydrocarbon, C 19 Hydrocarbon, C 20 Hydrocarbon or C 21 hydrocarbon group), unsubstituted unsaturated C 8-21 Hydrocarbyl (e.g. C8 hydrocarbyl, C9 hydrocarbyl, C 10 Hydrocarbon, C 11 Hydrocarbon, C 12 Hydrocarbon, C 13 Hydrocarbon, C 14 Hydrocarbon, C 15 Hydrocarbon, C 16 Hydrocarbon, C 17 Hydrocarbon, C 18 Hydrocarbon, C 19 Hydrocarbon, C 20 Hydrocarbon or C 21 Hydrocarbon), one or more R 10 Substituted saturated C 8-21 Hydrocarbyl (e.g. C8 hydrocarbyl, C9 hydrocarbyl, C 10 Hydrocarbon, C 11 Hydrocarbon, C 12 Hydrocarbon, C 13 Hydrocarbon, C 14 Hydrocarbon, C 15 Hydrocarbon, C 16 Hydrocarbon, C 17 Hydrocarbon, C 18 Hydrocarbon, C19 Hydrocarbon, C 20 Hydrocarbon or C 21 hydrocarbon group).

[0083] In some embodiments, R 1 and R 2 are independently -(CH2)7CH3, -(CH2) 12 CH3, -(CH2) 13 CH3, -(CH2) 14 CH3, -(CH2) 15 CH3, -(CH2) 16 CH3, -(CH2) 18 CH3, -(CH2) 20 CH3,

[0084] In some embodiments, R 1 and R 2 same.

[0085] In some embodiments, R 1 and R 2 different.

[0086] In some embodiments, the conjugate represented by formula I is a conjugate represented by formula I-1 or I-2,

[0087] wherein m3 and m4 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R 1 、R 2 , A, L 5 , L 6 The definition and selection range of n are as described in any scheme in the context of this disclosure.

[0088] In some embodiments, the 3' end of the nucleic acid chain of the nucleic acid is connected to -OL 1 -Connect, L 5 , L 6 、R 1 and R 2 The definition and selection range of are as described in any embodiment of the present disclosure. In some embodiments, the conjugate represented by formula I is a conjugate represented by formula I-3 or I-4,

[0089] Wherein, m2 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R 1 、R 2 , A, L 5 , L 6The definition and selection range of n are as described in any embodiment of the present disclosure. In some embodiments, the 5' end of the nucleic acid chain of the nucleic acid is connected to -OL 1 -Connect, L 5 , L 6 、R 1 and R 2 The definition and selection range of are as described in any embodiment in the context of this disclosure.

[0090] In some embodiments, n is 1, 2, or 3.

[0091] In some embodiments, n is 1 or 2.

[0092] In some embodiments, n is 1.

[0093] In some embodiments, n is 2.

[0094] In some embodiments, n is 3.

[0095] In some embodiments, the nucleic acid includes, but is not limited to, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, siRNA, dsRNA, miRNA, shRNA, and Dicer substrates. In some embodiments, the nucleic acid further comprises a targeting ligand that mediates delivery to a location where target mRNA is expressed, wherein the targeting ligand is covalently or non-covalently linked to a position in the nucleic acid other than the position linked to -O-L1-.

[0096] In some embodiments, the nucleic acid is single-stranded nucleic acid.

[0097] In some embodiments, the single-stranded nucleic acid is the sense strand of an siRNA.

[0098] In some embodiments, the single-stranded nucleic acid is the antisense strand of a siRNA.

[0099] In some embodiments, the nucleic acid is a double-stranded nucleic acid.

[0100] In some embodiments, the nucleic acid is siRNA. In some embodiments, the 3' end of the SS strand or AS strand in the siRNA is connected to -OL 1 In some embodiments, the 5' end of the SS chain or AS chain in the siRNA is connected to -OL 1 In some embodiments, the 3' end and 5' end of the SS chain or AS chain in the siRNA are both connected to -OL 1 -connect.

[0101] In some embodiments, the nucleic acid includes one or more modified nucleotides.

[0102] In some embodiments, the nucleic acid is 1 They are connected by phosphodiester groups, where -OL 1 The O atom in - is included in the phosphodiester group.

[0103] In some embodiments, the nucleic acid is 1 They are connected by phosphorothioate diester groups, where -OL 1 The O atom in - is included in the phosphorothioate diester group.

[0104] In some embodiments, the 3' end of the nucleic acid strand of the nucleic acid is conjugated to -OL 1 -connect.

[0105] In some embodiments, the 5' end of the nucleic acid strand of the nucleic acid is conjugated to -OL 1 -connect.

[0106] In some embodiments, both the 3' end and the 5' end of the nucleic acid chain of the nucleic acid are connected to -OL 1 -connect.

[0107] In some embodiments, the nucleic acid is a double-stranded nucleic acid comprising at least one duplex region in which a first strand of nucleic acid is at least partially complementary to a second strand of nucleic acid, wherein:

[0108] (1) The two ends of the first strand of nucleic acid do not have -OL 1 - connection; and the 5' end of the second strand nucleic acid is connected to -OL 1 - ligated, 3' end not with -OL 1 - ligation, or the 3' end of the second strand of nucleic acid is linked to -OL 1 - ligated, 5' end not with -OL 1 -linked, or both the 3' and 5' ends of the second strand of nucleic acid are linked to -OL 1 -connect;

[0109] (2) The two ends of the second strand of nucleic acid do not have -OL 1 - connection; and the 5' end of the first strand of nucleic acid is connected to -OL 1 - ligated, 3' end not with -OL 1 - ligation, or the 3' end of the first strand of nucleic acid is linked to -OL 1 - ligated, 5' end not with -OL 1 -linked, or both the 3' and 5' ends of the first strand of nucleic acid are linked to -OL 1 -connect;

[0110] (3) The 5' ends of the first and second strands of nucleic acid are both fused with -OL 1 - connection, the 3' end is not connected with -OL 1- connection; or the first strand nucleic acid and the second strand nucleic acid 3' end are connected with -OL 1 - connection, the 5' end is not connected with -OL 1 - connection; or the first strand nucleic acid and the second strand nucleic acid 3' end and 5' end are connected with -OL 1 - connection; or the first strand nucleic acid 3' end and the second strand nucleic acid 5' end are connected with -OL 1 - ligation, the 5' end of the first strand nucleic acid and the 3' end of the second strand nucleic acid are not connected to -OL 1 - connection; or the first strand nucleic acid 5' end and the second strand nucleic acid 3' end are connected with -OL 1 - ligation, the 3' end of the first strand nucleic acid and the 5' end of the second strand nucleic acid are not connected to -OL 1 -connect;

[0111] (4) Both the 3' and 5' ends of the first strand of nucleic acid are stained with -OL 1 - ligation, one of the 5' end or 3' end of the second strand nucleic acid is connected to -OL 1 - connection; or the second strand nucleic acid 3' end and 5' end are connected to -OL 1 -ligation, one of the 5' end or 3' end of the first strand nucleic acid is connected to -OL 1 -connect.

[0112] The present disclosure provides a conjugate of the following structure or a pharmaceutically acceptable salt thereof, wherein A is a nucleic acid, and the definition and selection range of the nucleic acid are as described in any embodiment of the present disclosure,

[0113] The nucleic acid compound comprising a lipophilic portion as shown in Formula I of the present disclosure has increased hydrophobicity and membrane permeability, and can be effectively delivered into the cytoplasm. It not only improves the membrane permeability of the oligonucleotide, but also the oligonucleotide containing the modification can exert RNA interference effects in non-hepatic tissues.

[0114] The present disclosure provides a compound represented by Formula II or a pharmaceutically acceptable salt thereof,

[0115] Among them, L 5 , L 6 、R 1 and R 2 The definitions and selection ranges are described in any of the embodiments in the context of this disclosure.

[0116] G is hydrogen,

[0117] R 5 、R 6 and R 7Each independently is unsubstituted or substituted with one or more substituents C 1-6 Alkyl, the substituent is halogen, -OH or -CN;

[0118] L 7 , L 8 , L 9 and L 10 Each is independently a bond, -O-, -S-, -NH-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, unsubstituted or replaced by R 8 Substituted C 1-10 alkyl;

[0119] R 8 It is a hydroxyl protecting group.

[0120] In some embodiments,

[0121] G is hydrogen,

[0122] R 5 、R 6 and R 7 Each independently is unsubstituted or substituted with one or more substituents C 1-6 Alkyl, the substituent is halogen, -OH or -CN;

[0123] L 7 , L 8 , L 9 and L 10 Each independently represents a bond, -NH-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, unsubstituted or replaced by R 8 Substituted C 1-10 alkyl;

[0124] R 8 is a hydroxyl protecting group;

[0125] L 5 -L 5A -L 5B -L 5C -L 5D -;

[0126] L 6 -L 6A -L 6B -L 6C- L 6D -;

[0127] L 5A , L 5B , L 5C , L 5D, L 6A , L 6B , L 6C and L 6D are each independently a bond, -O-, -NH-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -(CH2) m1 -, unsubstituted 3-10 membered heteroaryl, unsubstituted 6-14 membered aryl;

[0128] R 1 and R 2 Each independently is an unsubstituted saturated C 1-25 Hydrocarbon, unsubstituted unsaturated C 3-25 Hydrocarbon, one or more R 10 Substituted saturated C 1-25 hydrocarbon group;

[0129] R 10 is halogen or 3-6 membered cycloalkyl;

[0130] Alternatively, when a carbon atom is substituted with two R 10 When two R 10 Together with the carbon atom to which it is attached, it forms a 3-6 membered cycloalkyl group;

[0131] m1 is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0132] In some embodiments, G is H.

[0133] In some embodiments, G is

[0134] In some embodiments, G is

[0135] In some embodiments, the solid phase carrier is a solid phase carrier conventionally used in the art, including but not limited to controlled pore glass (CPG), macroporous amine methyl resin, and polystyrene (PS) type carriers.

[0136] In some embodiments, G is R 5 、R 6 and R 7 The definition and selection range of are as described in any embodiment in the context of this disclosure.

[0137] In some embodiments, R 5 、R 6 and R 7 wherein the number of the substituents is 1, 2 or 3.

[0138] In some embodiments, R5 、R 6 and R 7 Each independently is unsubstituted or substituted with one or more substituents C 1-4 Alkyl, the substituent is halogen, hydroxyl or cyano.

[0139] In some embodiments, R 5 、R 6 and R 7 Each is independently methyl, ethyl, propyl, isopropyl, which is unsubstituted or substituted by one or more substituents, wherein the substituent is fluorine, chlorine, bromine, hydroxyl or cyano.

[0140] In some embodiments, R 5 and R 6 It is isopropyl.

[0141] In some embodiments, R 7 for

[0142] In some embodiments, G is

[0143] In some embodiments, the hydroxyl protecting group is an ester protecting group, an alkoxymethyl protecting group, an alkyl protecting group, a silyl protecting group, or an aryl protecting group.

[0144] In some embodiments, the hydroxy protecting group is an aryl protecting group.

[0145] In some embodiments, the hydroxy protecting group is MMTr and DMTr.

[0146] In some embodiments, the hydroxy protecting group is DMTr.

[0147] In some embodiments, L 7 , L 8 , L 9 and L 10 In the C 1-10 The alkyl group is C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl or C 10 alkyl.

[0148] In some embodiments, L 7 , L 8 , L 9 and L 10 In the C 1-10 Alkyl is C 4-8 alkyl.

[0149] In some embodiments, L 7 , L8 , L 9 and L 10 In the C 1-10 Alkyl is C 1-4 alkyl.

[0150] In some embodiments, L 7 , L 8 , L 9 and L 10 In the C 1-10 Alkyl is a straight chain or branched C 1-10 alkyl.

[0151] In some embodiments, L 7 , L 8 , L 9 and L 10 Each independently represents a bond, -NH-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, unsubstituted or replaced by R 8 Substituted C 1-10 Alkyl, R 8 The definition and selection range of are as described in any embodiment of the present disclosure.

[0152] In some embodiments, -L 7 -L 8 -L 9 -L 10 -for-by R 8 Substituted C 1-10 Alkyl-C(=O)-, - is R 8 Substituted C 1-10 Alkyl-, R 8 The definition and selection range of are as described in any scheme in the context of this disclosure. In some embodiments, G is In some embodiments, G is hydrogen.

[0153] In some embodiments, -L 7 -L 8 -L 9 -L 10 -for-by R 8 Substituted C 4-8 Alkyl-C(=O)-, - is R 8 Substituted C 4-8 Alkyl-, R 8 The definition and selection range of are as described in any scheme in the context of this disclosure. In some embodiments, G is In some embodiments, G is hydrogen.

[0154] In some embodiments, -L 7 -L 8 -L9 -L 10 -for-by R 8 Substituted C8 alkyl-NH-C(=O)-, - is R 8 Substituted C5 alkyl -C(=O)-, - is R 8 Substituted C6 alkyl -C(=O)-, - is R 8 Substituted C7 alkyl -C(=O)-, - is R 8 Substituted C8 alkyl-C(=O)-, - is R 8 Substituted C5 alkyl-, - by R 8 Substituted C6 alkyl-, - by R 8 Substituted C7 alkyl-, - by R 8 Substituted C8 alkyl-, R 8 The definition and selection range of are as described in any scheme in the context of this disclosure. In some embodiments, G is In some embodiments, G is hydrogen.

[0155] In some embodiments, L 7 , L 8 , L 9 and L 10 Each is independently a bond, -NH-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -(CH2) m5 -、-(CH2) m6 -CH(CH2R 8 )-(CH2) m7 -, m5, m6 and m7 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, R 8 The definition and selection range of are as described in any scheme in the context of this disclosure. In some embodiments, G is In some embodiments, G is hydrogen.

[0156] In some embodiments, -L 7 -L 8 -L 9 -L 10 - is -(CH2) m6 -CH(CH2R 8 )-(CH2) m7 -C(=O)-, -(CH2) m6 -CH(CH2R 8 )-(CH2) m7 -, m6 and m7 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, R 8 The definition and selection range of are as described in any scheme in the context of this disclosure. In some embodiments, G is In some embodiments, G is hydrogen.

[0157] In some embodiments, -L 7 -L 8 -L 9 -L 10 -for In some embodiments, G is In some embodiments, G is hydrogen.

[0158] In some embodiments, R 8 The number of is 1, 2 or 3.

[0159] In some embodiments, -L 7 -L 8 -L 9 -L 10 - is - unsubstituted C 1-10 Alkyl-C(=O)-, -unsubstituted C 1-10 In some embodiments, G is In some embodiments, G is hydrogen.

[0160] In some embodiments, -L 7 -L 8 -L 9 -L 10 - is - unsubstituted C 4-8 Alkyl-C(=O)-, -unsubstituted C 4-8 In some embodiments, G is In some embodiments, G is hydrogen.

[0161] In some embodiments, -L 7 -L 8 -L 9 -L 10 - is -unsubstituted C5 alkyl-C(=O)-, -unsubstituted C6 alkyl-C(=O)-, -unsubstituted C7 alkyl-C(=O)-, -unsubstituted C8 alkyl-C(=O)-, -unsubstituted C5 alkyl-, -unsubstituted C6 alkyl-, -unsubstituted C7 alkyl-, -unsubstituted C8 alkyl-. In some embodiments, G is In some embodiments, G is hydrogen.

[0162] In some embodiments, -L 7 -L 8 -L 9 -L 10 - is -(CH2) m5 -C(=O)-, -(CH2)m5 -, m5 is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, G is In some embodiments, G is hydrogen.

[0163] In some embodiments, -L 7 -L 8 -L 9 -L 10 -for In some embodiments, G is In some embodiments, G is hydrogen.

[0164] In some embodiments, the compound represented by formula II is a compound represented by II-1 or II-2,

[0165] wherein m6 and m7 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, R 1 、R 2 , G, L 5 and L 6 The definition and selection range of are as described in any scheme in the context of this disclosure. In some embodiments, G is In some embodiments, G is hydrogen.

[0166] In some embodiments, the compound represented by formula II is a compound represented by formula II-3, II-4, or II-5.

[0167] wherein m5 is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, R 1 、R 2 , G, L 5 and L 6 The definition and selection range of are as described in any scheme in the context of this disclosure. In some embodiments, G is In some embodiments, G is hydrogen.

[0168] In some embodiments, the compound represented by Formula II is a compound represented by Formula II-1A or II-2A,

[0169] wherein m6 and m7 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, R 1 、R 2 , L 5 and L 6 The definition and selection range of are as described in any embodiment of the present disclosure.

[0170] In some embodiments, the compound represented by formula II is a compound represented by formula II-3A or II-4A,

[0171] wherein m5 is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, R 1 、R 2 , L 5 and L 6 The definition and selection range of are as described in any scheme in the context of this disclosure.

[0172] The present disclosure provides a compound of the following structure or a pharmaceutically acceptable salt thereof, wherein: is a solid phase carrier, and the solid phase carrier is defined as any one of the schemes in formula II,

[0173] The present disclosure provides an RNAi agent comprising the above conjugate or a pharmaceutically acceptable salt thereof.

[0174] The present disclosure provides a pharmaceutical composition comprising the conjugate or a pharmaceutically acceptable salt thereof, or the RNAi agent described above, and one or more pharmaceutically acceptable excipients. The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically pharmaceutically acceptable salts or prodrugs, and other chemical components, as well as other components such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient, and thereby exert its biological activity.

[0175] In some embodiments, the conjugate or RNAi agent can be in a therapeutically effective amount.

[0176] In some embodiments, the pharmaceutically acceptable excipient may be an excipient commonly used in the art, such as a carrier, a vehicle, a diluent, and / or a delivery polymer.

[0177] In some embodiments, the unit dose of the pharmaceutical composition may be 0.001 mg-1000 mg.

[0178] In some embodiments, based on the total weight of the pharmaceutical composition, the content of the pharmaceutically acceptable excipient may be 0.01-99.99%, further 0.1-99.9%, further 0.5%-99.5%, further 1%-99%, and further 2%-98%.

[0179] In certain embodiments, the pharmaceutical composition contains 0.01-99.99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution, based on the total weight of the pharmaceutical composition. In certain embodiments, the pharmaceutical composition contains 0.1-99.9% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned compound or its pharmaceutically acceptable salt or its isotopic substitution.

[0180] The pharmaceutical composition can be conveniently presented in unit dosage form. Generally, the pharmaceutical composition can be formulated into any suitable dosage form, such as, but not limited to, injection, tablet, capsule, gel, etc. Pharmaceutical compositions include, but are not limited to, solutions, emulsions, and liposome-containing formulations.

[0181] The present disclosure provides a use of a conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, an RNAi agent or a pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease, including but not limited to a disease or disorder of the brain, eye, liver, kidney, heart, adipose tissue, lung, muscle or spleen.

[0182] The present disclosure provides a use of a conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, an RNAi agent or a pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease, including but not limited to a neurological disease or disorder, a metabolic disease or disorder, an inflammatory disease or disorder, or cancer.

[0183] The present disclosure provides a method for preventing and / or treating a disease, comprising administering the conjugate of the present disclosure or a pharmaceutically acceptable salt thereof, an RNAi agent, or a pharmaceutical composition to a patient.

[0184] The present disclosure provides a use of the conjugate or its pharmaceutically acceptable salt, RNAi agent or pharmaceutical composition of the present disclosure for preventing and / or treating a disease, which comprises administering the conjugate or its pharmaceutically acceptable salt, RNAi agent or pharmaceutical composition of the present disclosure to a patient.

[0185] In some embodiments, the disease includes, but is not limited to, a disease or disorder of the brain, eye, liver, kidney, heart, adipose tissue, lung, muscle, or spleen.

[0186] In some embodiments, the disease includes, but is not limited to, a neurological disease or disorder, a metabolic disease or disorder, an inflammatory disease or disorder, or cancer.

[0187] The present disclosure provides the above-mentioned conjugate or a pharmaceutically acceptable salt thereof, the above-mentioned RNAi agent or the above-mentioned pharmaceutical composition for preventing and / or treating a disease.

[0188] In some embodiments, the disease includes, but is not limited to, a disease or disorder of the brain, eye, liver, kidney, heart, adipose tissue, lung, muscle, or spleen.

[0189] In some embodiments, the disease includes, but is not limited to, a neurological disease or disorder, a metabolic disease or disorder, an inflammatory disease or disorder, or cancer.

[0190] The present disclosure provides a cell comprising the above-mentioned conjugate or RNAi agent.

[0191] In some embodiments, the cell is a primary cell. In some embodiments, the cell is a fat cell, a hepatocyte, a fibroblast, an endothelial cell, a kidney cell, a human umbilical vein endothelial cell (HUVEC), a fat cell, a macrophage, a neuronal cell, a myocyte or a differentiated primary human skeletal muscle cell.

[0192] In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is a NIH3T3 cell, a differentiated 3T3L1 cell, a RAW264.7 cell, or a SH-SY5Y cell.

[0193] In some embodiments, the cell is an adipocyte or a hepatocyte.

[0194] The present disclosure provides a method for introducing a nucleic acid into a cell, the method comprising contacting the cell with the above-mentioned conjugate, RNAi agent or pharmaceutical composition.

[0195] In some embodiments, the cell is a primary cell. In some embodiments, the cell is a fat cell, a hepatocyte, a fibroblast, an endothelial cell, a kidney cell, a human umbilical vein endothelial cell (HUVEC), a fat cell, a macrophage, a neuronal cell, a myocyte or a differentiated primary human skeletal muscle cell.

[0196] In some embodiments, the cell is an immortalized cell. In some embodiments, the cell is a NIH3T3 cell, a differentiated 3T3L1 cell, a RAW264.7 cell, or a SH-SY5Y cell.

[0197] In some embodiments, the cell is an adipocyte or a hepatocyte.

[0198] The present disclosure provides a method for preparing the above-mentioned conjugate, which is method 1, method 2 or method 3.

[0199] Method 1 comprises: starting with the compound represented by formula II-1A or II-2A, linking nucleoside monomers one by one from the 3'-5' direction according to the nucleotide arrangement order to obtain the conjugate;

[0200] Method 2 comprises: starting with a universal solid phase support, linking nucleoside monomers one by one from the 3'-5' direction according to the nucleotide arrangement order, and then linking the compound represented by formula II-3A or II-4A to obtain the conjugate;

[0201] Method three comprises: starting with the compound shown in formula II-1A or II-2A, linking nucleoside monomers one by one from the 3'-5' direction according to the nucleotide arrangement order, and then linking the compound shown in formula II-3A or II-4A to obtain the conjugate.

[0202] In some embodiments, each linking of a nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfurization.

[0203] In some embodiments, in method 1, after the linking of nucleoside monomers is completed, an annealing step may be further included.

[0204] In some embodiments, in methods 2 and 3, after the connection of the compound represented by formula II-3A or II-4A is completed, an annealing step may be further included.

[0205] Terminology

[0206] On the other hand, where the present disclosure does not limit a particular configuration, the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All of these isomers and their mixtures are included within the scope of the present disclosure.

[0207] Additionally, the compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are encompassed within the scope of the present disclosure.The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier.

[0208] The compounds of the present disclosure may be asymmetric, for example, having one or more stereoisomers. Unless otherwise indicated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds of the present disclosure containing asymmetric carbon atoms can be isolated in optically pure forms or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

[0209] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary groups are cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), diastereomeric salts are formed with an appropriate optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and then the pure enantiomers are recovered. In addition, separation of enantiomers and diastereoisomers is typically accomplished using chromatography using a chiral stationary phase, optionally combined with chemical derivatization (e.g., to form carbamates from amines).

[0210] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 30 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, and further preferably an alkyl group containing 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. More preferred are alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.

[0211] The term "alkylene" refers to the portion remaining after removing two hydrogen atoms from an alkane molecule, including straight and branched subgroups of 1 to 20 carbon atoms. Non-limiting examples of alkylene groups containing 1 to 6 carbon atoms include methylene (-CH2-), ethylene (such as -CH2CH2- or -CH(CH3)-), propylene (such as -CH2CH2CH2- or -CH(CH2CH3)-), and butylene (such as -CH2CH2CH2CH2-). Unless otherwise specified, alkylene groups may be substituted or unsubstituted. When substituted, the substituents may be substituted at any available point of attachment, and the substituents are preferably one or more of the following groups, independently selected from aryl, heteroaryl, and halogen.

[0212] The term "cycloalkyl" or "carbocycle" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls. Cycloalkyls may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from halogen, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 The cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl group is optionally substituted by one or more groups selected from halogen, hydroxy, oxo, nitro, and cyano.

[0213] The cycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a cycloalkyl, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, etc. The cycloalkyl may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups, which are independently selected from halogen, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 The cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl group is optionally substituted by one or more groups selected from halogen, hydroxy, oxo, nitro, and cyano.

[0214] The term "heterocycloalkyl" or "heterocycle" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is a heteroatom selected from nitrogen, oxygen or S(O)m (wherein m is an integer from 0 to 2), but excluding the ring portion of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 7 ring atoms. Non-limiting examples of monocyclic heterocycloalkyls include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocycloalkyls include spirocyclic, fused ring, and bridged ring heterocycloalkyls. Non-limiting examples of "heterocycloalkyls" include:

[0215] etc.

[0216] The heterocycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a heterocycloalkyl, non-limiting examples of which include:

[0217] wait.

[0218] Heterocycloalkyl may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 The cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl group is optionally substituted by one or more groups selected from halogen, hydroxy, oxo, nitro, and cyano.

[0219] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 12-membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocycloalkyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring, non-limiting examples of which include:

[0220] Aryl may be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 The cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl group is optionally substituted by one or more groups selected from halogen, hydroxy, oxo, nitro, and cyano.

[0221] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 12-membered, more preferably 5-membered or 6-membered. For example, non-limiting examples include: imidazolyl, furyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazinyl, triazolyl, indazolyl, benzimidazolyl, wait.

[0222] The heteroaryl ring may be fused to an aryl, heterocycloalkyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring, non-limiting examples of which include:

[0223] Heteroaryl may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, oxo, nitro, cyano, C1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 The cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl group is optionally substituted by one or more groups selected from halogen, hydroxy, oxo, nitro, and cyano.

[0224] The term "spirocyclic" refers to a compound in which two rings share one atom.

[0225] The term "spiroalkyl" refers to a polycyclic group that shares a carbon atom (called a spiro atom) between 5 to 20 monocyclic rings, which may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of spiro atoms shared between the rings, the spiroalkyl is divided into a single spiroalkyl, a double spiroalkyl or a multi-spiroalkyl, preferably a single spiroalkyl and a double spiroalkyl. More preferably, it is 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6 yuan single spiroalkyl. "Spiro carbocycle" refers to the ring system in the spiroalkyl. Non-limiting examples of spiroalkyl include:

[0226] The term "spiroheterocyclyl" refers to a polycyclic heterocyclic group in which the monocyclic rings of 5 to 20 members share one atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer 0 to 2) heteroatom, and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated π electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members. According to the number of shared spiral atoms between the rings, the spiro heterocyclic group is divided into a monospiro heterocyclic group, a dispiro heterocyclic group or a polyspiro heterocyclic group, preferably a monospiro heterocyclic group and a dispiro heterocyclic group. More preferably, it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered or 5-membered / 6-membered monospiro heterocyclic group. "Spiro heterocycle" refers to the ring system in the spiro heterocyclic group. Non-limiting examples of spiro heterocyclic groups include:

[0227] The term "fused ring" refers to a compound in which two or more rings are fused by sharing two adjacent atoms.

[0228] The term "fused cycloalkyl" refers to a 5 to 20-membered, all-carbon polycyclic group in which each ring in the system shares a pair of adjacent carbon atoms with the other rings in the system, wherein one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused cycloalkyl, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl. Non-limiting examples of fused cycloalkyl include:

[0229] The term "fused heterocyclyl" refers to a polycyclic heterocyclic group of 5 to 20 members, wherein each ring in the system shares a pair of adjacent atoms with other rings in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, the remaining ring atoms being carbon. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic groups. "Fused heterocycle" refers to the ring system in a fused heterocyclic group. Non-limiting examples of fused heterocyclic groups include:

[0230] The term "fused heteroaryl" may be an unsaturated aromatic fused ring structure containing 5-14 ring atoms (including at least one heteroatom), which is formed by two or more ring structures sharing two adjacent atoms, and includes the case where the carbon atoms, nitrogen atoms and sulfur atoms may be oxidized. Preferably, "5-12 membered fused heteroaryl", "7-12 membered fused heteroaryl", "9-12 membered fused heteroaryl" and the like are used, such as benzofuranyl, benzisofuranyl, benzothiophenyl, indolyl, isoindolyl, benzoxazolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, 2-quinolinonyl, 4-quinolinonyl, 1-isoquinolinonyl, isoquinolyl, acridinyl, phenanthridinyl, benzopyridazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, phenolazinyl, pteridinyl, purinyl, naphthyridinyl, phenazinyl, phenothiazinyl and the like. "Fused heteroaryl ring" refers to the ring system within a fused heteroaryl group.

[0231] The fused heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0232] The term "bridged ring" refers to a structure formed by two or more cyclic structures sharing two non-adjacent ring atoms.

[0233] The term "bridged cycloalkyl" refers to a 5-20 membered, all-carbon polycyclic group in which any two rings share two carbon atoms that are not directly connected, which may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6-14 members, more preferably 7-10 members. Depending on the number of constituent rings, it can be classified as a bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl group, preferably a bicyclic, tricyclic or tetracyclic group, more preferably a bicyclic or tricyclic group. Non-limiting examples of bridged cycloalkyl groups include:

[0234] The term "bridged heterocyclyl" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two atoms that are not directly connected, which may contain one or more double bonds but no ring has a completely conjugated π electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, the remaining ring atoms being carbon. Preferably, it is 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it can be classified as a bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic group, preferably a bicyclic, tricyclic or tetracyclic group, more preferably a bicyclic or tricyclic group. Non-limiting examples of bridged heterocyclic groups include:

[0235] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from halogen, hydroxy, oxo, nitro, cyano, C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8 Cycloalkenyloxy, 5 to 6 membered aryl or heteroaryl, the C 1-6 Alkyl, C 1-6 Alkoxy, C 2-6 Alkenyloxy, C 2-6 Alkynyloxy, C 3-6 Cycloalkoxy, 3 to 6 membered heterocycloalkoxy, C 3-8The cycloalkenyloxy, 5- to 6-membered aryl or heteroaryl groups are optionally substituted with one or more halogen, hydroxy, oxo, nitro, and cyano groups. Similarly, the definitions of "alkynyloxy," "alkenyloxy," "cycloalkoxy," "heterocycloalkoxy," and "cycloalkenyloxy" are the same as those for "alkoxy" above.

[0236] The term "hydroxy" refers to -OH.

[0237] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0238] The term "haloalkyl" refers to an alkyl group substituted with a halogen, wherein alkyl is as defined above.

[0239] The term "cyano" refers to -CN.

[0240] The term "nitro" refers to -NO2.

[0241] The term "oxo" refers to a =0 group, for example, a carbon atom connected to an oxygen atom via a double bond, whereby a ketone or aldehyde group is formed.

[0242] The term "amino" refers to -NH2.

[0243] The term "carboxy" refers to -C(O)OH.

[0244] The term "aldehyde" refers to -CHO.

[0245] The term "benzoyl" refers to -Bz.

[0246] The term "methoxyphenylbenzhydryl" refers to -MMTr.

[0247] The term "dimethoxytrityl" refers to -DMTr.

[0248] "Optionally" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "C 1-6 The term "alkyl" means that halogen or cyano may but need not be present, and the description includes both the case where the alkyl is substituted by halogen or cyano and the case where the alkyl is not substituted by halogen and cyano.

[0249] The term "substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in a group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions and a person skilled in the art can determine (by experiment or theory) which substitutions are possible or impossible without undue effort.

[0250] "Substituted by one or more..." means that the compound may be substituted by a single or multiple substituents. When substituted by multiple substituents, the substituents may be multiple identical substituents or a combination of one or more different substituents.

[0251] The term "linked," when referring to a connection between two molecules, means that the two molecules are connected by a covalent bond or the two molecules are associated via a non-covalent bond (eg, a hydrogen bond or an ionic bond), including direct and indirect connections.

[0252] The term "directly linked" refers to a first compound or group being linked to a second compound or group without any intervening atoms or groups of atoms. The term "indirectly linked" refers to a first compound or group being linked to a second compound or group through an intermediate group, compound, or molecule (e.g., a linking group).

[0253] In the chemical structures of the compounds disclosed herein, the bond Indicates that the configuration is not specified, that is, if chiral isomers exist in the chemical structure, the bond Can be or include both Although all the above structural formulas are drawn as certain isomers for the sake of simplicity, the present disclosure may include all isomers, such as tautomers, rotational isomers, geometric isomers, diastereomers, racemates and enantiomers. In the chemical structures of the compounds described in the present disclosure, the bonds No configuration is specified, i.e., the bond The configuration can be E-type or Z-type, or include both E and Z configurations.

[0254] Unless otherwise specified, the symbols used in this paper are It means that it can be linked to one or more any groups according to the disclosure described herein.

[0255] Unless otherwise indicated, the variables shown can be oriented so that any one point of attachment on the variable is connected to any one point of attachment on the compound of Formula I. For example, the variable L1 has two points of attachment to the compound of Formula I. Although L1 is shown as -(CH2)6-O-CH2- in one embodiment, this embodiment should also be understood to encompass compounds where L1 is -CH2-O-(CH2)6-.

[0256] In the present disclosure, the terms "comprising" and "including" can be replaced with "consisting of".

[0257] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein, or their physiologically acceptable salts or precursors, with other chemical components, as well as other components such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0258] The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that has been approved by the U.S. Food and Drug Administration as acceptable for use in humans or domestic animals.

[0259] Unless otherwise specified, the "compounds," "conjugates," and "nucleic acids" of the present disclosure may independently exist in the form of salts, mixed salts, or non-salt forms (e.g., free acids or free bases). When present in the form of salts or mixed salts, they may be pharmaceutically acceptable salts or pharmaceutically usable salts.

[0260] The terms "pharmaceutically acceptable salts" and "pharmaceutically usable salts" are used interchangeably and are meant to include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0261] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, octanoates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamate, pyroglutamate, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbate, salicylates, 4-aminosalicylates, and naphthalene disulfonates. These salts can be prepared by methods known in the art.

[0262] "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts, with sodium salts being preferred. The salt derived from organic base includes but is not limited to following salt: primary amines, secondary amines and tertiary amines, substituted amines, comprise natural substituted amines, cyclic amines and basic ion exchange resins, for example ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidines, N-ethylpiperidine, polyamine resins etc.Preferred organic base comprises isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.These salts can be prepared by methods known in the art.

[0263] As used herein, the term "inhibit" can be used interchangeably with "reduce", "silence", "downregulate", "suppress" and other similar terms, and includes any level of inhibition. Inhibition can be assessed by the reduction of the absolute or relative level of one or more of these variables compared to the control level. The control level can be any type of control level used in the art, such as baseline level before administration or the level determined from a similar untreated or control-treated subject, cell, or sample (such as only a buffer control or an inert agent control). For example, the remaining expression of mRNA can be used to characterize the degree of inhibition of siRNA on target gene expression, such as the remaining expression of mRNA being no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%. The inhibition rate of target gene expression can be measured using The Luciferase Assay System measures the chemiluminescence values ​​of firefly and Renilla, respectively, and calculates the relative value (Ratio = Ren / Fir). In this disclosure, the ratio of remaining mRNA expression (or remaining activity %) = Ratio (siRNA-treated group) / Ratio (no siRNA control group), and the inhibition rate (%) = 100% - remaining mRNA expression (%).

[0264] "Effective amount," "effective dose," "effective therapeutic amount," or "therapeutically effective amount" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic results. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, reducing the severity, or delaying the onset of a condition, including the biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes that present during the progression of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various target gene, target mRNA, or target protein-related conditions of the present invention or improving one or more symptoms of the condition, reducing the dose of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of a target gene, target mRNA, or target protein-related condition of the present invention in a patient. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on, for example, the condition to be treated, the patient's overall health, the method, route, and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0265] As used herein, "subject," "patient," "subject," or "individual" are used interchangeably and include humans or non-human animals, such as mammals, eg, humans or monkeys.

[0266] In this disclosure, "phosphate group," "phosphate group," and "phosphate bond" are used interchangeably to include monoesters, phosphodiesters, or phosphotriesters. "Phosphate group" in "phosphorothioate group" also has the same meaning. Unless otherwise specified, a natural internucleotide phosphate group is a phosphodiester group.

[0267] As used herein, in the context of RNA-mediated gene silencing, the positive strand of siRNA (also referred to as SS, SS strand or sense strand) refers to a strand comprising a sequence identical or substantially identical to the target mRNA sequence; the antisense strand of siRNA (also referred to as AS or AS strand) refers to a strand having a sequence complementary to the target mRNA sequence.

[0268] As used herein, the first strand may be referred to as the antisense strand and the second strand may be referred to as the sense strand. The terms first strand and antisense strand or second strand and sense strand should be considered interchangeable.

[0269] In this disclosure, the "5' region," also known as the "5' end," or "5' terminus" of the sense or antisense strand may be used interchangeably. For example, nucleotides 2 through 8 in the 5' region of the antisense strand may be replaced with nucleotides 2 through 8 at the 5' terminus of the antisense strand. Similarly, the "3' region," "3' terminus," and "3' terminus" of the sense or antisense strand may be used interchangeably.

[0270] As used herein, the term "base" encompasses any known DNA and RNA base, base analogs such as purine or pyrimidine, and also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs.

[0271] Unless otherwise specified, in the context of this disclosure, capital letters C, G, U, A, and T represent the base composition of a nucleotide; a lowercase letter d indicates that the nucleotide adjacent to the right of the letter d is a deoxyribonucleotide; a lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; a lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; and a lowercase letter s indicates that the two nucleotides adjacent to the left and right of the letter s are connected by a phosphorothioate group.

[0272] As used herein, the terms "complementary" and "reverse complement" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one chain are paired with bases on the other chain in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair consists of a purine and a pyrimidine. When adenine on one chain is always paired with thymine (or uracil) on the other chain, and guanine is always paired with cytosine, the two chains are considered to be complementary to each other, and the sequence of the chain can be inferred from the sequence of its complementary chain. Accordingly, "mismatch" means in the art that the bases at corresponding positions in a double-stranded nucleic acid are not paired in a complementary manner.

[0273] As used herein, "chemically modified" or "modification" means a structure that is chemically different when compared to a naturally occurring counterpart, including all changes by chemical means, such as the addition or removal of a chemical moiety, or the substitution of one chemical moiety for another.

[0274] As used herein, the term "fluoro-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by fluorine; the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0275] "Nucleic acids" in the present disclosure include, but are not limited to, oligonucleotides, single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), ribozymes, interfering RNA molecules, and Dicer enzyme substrates. "RNAi agents" in the present disclosure refer to agents containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that can degrade or inhibit the transcription and translation of target messenger RNA (mRNA) in a sequence-specific manner. The RNAi agents in the present disclosure can be manipulated by an RNA interference mechanism (i.e., by inducing RNA interference by interacting with the RNA interference pathway-constituting mechanism of mammalian cells (RNA-induced silencing complex or RISC)), or act through any other mechanism or pathway.

[0276] The RNAi agents described herein include nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0277] Figure 1 shows the inhibitory activity of DS-01, DS-04, and DS-05 on mouse APP mRNA expression. Data are presented as mean ± SD. **** in the figure indicates p < 0.0001 compared with the blank vector.

[0278] Figure 2 shows the inhibitory activity of DS-07, DS-10, DS-11, and DS-12 on mouse MAPT mRNA expression. Data are presented as mean ± SD. **** indicates p < 0.0001 compared to the blank vector, *** indicates p < 0.001 compared to the blank vector, ** indicates p < 0.01 compared to the blank vector, ns indicates no significance compared to the blank vector, #### indicates p < 0.0001 compared to DS-12, # indicates p < 0.05 compared to DS-12, &&& indicates p < 0.001 compared to DS-12, &&&& indicates p < 0.0001 compared to DS-12, $$ indicates p < 0.01 compared to DS-12, and $$$$$ indicates p < 0.0001 compared to DS-12. Specific implementation plan

[0279] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples disclosed herein, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the raw material or commercial manufacturer. Reagents whose sources are not specified can be obtained from any molecular biology reagent supplier of a quality / purity suitable for molecular biology applications.

[0280] Unless otherwise specified, the reagents used in the following examples are commercially available products.

[0281] Example 1: Synthesis of Compound A01

[0282] The synthetic route of compound A01 is as follows:

[0283] The synthesis and identification of the specific intermediates and final products involved in the above route are as follows:

[0284] Compound 1-2:

[0285] Compound 1-1 (10.0 g, 62.4 mmol) and 1,4-dibromobutane (47.18 g, 218.5 mmol) were added to dimethyl sulfoxide (30 mL). Under a nitrogen atmosphere, the temperature was lowered to below 10°C, and sodium hydride (2.5 g, 62.4 mmol) was added portionwise. The reaction was incubated for 2 hours. After LCMS analysis showed the reaction was complete, water (100 ml) was slowly added to quench the reaction, and the mixture was extracted with ethyl acetate (50 mL x 2). The organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The concentrated crude product was purified and isolated by normal silica gel column chromatography to obtain compound 1-2 (9.50 g, 51% yield). LCMS: MS (ESI) m / z = 296.2 [M+H] + .

[0286] Compounds 1-3:

[0287] N-Benzyloxycarbonyl-1,4-diaminobutane hydrochloride (4.38 g, 16.9 mmol) and potassium carbonate (3.28 g, 23.7 mmol) were added to N,N-dimethylformamide (20 mL). The temperature was raised to 70°C and stirred for 30 minutes. Compound 1-2 (2.0 g, 6.8 mmol) was then added dropwise to the reaction mixture. After the addition was complete, the mixture was stirred for 30 minutes. LCMS analysis indicated that the reaction was complete. The reaction mixture was cooled to room temperature and quenched by pouring into water (80 mL). The mixture was extracted with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to afford crude compound 1-3 (2.0 g, 67% yield). LCMS: MS (ESI) m / z = 437.6 [M+H] + .

[0288] Compounds 1-4:

[0289] Compound 1-3 (2.0 g, 4.6 mmol) was dissolved in dichloromethane (20 mL) and benzyl chloroformate (0.86 g, 5.0 mmol) was slowly added dropwise at room temperature. After 10 minutes of reaction at room temperature, LCMS analysis indicated the reaction was complete. The reaction solution was concentrated, and the crude product was purified and isolated by normal phase silica gel column chromatography to obtain compound 1-4 (0.52 g, 20% yield). LCMS: MS (ESI) m / z = 571.8 [M+H] + .

[0290] Compounds 1-5:

[0291] Compound 1-4 (1.19 g, 2.1 mmol) was dissolved in ethanol (10 mL). Under a nitrogen atmosphere, sodium borohydride (0.29 g, 8.4 mmol) and lithium chloride (0.71 g, 42.4 mmol) were added, and the mixture was stirred at 70°C for 5 hours. LCMS analysis indicated that the reaction was complete. The reaction mixture was cooled to room temperature and quenched by the slow dropwise addition of saturated aqueous ammonium chloride (30 ml). The mixture was then extracted with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The concentrated crude product was purified and isolated by normal phase silica gel column chromatography to afford compound 1-5 (0.80 g, 78% yield). LCMS: MS (ESI) m / z = 486.6 [M+Na] + .

[0292] Compounds 1-6:

[0293] Compound 1-5 (1.40 g, 2.9 mmol) was dissolved in pyridine (14 mL) and a few molecular sieves were added. The mixture was stirred at room temperature for 10 minutes. 4,4'-bis(methoxytrityl) chloride (1.07 g, 3.2 mmol) was then added and allowed to react at room temperature for 5 hours. LCMS analysis indicated the reaction was complete. The crude product obtained by concentration was separated by reverse phase column chromatography (acetonitrile / water) to yield compound 1-6 (1.50 g, 66% yield). LCMS: MS (ESI) m / z = 812.1 [M+Na] + .

[0294] Compounds 1-7:

[0295] Compound 1-6 (1.60 g, 2.0 mmol) was dissolved in methanol (16 mL) and palladium / carbon (0.20 g) was added. The mixture was allowed to react overnight at room temperature under a hydrogen atmosphere. LCMS analysis indicated the reaction was complete, and the mixture was filtered through Celite. The filtrate was concentrated to afford compound 1-7 (1.0 g, 94% yield). LCMS: MS (ESI) m / z = 521.9 [M+H] + .

[0296] Compounds 1-8:

[0297] Compound 1-7 (1.20 g, 2.3 mmol) was dissolved in N,N-dimethylformamide (12 mL), and palmitic acid (1.18 g, 4.6 mmol), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid (1.55 g, 4.8 mmol), and N,N-diisopropylethylamine (0.89 g, 6.9 mmol) were added. The mixture was allowed to react at room temperature for 3 hours. LCMS analysis indicated that the reaction was complete. The reaction solution was quenched by pouring into water (50 mL) and extracted with ethyl acetate (50 mL x 2). The organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The concentrated crude product was purified by normal phase silica gel column chromatography to obtain compound 1-8 (0.70 g, 31% yield). LCMS: MS (ESI) m / z = 1020.1 [M+Na] + .

[0298] Compound A01

[0299] Compound 1-8 (0.10 g, 0.1 mmol) was dissolved in anhydrous pyridine (2 mL) and N,N-dimethylformamide (1 mL), and 4-dimethylaminopyridine (0.06 g, 0.5 mmol) and succinic anhydride (0.10 g, 1.0 mmol) were added sequentially. The reaction mixture was stirred at 50°C for 16 hours. After LCMS showed the reaction was complete, the reaction mixture was poured into water, extracted with ethyl acetate, washed with saturated brine, and dried over anhydrous sodium sulfate to obtain crude compound A01 (0.10 g, 95% yield). LCMS: MS (ESI) m / z = 1096.3 [M-1] + .

[0300] Example 2: Synthesis of Compound A02

[0301] The synthetic route of compound A02 is as follows:

[0302] Compound 2-2:

[0303] 1,9-Bis-BOC-1,5,9-triazanonane (10.00 g, 30.0 mmol) was dissolved in N,N-dimethylformamide (80.0 mL) at 0°C, followed by the addition of triethylamine (9.16 g, 90.0 mmol) and 9-fluorenylmethyl chloroformate (11.70 g, 45.0 mmol). The mixture was allowed to warm to room temperature and allowed to react for 18 hours. After LC-MS analysis indicated the reaction was complete, the mixture was quenched and diluted with water. The reaction solution was extracted three times with dichloromethane (80.0 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by normal silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) and concentrated under reduced pressure to afford compound 2-2 (11.60 g, 69% yield). LCMS: MS (ESI) m / z = 573.6 [M+Na] + .

[0304] Compound 2-3:

[0305] Compound 2-2 (11.60 g, 21.0 mmol) was dissolved in dioxane (40.0 mL) and a 4N hydrochloric acid-dioxane solution (40.0 mL) was added at room temperature. The mixture was allowed to react for 18 hours. LCMS monitoring indicated the reaction was complete. The reaction solution was concentrated and the resulting crude product was slurried (petroleum ether / ethyl acetate = 10 / 1) to afford compound 2-3 (7.41 g, 83% yield). LCMS: MS (ESI) m / z = 354.5 [M+H] + .

[0306] Compound 2-4:

[0307] Compound 2-3 (7.20 g, 17.0 mmol) was suspended in N,N-dimethylformamide (150 mL), and diisopropylethylamine (13.00 g, 102.0 mmol) and palmitoyl chloride (14.00 g, 51.0 mmol) were added at room temperature. The temperature was raised to 50°C for 18 hours. LCMS monitoring showed that the reaction was complete. Water was added to quench the reaction, and the reaction solution was extracted three times with dichloromethane (80 mL). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The crude product was concentrated and slurried (petroleum ether / ethyl acetate = 5 / 1) to obtain compound 2-4 (13.60 g, yield 97%). LCMS: MS (ESI) m / z = 831.4 [M+H] + .

[0308] Compound 2-5:

[0309] Compound 2-4 (13.60 g, 16.0 mmol) was suspended in N,N-dimethylformamide (130.0 mL) and triethylamine (40.0 mL) was added at room temperature. The reaction was allowed to proceed at room temperature for 18 hours. LCMS monitoring showed that approximately 30% of the starting material remained. 1,8-diazacyclo[5,4,0]undecene (10.0 mL) was added and the temperature was raised to 50°C for 2 hours. LCMS monitoring showed that the reaction was complete. Water was added to quench the reaction, and the reaction solution was extracted three times with dichloromethane (80 mL). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The crude product was concentrated and slurried (pure ethyl acetate) to obtain compound 2-5 (9.40 g, 94% yield).

[0310] Compound 2-7:

[0311] Dimethyl malonate (8.22 g, 62.2 mmol) was dissolved in anhydrous tetrahydrofuran (80.0 mL). Sodium hydride (2.33 g, 58.3 mmol, 60% in mineral oil) was added at 0°C. After reacting at 0°C for one hour, benzyl 4-bromobutyrate (10.0 g, 38.9 mmol) was added dropwise. The reaction solution was warmed to room temperature and stirred for 18 hours. LCMS monitoring indicated the reaction was complete. The reaction was quenched with water and extracted three times with ethyl acetate (50 mL). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product which was purified by normal phase silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to afford compound 2-7 (6.30 g, 52% yield). LCMS: MS (ESI) m / z = 309.3 [M+H] + .

[0312] Compound 2-8:

[0313] Compound 2-7 (8.77 g, 28.4 mmol) was dissolved in methanol (70.0 mL) and 10% palladium on carbon (900 mg) was added at room temperature. The mixture was allowed to react under a hydrogen atmosphere at room temperature for 18 hours. LCMS monitoring indicated the reaction was complete. The reaction solution was filtered and concentrated to afford compound 2-8 (6.15 g, 99% yield).

[0314] Compound 2-9:

[0315] Compound 2-8 (1.08 g, 4.9 mmol) was dissolved in thionyl chloride (6.0 mL). One drop of N,N-dimethylformamide was added at room temperature. After reflux for 2 hours, the reaction solution was concentrated to obtain a crude product. The crude product was then re-dissolved in anhydrous dichloromethane (10.0 mL) and added dropwise to a suspension of compound 2-5 (3.00 g, 4.9 mmol) and N,N-diisopropylethylamine (1.28 g, 9.8 mmol) in anhydrous dichloromethane (50.0 mL). The reaction was allowed to react at room temperature for 18 hours, and LCMS monitoring indicated the reaction was complete. The reaction solution was quenched with water and extracted three times with dichloromethane (50 mL). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The concentrated crude product was purified by normal phase silica gel column chromatography (dichloromethane / methanol = 20 / 1) to obtain compound 2-9 (3.19 g, 80% yield). LCMS: MS (ESI) m / z = 806.98 [MH] - .

[0316] Compound 2-10:

[0317] Compound 2-9 (3.19 g, 3.9 mmol) was dissolved in methanol (15.0 mL) and dichloromethane (15.0 mL), and lithium borohydride (1.74 g, 79.0 mmol) was added at room temperature. The reaction was allowed to proceed at room temperature for 18 hours, and LCMS monitoring indicated that the reaction was complete. The reaction solution was quenched with water and extracted three times with dichloromethane (50 mL). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by normal silica gel column chromatography (dichloromethane / methanol = 10 / 1) to obtain compound 2-10 (2.22 g, yield 75%). LCMS: MS (ESI) m / z = 750.96 [MH] - .

[0318] Compound 2-11:

[0319] Compound 2-10 (0.21 g, 0.3 mmol) was dissolved in pyridine (5.0 mL) and 4,4'-bismethoxytrityl chloride (0.12 g, 0.3 mmol) was added at room temperature. After stirring for 18 hours, LCMS monitoring showed that the reaction was complete. The reaction solution was quenched with water and extracted three times with dichloromethane (50 mL). The combined organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by normal silica gel column chromatography (0.5% triethylamine / dichloromethane / methanol = 10 / 1) to obtain compound 2-11 (0.07 g, yield 24%). LCMS: MS (ESI) m / z = 1053.2 [MH] - . 1H NMR(400MHz, CDCl3)δ7.29(d,5H),7.17(d,4H),6.87-6.79(m,4H),3.88-3.76(m,6H),3.71(dd,2H),3.40(t,2H),3.28(p,4H) ,3.16(d,2H),2.36(t,2H),2.19(t,4H),2.12-1.96(m,3H),1.82(p,2H),1.76-1.55(m,9H),1.42-1.19(m,50H),0.88(t,6H).

[0320] Compound A02:

[0321] Compound 2-11 (45 mg, 0.04 mmol) was dissolved in anhydrous pyridine (2 mL) and N,N-dimethylformamide (1 mL). 4-Dimethylaminopyridine (26 mg, 0.21 mmol) and succinic anhydride (43 mg, 0.43 mmol) were added sequentially. The reaction mixture was stirred at 50°C for 16 hours. LCMS showed that the reaction was complete. The crude product was concentrated and then triturated with ethyl acetate three times to obtain compound A02 (44 mg, 89% yield). LCMS: MS (ESI) m / z = 1153.2 [MH] - .

[0322] Example 3: Synthesis of Compound A03

[0323] The synthetic route of compound A03 is as follows:

[0324] Compound 3-2:

[0325] Dissolve hexadecanol (5.00 g, 20.6 mmol) in ethyl acetate (55 mL) and add 2-iodoacylbenzoic acid (17.32 g, 61.8 mmol) at room temperature. The reaction mixture was heated to 80°C and stirred for 16 hours. TLC monitoring indicated the reaction was complete. The reaction mixture was cooled to room temperature, filtered, and the filter cake was rinsed with dichloromethane (80 mL). The filtrate was concentrated to obtain the crude product, which was purified by normal phase silica gel column chromatography (petroleum ether / ethyl acetate = 90 / 10) to afford compound 3-2 (4.3 g, 78% yield). 1 H NMR (400MHz, CDCl3) δ9.77 (t, J = 1.6 Hz, 1H), 2.44-2.40 (m, 2H), 1.65-1.61 (m, 2H), 1.26 (s, 24H), 0.88 (t, J = 6.8 Hz, 3H).

[0326] Compound 3-4:

[0327] At 0°C, a catalytic amount of N,N-dimethylformamide (one drop) and oxalyl chloride (20.0 mL, 232.8 mmol) were added dropwise to a solution of palmitic acid (13.60 g, 53.1 mmol) in dichloromethane (50.0 mL). The reaction was allowed to warm to room temperature and stirred for 2 hours. After TLC analysis showed the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain a crude product (14.00 g). At 0°C, a solution of N-tert-butoxycarbonyl-1,4-butanediamine (10.00 g, 53.1 mmol) in tetrahydrofuran (100.0 mL) was added with triethylamine (10.75 g, 106.2 mmol) and a solution of palmitoyl chloride (14.00 g) in tetrahydrofuran (20.0 mL), respectively. The reaction solution was stirred at room temperature for 2 hours. LCMS monitoring showed the reaction was complete. The reaction was quenched by adding water and extracted three times with dichloromethane (80 mL). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The crude product obtained after concentration was purified by beating with ethyl acetate to obtain compound 3-4 (19.00 g, yield 75%). LCMS: MS (ESI) m / z = 449.5 [M+Na] + .

[0328] Compound 3-5:

[0329] To a solution of compound 3-4 (7.00 g, 16.4 mmol) in dioxane (60.0 mL) was added a 4N hydrochloric acid-dioxane solution (40.0 mL) at room temperature. The reaction was stirred for 16 hours, and LCMS monitoring indicated the reaction was complete. The reaction solution was concentrated to afford compound 3-5 (5.00 g, 75% yield). LCMS: MS (ESI) m / z = 349.5 [M+Na] + .

[0330] Compound 3-6:

[0331] To a suspension of compound 3-5 (4.00 g, 12.2 mmol) in methanol (80 mL) at room temperature was added compound 3-2 (3.24 g, 13.4 mmol), a few drops of acetic acid, and sodium triacetoxyborohydride (5.19 g, 24.5 mmol). The reaction was stirred for 16 hours. TLC monitored the reaction for completion. The reaction mixture was filtered and rinsed with methanol to afford compound 3-6 (5.60 g, 78% yield). 1 H NMR (400MHz, MeOD) δ3.20 (t, J = 6.8 Hz, 2H), 3.02-2.95 (m, 4H), 2.17 (t, J = 7.2 Hz, 2H), 1.70-1.54 (m, 8H), 1.29 (s, 52H), 0.90 (t, J = 6.8 Hz, 6H).

[0332] Compound 3-7:

[0333] At room temperature, a solution of compound 2-8 (0.74 g, 3.4 mmol) in thionyl chloride (5.0 mL) was added with one drop of N,N-dimethylformamide, and the reaction was refluxed for 2 hours. TLC monitoring showed that the reaction was complete. After the reaction solution was concentrated, the crude product was redissolved in anhydrous dichloromethane (10.0 mL). To this solution, a suspension of compound 3-6 (1.50 g, 2.7 mmol) in anhydrous dichloromethane (50.0 mL) and N,N-diisopropylethylamine (0.88 g, 6.8 mmol) were added dropwise. The reaction was stirred at room temperature for 18 hours, and LCMS monitoring showed that the reaction was complete. The reaction was quenched by adding water and extracted three times with dichloromethane (80 mL). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The crude product obtained after concentration was purified by normal phase silica gel column chromatography to obtain compound 3-7 (1.53 g, yield 60%). LCMS: MS (ESI) m / z = 748.0 [MH] - .

[0334] Compound 3-8:

[0335] Lithium borohydride (0.56 g, 24.4 mmol) was added to a mixed solution of compound 3-7 (1.53 g, 2.0 mmol) in methanol (15.0 mL) and dichloromethane (15.0 mL) at room temperature. The reaction was stirred for 18 hours, and LCMS monitoring indicated the reaction was complete. The reaction was quenched by adding water and extracted three times with dichloromethane (80 mL). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The crude product obtained after concentration was purified by normal silica gel column chromatography (dichloromethane / methanol = 10 / 1) to obtain compound 3-8 (0.91 g, 64% yield). LCMS: MS (ESI) m / z = 693.9 [MH] - .

[0336] Compound 3-9:

[0337] To a solution of compound 3-8 (0.91 g, 1.3 mmol) in pyridine (10.0 mL) at room temperature was added 4,4'-bismethoxytrityl chloride (0.53 g, 1.6 mmol). The reaction was stirred for 18 hours, and LCMS monitoring indicated completion. The reaction was quenched by addition of water and extracted three times with dichloromethane (50 mL). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The crude product obtained after concentration was purified by normal phase silica gel column chromatography (0.5% triethylamine / dichloromethane / methanol = 10 / 1) to give compound 3-9 (0.55 g, 42% yield). LCMS: MS (ESI) m / z = 996.2 [MH] -. 1H NMR (400MHz, DMSO) δ7.33-7.13(m,9H),6.90-6.83(m,4H),4.35(td,1H),3.72(d,6H),3.42(tt,2H),3.15 (h,4H),2.98(m,4H),2.43(d,1H),2.22-2.11(m,2H),2.02(t,2H),1.52-1.10(m,62H),0.89-0.78(m,6H).

[0338] Compound A03:

[0339] Compound 3-9 (0.15 g, 0.2 mmol) was dissolved in anhydrous pyridine (2 mL) and N,N-dimethylformamide (1 mL). 4-Dimethylaminopyridine (0.19 g, 1.6 mmol) and succinic anhydride (0.08 g, 0.8 mmol) were added sequentially. The reaction mixture was stirred at 50°C for 16 hours. After LCMS showed the reaction was complete, the crude product was concentrated and slurried three times with ethyl acetate to obtain compound A03 (77 mg, 45% yield). LCMS: MS (ESI) m / z = 1096.2 [MH] - .

[0340] Example 4: Synthesis of Compound A04

[0341] The synthetic route of compound A04 is as follows:

[0342] The synthesis and identification of the specific intermediates and final products involved in the above route are as follows:

[0343] Compound 4-2:

[0344] To a solution of compound 4-1 (5.00 g, 27.6 mmol) in N,N-dimethylformamide (40 mL) at 0°C under a nitrogen atmosphere, tert-butyldiphenylsilyl chloride (11.38 g, 41.4 mmol) and imidazole (2.82 g, 41.4 mmol) were added. The reaction was warmed to room temperature and stirred for 4 hours. TLC indicated completion of the reaction, and the mixture was extracted with petroleum ether (40 mL x 3). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The crude product obtained after concentration was purified by normal phase silica gel column chromatography (pure petroleum ether) to afford compound 4-2 (10.0 g, 86% yield). 1 H NMR (400MHz, DMSO-d6) δ7.62(dd,4H),7.49-7.38(m,6H),3.64(t,2H),3.48(t,2H),1.76(q,2H),1.52(q,2H),1.35(p,4H),0.99(s,9H).

[0345] Compound 4-3:

[0346] To a solution of compound 4-2 (10.0 g, 23.8 mmol) in anhydrous acetonitrile (150 mL) were added N-Boc-butanediamine (13.46 g, 71.52 mmol) and potassium carbonate (9.88 g, 71.5 mmol). The reaction was heated to 85°C and stirred for 16 hours. LCMS analysis indicated the reaction was complete. The concentrated crude product was purified by normal phase silica gel column chromatography (dichloromethane / methanol = 20 / 1) to afford compound 4-3 (5.00 g, 40% yield). 1 H NMR(400MHz,DMSO-d6)δ7.64-7.57(m,4H),7.48-7.38(m,6H),6.84(t,1H),3.63(t, 2H),2.90(q,2H),2.51(p,1H),2.43(q,4H),1.52(m,2H),1.37(s,19H),0.99(s,9H).

[0347] Compound 4-4:

[0348] A 1N hydrochloric acid / dioxane solution (5 mL) was added to a solution of compound 4-3 (5.00 g, 9.5 mmol) in dichloromethane (40 mL) at 0°C. The reaction was allowed to warm to room temperature and stirred for 6 hours. LCMS analysis indicated the reaction was complete. The crude product was concentrated and slurried with petroleum ether to afford compound 4-4 (3.20 g, 79% yield). LCMS: MS (ESI) m / z = 427.5 [M+H] + .

[0349] Compound 4-5:

[0350] To a solution of palmitic acid (1.89 g, 7.4 mmol) in N,N-dimethylformamide (20 mL) at 0°C under a nitrogen atmosphere were added 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.81 g, 7.4 mmol) and N,N-diisopropylethylamine (2.73 g, 21.1 mmol). After stirring at room temperature for 0.5 hour, compound 4-4 (1.50 g, 3.5 mmol) was added. The reaction mixture was heated to 50°C and stirred for 16 hours. LCMS analysis indicated the reaction was complete. The reaction was quenched with water and extracted with dichloromethane (20 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product which was purified by reverse-phase column chromatography (acetonitrile / water) to afford compound 4-5 (1.60 g, 50% yield). LCMS: MS (ESI) m / z = 903.7 [M+H] +

[0351] Compounds 4-6:

[0352] To a solution of compound 4-5 (1.50 g, 1.7 mmol) in tetrahydrofuran (15 mL) was added a pyridine hydrogen fluoride solution (3 mL). The reaction was stirred under a nitrogen atmosphere for 16 hours. LCMS analysis showed that the reaction was complete. The crude product obtained by concentration was slurried with acetonitrile, and the filter cake obtained after filtration was purified by normal silica gel column chromatography (dichloromethane / methanol = 20 / 1) to obtain compound 4-6 (0.78 g, yield 71%). LCMS: MS (ESI) m / z = 663.9 [MH] - .

[0353] Compound A04:

[0354] Under a nitrogen atmosphere, 4A molecular sieves were added to a solution of compound 4-6 (0.65 g, 0.6 mmol) in dry dichloromethane (10 mL). After stirring at room temperature for 5 minutes, a pre-prepared solution of 1H-tetrazole (0.45 M) and N-methylimidazole (0.18 M) in dry acetonitrile (2.3 mL) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.70 g, 2.4 mmol) were added sequentially and allowed to react at room temperature for 1 hour. After LCMS analysis showed the reaction was complete, saturated aqueous sodium bicarbonate (10 mL) was added, and the mixture was extracted with dichloromethane (10 mL × 3). The combined organic phases were washed once with saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was slurried with acetonitrile, filtered, and the filter cake was washed three times with acetonitrile to obtain compound A04 (0.70 g, 82% yield). LCMS:MS(ESI)m / z=727.8[M-137] - . 31 P NMR: (400MHz, CDCl3) δ147.31,147.16.

[0355] Example 5: Synthesis of Compound A05

[0356] The synthetic route of compound A05 is as follows:

[0357] The synthesis and identification of the specific intermediates and final products involved in the above route are as follows:

[0358] Compound 5-2:

[0359] To a solution of 8-hydroxyoctanoic acid (5.00 g, 31.2 mmol) in dichloromethane (150.0 mL) were added imidazole (3.19 g, 46.8 mmol) and tert-butyldiphenylsilyl chloride (9.01 g, 32.8 mmol). The reaction was stirred for 2 hours, and TLC monitoring indicated completion. The reaction was quenched with water, and the separated organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The crude product obtained by concentration was purified by normal silica gel column chromatography (petroleum ether / ethyl acetate = 6 / 1) to obtain compound 5-2 (4.50 g, 36% yield). 1 H NMR (400MHz, CDCl3) δ7.69-7.65(m,4H),7.46-7.36(m,6H),3.63(t,2H),2.47(t,2H),1.87(s, 2H),1.72-1.63(m,2H),1.62-1.52(m,2H),1.39-1.35(m,2H),1.34-1.32(m,2H),1.10(s,9H).

[0360] Compound 5-3:

[0361] To a solution of compound 5-2 (2.89 g, 1.2 mmol) in dichloromethane (60 mL) at 0°C were added N,N-diisopropylethylamine (3.0 mL, 18.1 mmol), 1-hydroxybenzotriazole (1.47 g, 10.9 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.09 g, 10.9 mmol). The reaction mixture was stirred for 0.5 hours before compound 3-6 (2.00 g, 3.6 mmol) was added. The temperature was then raised to 50°C and stirred for 16 hours. LCMS monitoring indicated the reaction was complete. The reaction was quenched with water, and the separated organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The crude product obtained after concentration was purified by normal phase silica gel column chromatography (dichloromethane / methanol = 50 / 1) to afford compound 5-3 (1.80 g, 53% yield). LCMS: MS (ESI) m / z = 932.1 [M+H] + .

[0362] Compound 5-4:

[0363] At 0°C, pyridine hydrofluoride (4.00 mL, 44.4 mmol) was added to a solution of compound 5-3 (1.80 g, 1.9 mmol) in dichloromethane (20 mL). The reaction was allowed to warm to room temperature and stirred for 16 hours. After LCMS monitoring showed that the reaction was complete, saturated sodium bicarbonate solution was added to quench the reaction. The separated organic phase was washed with saturated sodium bicarbonate solution (50 mL × 3) and dried over anhydrous sodium sulfate. The crude product obtained after concentration was purified by normal silica gel column chromatography (dichloromethane / methanol = 25 / 1) to obtain compound 5-4 (0.89 g, yield 66%). LCMS: MS (ESI) m / z = 693.9 [M+H] + . 1 H NMR(400MHz, CDCl3)δ3.64(t,2H),3.32-3.19(m,6H),2.33(t,2H),2.25-2.1 7(m,2H),1.98(s,4H),1.67-1.50(m,12H),1.30-1.25(m,52H),0.88(t,6H).

[0364] Compound A05:

[0365] Under a nitrogen atmosphere, 4A molecular sieves were added to a solution of compound 5-4 (0.59 g, 0.8 mmol) in dry dichloromethane (18 mL). The mixture was stirred at room temperature for 5 minutes, followed by the addition of a pre-prepared solution of 1H-tetrazole (0.45 M) and N-methylimidazole (0.18 M) in dry acetonitrile (1.9 mL) and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.58 g, 1.9 mmol). The mixture was allowed to react at room temperature for 1 hour. LCMS analysis indicated the reaction was complete. Saturated aqueous sodium bicarbonate (10 mL) was added, and the mixture was extracted with dichloromethane (10 mL × 3). The combined organic phases were washed once with saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated to afford compound A05 (0.60 g, 79% yield). LCMS: MS (ESI) m / z = 755.9 [M-137] - . 31 P NMR: (400MHz, CDCl3) δ147.17,147.14.

[0366] Example 6: Synthesis of siRNA conjugates of the present disclosure

[0367] 6-1. Synthesis of siRNA Conjugates Containing Lipid Compound A04″ or A05″

[0368] The synthesis of the siRNA conjugates disclosed herein is no different from the conventional phosphoramidite solid phase synthesis method. The synthesis process is briefly described as follows: on a Dr.Oligo48 synthesizer (Biolytic), starting with a universal solid phase carrier, unmodified nucleoside phosphoramidite monomers or nucleoside phosphoramidite monomers with modifications at corresponding positions in the sequence are connected one by one according to the synthesis procedure. Nucleoside phosphoramidite monomers such as 2'-F RNA, 2'-O-methyl RNA and VPUm monomers are purchased from Shanghai Zhaowei or Jiangsu Shenji Biotechnology Co., Ltd. For siRNA conjugates containing lipid compounds A04" or A05", the phosphoramidite monomers A04 and A05 prepared in Example 4 and Example 5 are used at their corresponding positions, respectively. 5-Ethylthio-1H-tetrazole (ETT) was used as an activator (0.6 M acetonitrile solution), 0.22 M PADS dissolved in 1:1 volume ratio of acetonitrile and 3-methylpyridine (Shanghai Lingjiang) solution was used as a sulfurization reagent, and iodine pyridine / water solution (Shanghai Lingjiang) was used as an oxidant.

[0369] After solid-phase synthesis, the oligoribonucleotides were cleaved from the solid support and soaked in a 3:1 solution of 28% ammonia and ethanol at 50°C for 16 hours. The mixture was then centrifuged, and the supernatant was transferred to another centrifuge tube. After evaporation, the supernatant was purified using C8 reverse-phase chromatography with a mobile phase of 0.1 M TEAB and water. The target oligonucleotides were collected, lyophilized, and identified as the desired product by LC-MS and quantified by UV light (260 nm).

[0370] The obtained single-stranded oligonucleotides were annealed according to the complementary pairing in an equal molar ratio, and the resulting double-stranded RNAi agent was dissolved in 1×PBS and adjusted to the required concentration for the experiment.

[0371] 6-2. Synthesis of siRNA Conjugates Containing Lipid Compound A01", A02", A03", or A06"

[0372] Compound A01, A02, or A03 containing a carboxylic acid group (0.1 mmol) was dissolved in anhydrous N,N-dimethylformamide (9.7 mL). Once the substrate was nearly dissolved, anhydrous acetonitrile (6.5 mL), N,N-diisopropylethylamine (3.0 eq), and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.5 eq) were added sequentially. After the reaction mixture was thoroughly mixed, macroporous amine methyl resin (800 mg, target loading 0.1 mmol / g) was added. The reaction mixture was shaken overnight on a shaker (temperature: 25°C, speed: 200 rpm). The reaction mixture was filtered, and the filter cake was washed with dichloromethane and anhydrous acetonitrile. The solid was collected and dried under vacuum overnight.

[0373] The solid from the previous step was dispersed in anhydrous acetonitrile (5 mL), and the CapB1 mixed solution (5.0 mL) was added. The reaction solution was placed in a shaker (temperature: 25°C, speed: 200 rpm) and shaken for 2 hours. The reaction solution was filtered, the filter cake was washed with anhydrous acetonitrile, and the solid was collected and vacuum dried overnight to obtain resin A01', A02', or A03' with a carrier. The loading was measured to be 0.1 mmol / g. Resin A06' with a carrier can be prepared according to the method disclosed in patent WO2019232255(A1).

[0374] siRNA conjugates containing lipid compounds A01", A02", A03", or A06" were prepared using the same method as described in Example 6-1. The only difference was that the resin A01', A02', A03', or A06' with a carrier prepared in the previous step was used instead of the conventional solid phase carrier.

[0375] Table 1

[0376] In the structure shown in Table 1 above Denotes macroporous amine methyl resin.

[0377] Table 2. Sequences of siRNA conjugates targeting the APP gene

[0378] Table 3. Sequences of siRNA conjugates targeting the MAPT gene

[0379] Table 4. Sequences of siRNA conjugates targeting the SARM1 gene

[0380] Table 5. Sequences of siRNA conjugates targeting the PMP22 gene

[0381] In Tables 2 to 5 above, the sequences are from left to right in the direction from 5' to 3' end, and "G", "C", "A", "T" and "U" represent nucleosides, which respectively include the bases guanine, cytosine, adenine, thymine and uracil. A lowercase letter m indicates that the nucleoside adjacent to the left of the letter m is a 2'-methoxy-modified nucleoside; a lowercase letter f indicates that the nucleoside adjacent to the left of the letter f is a 2'-fluoro-modified nucleoside; VP indicates that the 5' end of the nucleoside adjacent to the left / right of the letter is a trans-vinyl phosphodiester group (5'-E-VP); A01", A02', A03", A04", A05", and A06" respectively have lipid compounds with corresponding structures as shown in Table 1; a lowercase letter s indicates that the two nucleosides adjacent to the letter s, or the nucleoside and the lipid compound, are linked by a thiophosphate diester group. Unless otherwise specified, the two adjacent nucleosides or nucleosides, or the nucleoside and the lipid compound are linked by a phosphodiester group. Unless otherwise specified, the 3' position of the first nucleotide at the 3' end of each chain is a hydroxyl group; the 5' position of the first nucleotide at the 5' end of each chain is a hydroxyl group.

[0382] The structures of the 2'-methoxy modified nucleosides, 2'-fluoro modified nucleosides, trans-vinyl phosphodiester groups (5'-E-VP), phosphorothioate diester groups, and phosphodiester groups are shown in the following table. When the siRNA of the present disclosure exists in salt form, for example, in sodium salt form, the salt forms corresponding to the structures in Table 6 below are also within the scope of protection of the present disclosure. In Table 6, Base represents the base at the corresponding position:

[0383] Table 6

[0384] Example 7: Evaluation of the in vitro knockout efficiency of the conjugates of the present disclosure in human A549 and Hek-293A cells

[0385] The siRNA conjugates listed in Table 2 were used to evaluate the in vitro APP knockdown efficiency in A549 cells or Hek-293A cells using five concentration gradients. The specific procedures are as follows:

[0386] A549 cells (purchased from Kebai Biotechnology, catalog number: CRM-CCL-185) and Hek-293A cells (purchased from Kebai Biotechnology, catalog number: CBP60436) were cultured in F-12K and DMEM medium containing 10% fetal bovine serum, respectively, at 37°C and 5% CO2. 24 hours before the free uptake experiment, A549 and Hek-293A cells were seeded in 96-well plates at a seeding density of 2×10 cells per well. 4 and 1×10 4Cells were plated with 100 μL of culture medium per well. After 48 hours of treatment, total RNA was extracted using the high-throughput cell RNA extraction kit FG0417-L / FG0418-XL (Zhifan Medical, magnetic bead method). Human APP mRNA levels were measured by RNA reverse transcription (Takara, RR037A) and real-time quantitative PCR (Thermo, 4444557). Human APP mRNA levels were corrected based on the level of the GAPDH internal reference gene.

[0387] The instruments involved in this experiment are shown in Table 7.

[0388] Table 7. Experimental instruments

[0389] Among them, in the real-time quantitative PCR detection, the probe Q-PCR detection experiment was used, and its primer information is shown in Table 8.

[0390] Table 8. Taqman primer information table

[0391] Result analysis method

[0392] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct values. The comparative Ct refers to calculating the difference in gene expression by comparing the Ct value with the internal reference gene, also known as 2-△△Ct, where △△Ct = [(Ct experimental group target gene - Ct experimental group internal reference) - (Ct control group target gene - Ct control group internal reference)]. Inhibition rate (%) = (1 - residual expression of target gene) × 100%.

[0393] Results are expressed as the percentage of human APP mRNA expression remaining relative to that in cells treated with the siRNA conjugates.

[0394] The data results in Table 9 indicate that the siRNA conjugates disclosed herein have good inhibitory activity against the APP gene in both A549 cells and Hek-293A cells.

[0395] Table 9. APP knockdown activity of siRNA conjugates in A549 cells and Hek-293A cells

[0396] Example 8. Evaluation of the Inhibitory Activity of siRNA Conjugates in Mouse Neuro2α Cells in Vitro

[0397] The siRNA conjugates listed in Table 2 were used to evaluate the in vitro knockdown efficiency of APP in Neuro2α cells using two concentration gradients as follows:

[0398] Neuro2α cells (purchased from ATCC Cell Bank, catalog number: CCL-131) were cultured in MEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. 24 hours before the free uptake experiment, Neuro2α cells were seeded in 96-well plates at a seeding density of 1.5×10 per well. 4 Cells were plated with 100 μL of culture medium per well. After 72 hours of treatment, total RNA was extracted using the MNTR / FX96 (Lr) high-throughput cell RNA extraction kit (Zhiang Biotechnology, magnetic bead method). RNA reverse transcription (Takara, RR037A) and real-time quantitative PCR (Thermo, 4444557) were used to measure mouse APP mRNA levels. Approximately 500 mRNA levels were corrected for those of the GAPDH internal reference gene.

[0399] The instruments involved in this experiment are shown in Table 7.

[0400] Among them, in the real-time quantitative PCR detection, the probe Q-PCR detection experiment was used, and its primer information is shown in Table 10.

[0401] Table 10. Taqman primer information table

[0402] Result analysis method

[0403] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value: Comparative Ct refers to calculating the gene expression difference by the difference between the Ct value and the internal reference gene, also known as 2 -△△Ct , ΔΔCt=[(target gene in Ct experimental group - internal reference in Ct experimental group)-(target gene in Ct control group - internal reference in Ct control group)]. Inhibition rate (%)=(1-remaining amount of target gene expression)×100%.

[0404] The results are expressed as the remaining percentage of mouse APP mRNA expression relative to cells treated with the siRNA conjugates, where "+" indicates that the remaining percentage of expression is less than or equal to 50%, i.e., it has a high level of knockout activity. The data results in Table 11 show that the siRNA conjugates of the present disclosure have a high level of knockout activity against the APP gene in Neuro2α cells.

[0405] Table 11. Inhibitory activity against APP mRNA in Neuro2α cells

[0406] Example 9: Inhibitory activity of the conjugate of the present disclosure on mouse APP gene

[0407] Twenty-four 8-week-old C57BL / 6J male mice (purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) were acclimated for one week before the experiment. The compound was prepared with normal saline before administration on day 0, and the dose for each mouse was 5 mg / kg. The mice were weighed and divided into 4 groups, 6 mice / group, and administered with normal saline (blank vehicle group), siRNA conjugate DS-01, siRNA conjugate DS-04, and siRNA conjugate DS-05, respectively. After anesthesia with Zota, intracerebroventricular (ICV) administration was performed using a stereotactic-guided infusion system (Rayward). Using standard aseptic surgical procedures, a single 2ul injection was performed in the right lateral ventricle at the following positions: AP = -0.2mm (posterior to bregma), ML = -1.0mm, DV = +2.4mm. After a midline incision of the scalp skin, burr holes were created at the selected coordinates using stereotactic coordinates and a cranial drill. Afterwards, the dura mater was punctured at the center of the burr hole, and the needle was lowered to the designated depth to reach the lateral ventricle. The glass electrode was filled with a volume slightly larger than 2 μL. The administration volume was 2 μL, and the injection rate was 1 μL / min. After infusion, the needle was left in place for 2 minutes and then slowly withdrawn, pausing for 30 seconds when the needle tip was in the cortical area. On day 28, the mice were euthanized, and the hippocampus, striatum, and spinal cord tissues of the mice were obtained, snap-frozen in liquid nitrogen, and stored at −80°C. RT-PCR was used to detect the expression level of APP mRNA in the mouse cerebral cortex.

[0408] The experimental results are shown in Table 12 and Figure 1. Table 12 records the inhibition rate of APP mRNA in mice administered with the siRNA conjugates of the present disclosure compared to the blank vector group on day 28. As shown in Table 12 and Figure 1, the siRNA conjugates DS-01, DS-04, and DS-05 of the present disclosure all exhibited good inhibitory activity against APP mRNA in different brain regions of mice.

[0409] Table 12. Inhibitory activity of the siRNA conjugates disclosed herein on APP mRNA in mice

[0410] Example 10: Inhibitory activity of the conjugate of the present disclosure on the mouse MAPT gene

[0411] Thirty 8-week-old C57BL / 6J male mice (purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.) were acclimated for one week before the experiment. On day 0, the compound was prepared with normal saline before administration, and the dose for each mouse was 5 mg / kg. The mice were weighed and divided into 5 groups, with 6 mice per group, and were given normal saline (blank vehicle group), siRNA conjugate DS-07, siRNA conjugate DS-10, siRNA conjugate DS-11, and siRNA conjugate DS-12, respectively. After anesthesia with Zota, intracerebroventricular (ICV) administration was performed using a stereotaxic guided infusion system (Rayward). Using standard aseptic surgical procedures, a single 2 μl injection was performed into the right lateral ventricle at the following locations: AP = -0.2 mm (posterior to bregma), ML = -1.0 mm, DV = +2.4 mm. After a midline incision of the scalp skin, burr holes were created at the selected coordinates using stereotaxic coordinates and a cranial drill. Afterwards, the dura mater was punctured at the center of the burr hole, and the needle was lowered to the designated depth to reach the lateral ventricle. The glass electrode was filled with a volume slightly larger than 2 μL. The administration volume was 2 μL, and the injection rate was 1 μL / min. After infusion, the needle was left in place for 2 minutes and then slowly withdrawn, pausing for 30 seconds when the needle tip was in the cortical area. On day 28, the mice were euthanized, and the hippocampus, striatum, and spinal cord tissues were obtained, snap-frozen in liquid nitrogen, and stored at −80°C. RT-PCR was used to detect MAPT mRNA levels in the mouse cerebral cortex. The experimental results are shown in Table 13 and Figure 2.

[0412] Table 13 records the inhibition rate of MAPT mRNA in mice in each group administered with the siRNA conjugates of the present invention and the reference siRNA conjugate compared to the blank vector group on day 28. The results in Table 13 and Figure 2 show that the siRNA conjugates DS-07, DS-10, DS-11, and DS-12 of the present invention all exhibited significantly better inhibitory activity against MAPT mRNA in mice than the reference siRNA conjugate DS-12.

[0413] Table 13. Inhibitory activity of siRNA conjugates against MAPT mRNA in mice

[0414] Example 11: In vitro inhibitory activity of siRNA conjugates against target mRNA in human A172 cells

[0415] The SARM1 inhibitory activity of the siRNA disclosed herein was evaluated in vitro in A172 cells using two concentration gradients, as follows:

[0416] A172 cells (purchased from Kebai Biotechnology, catalog number: CBP60575) were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 24 hours before the free uptake experiment, A172 cells were seeded in 96-well plates at a seeding density of 0.7 × 10 cells per well. 4 Cells were plated with 100 μL of culture medium per well. After 72 hours of treatment, total RNA was extracted using the MNTR / FX96 (Lr) high-throughput cell RNA extraction kit (Zhiang Biotechnology, magnetic bead method). Human SARM1 mRNA levels were measured by RNA reverse transcription (Takara, RR037A) and real-time quantitative PCR (Thermo, 4444557). Human SARM1 mRNA levels were corrected for GAPDH levels.

[0417] The instruments involved in this experiment are shown in Table 7. In the real-time quantitative PCR detection, a probe Q-PCR detection experiment was used, and its primer information is shown in Table 14.

[0418] Table 14. Taqman primer information table

[0419] Result analysis method

[0420] After the Q-PCR test is completed, the corresponding Ct value is obtained according to the threshold value automatically set by the system. The expression of a gene can be relatively quantified by comparing the Ct value: Comparative Ct refers to calculating the gene expression difference by the difference between the Ct value and the internal reference gene, also known as 2 -△△Ct , ΔΔCt=[(target gene in Ct experimental group - internal reference in Ct experimental group)-(target gene in Ct control group - internal reference in Ct control group)]. Inhibition rate (%)=(1-remaining amount of target gene expression)×100%.

[0421] The results are expressed as the percentage of human SARM1 mRNA expression remaining relative to cells treated with the siRNA conjugates, where "+" indicates that the remaining percentage of expression is less than or equal to 50%, indicating a high level of knockdown activity.

[0422] The data results in Table 15 indicate that the siRNA conjugates disclosed herein have good inhibitory activity against the SARM1 gene in A172 cells.

[0423] Table 15. Inhibitory activity of siRNA conjugates in A172 cells

[0424] Example 12: In vitro inhibitory activity of siRNA conjugates in human HEK293A cells

[0425] The in vitro inhibitory activity of the siRNA conjugates of the present disclosure on PMP22 mRNA was evaluated in HEK293A cells using two concentration gradients. The specific procedures are as follows:

[0426] HEK293A cells (purchased from Kebai Biotechnology, catalog number: CBP60436) were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C and 5% CO2. 24 hours before the free uptake experiment, HEK293A cells were seeded in 96-well plates at a seeding density of 0.7 × 10 cells per well. 4 Cells were plated with 100 μL of culture medium per well. After 72 hours of treatment, total RNA was extracted using the MNTR / FX96 (Lr) high-throughput cell RNA extraction kit (Zhiang Biotechnology, magnetic bead method). Human PMP22 mRNA levels were measured by RNA reverse transcription (Takara, RR037A) and real-time quantitative PCR (Thermo, 4444557). Human PMP22 mRNA levels were corrected for GAPDH levels.

[0427] The instruments, primer information, and result analysis methods involved in this experiment were the same as those in Example 11. The results are shown in Table 16, where "+" indicates that the remaining expression percentage is less than or equal to 50%, i.e., a high level of knockout activity.

[0428] Table 16. Inhibitory activity of siRNA conjugates in HEK293A cells

[0429] Example 13: Evaluation of metabolic stability of siRNA conjugates in various parts of mice

[0430] Fresh liver tissue from C57 male mice (Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.) was obtained, and 1000 μL of homogenate (100 mM Tris, 1.5 mM MgCl2, pH 5.9) was added to 200 mg of the tissue. The tissue was placed in a -40°C refrigerator for 10 minutes, and then ground in a cell tissue grinder at 4°C for 3 minutes. This was repeated three times to prepare a liver homogenate.

[0431] The siRNA conjugates DS-01, DS-07, DS-13 and comparative conjugates DS-06, DS-12, and DS-18 disclosed herein were dissolved in 30% methanol / water (containing 0.2% diisopropylethylamine) at a concentration of 50 μM. Take 150 μL of liver homogenate, add 4 μL of the above siRNA solution, mix well, and incubate at 37°C for 48 hours at a speed of 500 rpm. After 48 hours, take 150 μL of liver homogenate, add 4 μL of the above siRNA conjugate solution to obtain a sample incubated for 0 hours. Add 20 μL of internal standard (siRNA analog) to the sample incubated for 0 hours or 48 hours, and the sample is analyzed by LC-MS after solid phase extraction. The stability of siRNA conjugates in mouse liver homogenates is expressed as the percentage of AS chains remaining (% remaining), calculated as the ratio of the peak area of ​​the analyte to the internal standard after 48 hours of incubation / the peak area of ​​the analyte to the internal standard after 0 hours of incubation. DS-01 and DS-06 are both siRNA conjugates targeting APP, differing only in their conjugate structures; DS-07 and DS-12 are both siRNA conjugates targeting MAPT, differing only in their conjugate structures; and DS-13 and DS-18 are both conjugates targeting SARM1, differing only in their conjugate structures.

[0432] The results showed that the remaining percentage of the siRNA conjugate DS-01 of the present disclosure targeting APP was 1.28 times that of the comparative conjugate DS-06, the remaining percentage of the siRNA conjugate DS-07 of the present disclosure targeting MAPT was 1.30 times that of the comparative conjugate DS-12; and the remaining percentage of the siRNA conjugate DS-13 of the present disclosure targeting SARM1 was 1.24 times that of the comparative conjugate DS-18.

[0433] The above results show that the siRNA conjugates of the present disclosure targeting different targets all exhibit significantly better liver homogenate stability than the comparative siRNA conjugates. In addition, the stability of the siRNA conjugates of the present disclosure in mouse brain homogenate and mouse serum was also tested, and both showed excellent stability.

Claims

1. A conjugate of formula I or a pharmaceutically acceptable salt thereof, in, A is nucleic acid; -L 1 -L 2 -L 3 -L 4 -for L 5 and L 6 are the same or different, each independently a bond, R 1 and R 2 Each independently is -(CH2)7CH3, -(CH2) 12 CH3, -(CH2) 13 CH3, -(CH2) 14 CH3, -(CH2) 15 CH3, -(CH2) 16 CH3, -(CH2) 18 CH3, -(CH2) 20 CH3; n is 1 or 2; Preferably, n is 1.

2. The conjugate of formula I or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The conjugate represented by formula I is a conjugate represented by formula I-1 or I-2, In formula I-1 and formula I-2, m3 is 1, and m4 is 3 or 4; Alternatively, the conjugate represented by formula I is a conjugate represented by formula I-3 or I-4, In formula I-3 and formula I-4, m2 is 6 or 7; R 1 , R 2 , A, L 5 , L 6 , n as defined in claim 1.

3. The conjugate of formula I or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the nucleic acid is siRNA, and the 3' end of the SS chain or the AS chain in the siRNA is connected to -OL 1 - ligation, or, the 5' end of the SS strand or AS strand is ligated with -OL 1 - ligated, or, the 3' and 5' ends of the SS or AS strands are both ligated with -OL 1 -connect.

4. The conjugate of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the nucleic acid and L 1 are connected by phosphodiester groups, wherein -OL 1 The O atom in - is included in the phosphodiester group; Alternatively, the nucleic acid and L 1 They are connected by phosphorothioate diester groups, where -OL 1 The O atom in - is included in the phosphorothioate diester group.

5. The conjugate of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, which has a structure as shown in any one of the following: A is as defined in claim 1.

6. A compound of formula II or a pharmaceutically acceptable salt thereof, in, L 5 , L 6 , R 1 and R 2 As defined in any one of claims 1 to 5; G is hydrogen, R 5 , R 6 is isopropyl, R 7 for -L 7 -L 8 -L 9 -L 10 - Each independently 7. The compound of formula II or a pharmaceutically acceptable salt thereof according to claim 6, wherein the compound of formula II is a compound of formula II-1 or II-2, In formula II-1 and II-2, m6 is 1, and m7 is 3 or 4; Alternatively, the compound represented by formula II is a compound represented by formula II-3 or II-4, In formula II-3 and II-4, m5 is 6 or 7; R 1 , R 2 , G, L 5 , L 6 As defined in claim 6.

8. The compound of formula II or a pharmaceutically acceptable salt thereof according to claim 7, wherein the compound of formula II is a compound of formula II-1A or II-2A, In formula II-1A and II-2A, m6 is 1, and m7 is 3 or 4; Alternatively, the compound represented by formula II is a compound represented by formula II-3A or II-4A, In formula II-3A and II-4A, m5 is 6 or 7; R 1 , R 2 , L 5 , L 6 As defined in claim 6.

9. A compound of the following structure or a pharmaceutically acceptable salt thereof, wherein: As a solid phase carrier, 10. An RNAi agent comprising the conjugate represented by formula I according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof.

11. A pharmaceutical composition comprising the conjugate of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 or the RNAi agent according to claim 10, and one or more pharmaceutically acceptable excipients.

12. Use of a conjugate of formula I according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, an RNAi agent according to claim 10 or a pharmaceutical composition according to claim 11 in the preparation of a medicament for preventing and / or treating a disease, wherein the disease is a neurological disease or disorder, a metabolic disease or disorder, an inflammatory disease or disorder, or cancer.

13. A method for preparing the conjugate of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, which is method 1, method 2 or method 3, Method 1 includes: According to the compound represented by formula II-1A or II-2A according to claim 8 or according to the Starting from the compound described in claim 9, nucleoside monomers are linked one by one from the 3'-5' direction according to the nucleotide arrangement order to obtain the conjugate; Method 2 includes: Starting with a universal solid phase carrier, connecting nucleoside monomers one by one from the 3'-5' direction according to the nucleotide arrangement order, and then connecting the compound represented by formula II-3A or II-4A according to claim 8 to obtain the conjugate; Method three comprises: starting with the compound represented by formula II-1A or II-2A according to claim 8, connecting nucleoside monomers one by one from the 3'-5' direction according to the nucleotide arrangement order, and then connecting the compound represented by formula II-3A or II-4A according to claim 8 to obtain the conjugate.

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