CN118930449A

CN118930449A - A lipid compound, lipid nanoparticles, and preparation method and application thereof

Info

Publication number: CN118930449A
Application number: CN202410966064.0A
Authority: CN
Inventors: 张龙贵; 张跃豹; 刘晨; 王艳; 许秀婷; 梁梅桂; 宋文婷; 徐缘园; 陆颖欣; 廖泽豪; 朱亲影
Original assignee: Shenzhen Houcun Nano Pharmaceutical Co ltd
Current assignee: Shenzhen Houcun Nano Pharmaceutical Co ltd
Priority date: 2024-07-18
Filing date: 2024-07-18
Publication date: 2024-11-12

Abstract

The invention relates to a lipid compound, belonging to the field of biological medicine and biotechnology. The structure of the lipid compound is shown as a formula A, and the lipid compound, the lipid compound nanoparticle or the nucleic acid nanoparticle compound provided by the invention has the advantages of good biocompatibility, high transfection efficiency, good immune activity, good in vivo delivery effect, low toxicity and the like, and has excellent technical effects.

Description

Lipid compound, lipid nanoparticle, and preparation methods and application thereof

Technical Field

The invention belongs to the field of biological medicine and biotechnology, and in particular relates to a lipid compound, a lipid nanoparticle, a preparation method and application thereof.

Background

Gene transfection is a technique of transferring or transporting nucleic acid having a biological function into a cell and allowing the nucleic acid to maintain its biological function in the cell. A gene vector refers to a means for introducing an exogenous therapeutic gene into a biological cell. Gene vectors having industrial transformation potential are currently mainly viral vectors and nonviral vectors.

The virus vector is a gene delivery tool for transmitting the genome of the virus into other cells for infection, and has good application prospects in the prior art such as lentivirus, adenovirus, retrovirus vector, adeno-associated virus vector and the like. However, viral vectors have serious drawbacks due to their inherent physicochemical properties and biological activities, such as high production cost, limited load, poor targeting, insertion integration, teratogenic mutagenesis, etc., which are disadvantageous for developing general and universal therapies.

The non-viral vector mainly comprises: liposome nanoparticles, complex nanoparticles, cationic polymer nanoparticles, polypeptide nanoparticles, and the like. The liposome nanoparticle is a main non-viral vector applied to RNA drug development at present, and the first RNAi drug (PATISIRAN) and the first mRNA drug (BNT 162b2, comirnaty) are marketed sequentially at present, so that the clinical application value of the Liposome Nanoparticle (LNP) is fully verified. Compared with virus vectors, liposome nanoparticles have the advantages of low production cost, clear chemical structure, convenience in quality control, targeting drug delivery realized through targeting modification, unlimited theoretical inclusion capacity and the like, but most liposome lipid materials are undegradable and have larger toxicity, so that the clinical requirement of repeated drug delivery is difficult to meet, in addition, the liposome nanoparticles have the problems of poor in vivo transfection effect, metabolism or clearance of nucleic acid in serum, low bioavailability and the like.

Therefore, there is still a need for a nanoparticle with good biocompatibility and high transfection efficiency.

Disclosure of Invention

In order to solve the technical problems, the invention provides the following technical scheme.

In a first aspect, the present invention provides a lipid compound.

(1) A lipid compound has a structure shown in formula A,

Wherein,

Each n is independently selected from any positive integer from 1-20 (i.e., 1, 2, 3,4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and each m is independently selected from any positive integer from 1-20 (i.e., 1, 2, 3,4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20);

R ¹、R² and R ³ are each independently selected from hydrogen, substituted or unsubstituted C _1-20 alkyl (the C _1-20 alkyl may be C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl or C20 alkyl), substituted or unsubstituted C _2-20 alkenyl (the C _2-20 alkenyl may be C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C10 alkenyl, C11 alkenyl, C12 alkenyl, C13 alkenyl, C14 alkenyl, C15 alkenyl, C16 alkenyl, C17 alkenyl, C18 alkenyl, C19 alkenyl or C20 alkenyl), substituted or unsubstituted C _2-20 alkynyl (the C _2-20 alkynyl may be C2 alkenyl, C3 alkenyl, C5 alkenyl, C11 alkenyl, C12 alkynyl, C15 alkynyl, C17 alkynyl, C20 alkynyl, substituted or unsubstituted C _2-20 heteroalkyl (the C _2-20 heteroalkyl may be C2 heteroalkyl, C3 heteroalkyl, C4 heteroalkyl, C5 heteroalkyl, C6 heteroalkyl, C7 heteroalkyl, C8 heteroalkyl, C9 heteroalkyl, C10 heteroalkyl, C11 heteroalkyl, C12 heteroalkyl, C13 heteroalkyl, C14 heteroalkyl, C15 heteroalkyl, C16 heteroalkyl, C17 heteroalkyl, C18 heteroalkyl, C19 heteroalkyl or C20 heteroalkyl),

Wherein the substitution in the substituted OR unsubstituted C _1-20 alkyl, substituted OR unsubstituted C _2-20 alkenyl, substituted OR unsubstituted C _2-20 alkynyl, OR substituted OR unsubstituted C _2-20 heteroalkyl each independently represents that at least one hydrogen atom is substituted with 1-5 halogen, cyano, -OR ⁴、-SR⁴、N(R⁴)₂、-N(R⁴)₃ ⁺, oxo, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, OR C _1-6 haloalkyl;

Each R ⁴ is independently selected from hydrogen, substituted or unsubstituted C _1-12 alkyl (the C _1-12 alkyl may be C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, or C12 alkyl), substituted or unsubstituted C _2-12 alkenyl (the C _2-12 alkenyl may be C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C10 alkenyl, C11 alkenyl, or C12 alkenyl), or substituted or unsubstituted C _2-12 alkynyl (the C _2-12 alkynyl may be C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl, C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C10 alkynyl, C11 alkynyl, or C12 alkynyl); wherein the substitution in each substituted or unsubstituted C _1-12 alkyl, substituted or unsubstituted C _2-12 alkenyl, or substituted or unsubstituted C _2-12 alkynyl in each R ⁴ independently represents that at least one hydrogen atom is substituted with 1-5 halogens, cyano, -OH, -SR ⁵、N(R⁵)₂, or-N (R ⁵)₃ ⁺ is substituted with =o;

Each R ⁵ is independently selected from hydrogen, substituted or unsubstituted C _1-12 alkyl (the C _1-12 alkyl may be C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, or C12 alkyl), a substituted or unsubstituted C _2-12 alkenyl (the C _2-12 alkenyl may be C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C10 alkenyl, C11 alkenyl, or C12 alkenyl), or a substituted or unsubstituted C _2-12 alkynyl (the C _2-12 alkynyl may be C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl, C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C10 alkynyl, C11 alkynyl or C12 alkynyl); Wherein each substitution in each substituted or unsubstituted C _1-12 alkyl, substituted or unsubstituted C _2-12 alkenyl, or substituted or unsubstituted C _2-12 alkynyl in each R ⁵ independently represents at least one hydrogen atom substituted by 1-5 halogens, cyano, -OH, -SH, NH ₂、-NH(C_1-6 alkyl), a, -N (C _1-6 alkyl) ₂、-N(C_1-6 alkyl) ₃ ⁺ substituted or at least an even number of hydrogen atoms substituted with =o;

Each L is independently selected from C _1-10 alkylene (the C _1-10 alkylene may be C1 alkylene, C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene, C6 alkylene, C7 alkylene, C8 alkylene, C9 alkylene, C10 alkylene, C11 alkylene, or C12 alkylene) or C _3-10 heteroalkylene (the C _3-10 heteroalkylene may be C3 heteroalkylene, C4 heteroalkylene, C5 heteroalkylene, C6 heteroalkylene, C7 heteroalkylene, C8 heteroalkylene, C9 heteroalkylene, C10 heteroalkylene, C11 heteroalkylene, or C12 heteroalkylene);

each Y is independently selected from-O-or-NR ⁶ -;

Each R ⁶ is independently selected from hydrogen, substituted or unsubstituted C _1-6 alkyl (the C _1-6 alkyl may be C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl), substituted or unsubstituted C _2-6 alkenyl (the C _2-6 alkenyl may be C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, or C6 alkenyl), a substituted or unsubstituted C _2-6 alkynyl (the C _2-6 alkynyl may be C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl, or C6 alkynyl), or a substituted or unsubstituted C _1-6 haloalkyl; wherein each substitution in each substituted or unsubstituted C _1-6 alkyl, substituted or unsubstituted C _2-6 alkenyl, substituted or unsubstituted C _2-6 alkynyl, or substituted or unsubstituted C _1-6 haloalkyl in each R ⁶ independently represents at least one hydrogen atom substituted with 1-5 halogens, Cyano, -OH, -NH ₂、-NH(C_1-6 alkyl), -N (C _1-6 alkyl) ₂、-N(C_1-6 alkyl) ₃ ⁺、C_1-6 alkoxy or C _1-6 haloalkoxy substituted or at least 2 hydrogen atoms substituted by =o;

Each R is independently selected from hydrogen, -Z ¹-C_1-20 alkyl, -Z ¹-C_2-20 alkenyl, -Z ¹-C_2-20 alkynyl, -Z ¹ -heterocyclyl, -Z ¹-C_1-6 alkylene-Z-C _1-20 alkyl, -Z ¹-C_1-6 alkylene-Z-C _2-20 alkenyl, -Z ¹-C_1-6 alkylene-Z-C _2-20 alkynyl, -Z ¹-C_1-6 alkylene-Z-heterocyclyl, -Z ¹-C_2-6 alkenyl-Z-C _1-20 alkyl, -Z ¹-C_2-6 alkenyl-Z-C _2-20 alkenyl, -Z ¹-C_2-6 alkenyl-Z-C _2-20 alkynyl, -Z ¹-C_2-6 alkynyl-Z-C _1-20 alkyl, -Z ¹-C_2-6 alkynyl-Z-C _2-20 alkenyl or-Z ¹-C_2-6 alkynyl-Z-C _2-20 alkynyl (the C _1-20 alkyl groups in each R may each be independently selected from C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl or C20 alkyl; The C _2-20 alkenyl in each R is independently selected from C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C10 alkenyl, C11 alkenyl, C12 alkenyl, C13 alkenyl, C14 alkenyl, C15 alkenyl, C16 alkenyl, C17 alkenyl, C18 alkenyl, C19 alkenyl, or C20 alkenyl; the C _2-20 alkynyl group in each R may be C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl, C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C10 alkynyl, C11 alkynyl, C12 alkynyl, C13 alkynyl, C14 alkynyl, C15 alkynyl, C16 alkynyl, C17 alkynyl, C18 alkynyl, C19 alkynyl or C20 alkynyl; The C _1-6 alkyl in each R may be C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl; the C _1-6 alkylene in each R may be independently selected from C1 alkylene, C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene, or C6 alkylene; the C _2-6 alkenyl groups in each R may each be independently selected from C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, or C6 alkenyl; The C _2-6 alkynyl in each R may be independently selected from C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl or C6 alkynyl, respectively;

Each Z is independently selected from a carbon-carbon single bond 、-O-、-O-CH₂-O-、-O-CH₂(CH₃)-O-、-OC(O)-、-NR⁴C(O)-、-C(O)O-、-NR⁴-、-S-、-S-S-、-C(O)-、-C(O)NR⁴-、-S(O)-、-S(O)₂-、-NR⁴C(O)O-、-NR⁴C(O)NR⁴-、-NR⁴S(O)- or-S (O) ₂NR⁴ -;

Each Z ¹ is independently selected from a carbon-carbon single bond 、-O-、-NR⁴-、-S-、-S-S-、-C(O)-、-C(O)O-、-C(O)NR⁴-、-S(O)-、-S(O)₂-、-NR⁴C(O)-、-NR⁴C(O)O-、-NR⁴C(O)NR⁴-、-NR⁴S(O)- or-S (O) ₂NR⁴ -;

Each R ⁴ is independently selected from H or C _1-6 alkyl;

each of the compounds represented by the formula A Comprising at least 6 linear atoms or comprising 6-20 linear atoms.

In some embodiments, the C _2-20 heteroalkyl group contains at least one heteroatom, including at least one of N, S or O.

In some embodiments, each of the compounds of formula AComprising at least 6 linear atoms or comprising 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 linear atoms.

(2) In any lipid compound according to (1), R ¹、R² and R ³ are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C _1-20 alkyl,

Each R ⁶ is independently selected from hydrogen, substituted or unsubstituted C _1-6 alkyl;

Each R is independently selected from hydrogen, -Z ¹-C_1-20 alkyl, -Z ¹-C_2-20 alkenyl, -Z ¹-C_1-6 alkylene-Z-C _1-20 alkyl, -Z ¹-C_1-6 alkylene-Z-C _2-20 alkenyl, -Z ¹-C_2-6 alkenyl-Z-C _1-20 alkyl, -Z ¹-C_2-6 alkenyl-Z-C _2-20 alkenyl.

(3) In the lipid compound according to any one of (1) or (2), n is selected from any one of positive integers from 1 to 20, and m is selected from any one of positive integers from 1 to 20;

R ¹、R² and R ³ are each independently selected from hydrogen, unsubstituted C _1-20 alkyl or

R ⁶ is selected from hydrogen, substituted or unsubstituted C _1-6 alkyl;

each Y is independently selected from-O-or-NR ⁶ -;

R is selected from hydrogen, -Z ¹-C_1-20 alkyl, -Z ¹-C_2-20 alkenyl, -Z ¹-C_1-6 alkylene-Z-C _1-20 alkyl, -Z ¹-C_1-6 alkylene-Z-C _2-20 alkenyl, -Z ¹-C_2-6 alkenyl-Z-C _1-20 alkyl, -Z ¹-C_2-6 alkenyl-Z-C _2-20 alkenyl;

Each Z is independently selected from carbon-carbon single bond, -O-CH ₂-O-、-O-CH₂(CH₃)-O-、-OC(O)-、-NR⁴ C (O) -, -C (O) O-;

Each Z ¹ is independently selected from-O-, -NR ⁴ -;

Each R ⁴ is independently selected from H or C _1-6 alkyl.

(4) The lipid compound according to any one of (1) to (3), wherein the compound represented by the formula A comprises a compound B,

(5) The lipid compound according to any one of (1) to (4), wherein the compound represented by the formula B comprises: a compound C, a compound D,

(6) In the lipid compound according to any one of (1) to (5), n is selected from any one of positive integers (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) from 1 to 10, and m is selected from any one of positive integers (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) from 1 to 20; R is selected from hydrogen, -Z ¹-C_1-20 alkyl, -Z ¹-C_2-20 alkenyl, -Z ¹-C_1-6 alkylene-Z-C _1-20 alkyl, -Z ¹-C_1-6 alkylene-Z-C _2-20 alkenyl, -Z ¹-C_2-6 alkenyl-Z-C _1-20 alkyl, -Z ¹-C_2-6 alkenyl-Z-C _2-20 alkenyl (the C _1-20 alkyl groups in each R may each be independently selected from C1 alkyl groups, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl or C20 alkyl; The C _2-20 alkenyl in each R is independently selected from C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C10 alkenyl, C11 alkenyl, C12 alkenyl, C13 alkenyl, C14 alkenyl, C15 alkenyl, C16 alkenyl, C17 alkenyl, C18 alkenyl, C19 alkenyl, or C20 alkenyl; the C _2-20 alkynyl group in each R may be C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl, C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C10 alkynyl, C11 alkynyl, C12 alkynyl, C13 alkynyl, C14 alkynyl, C15 alkynyl, C16 alkynyl, C17 alkynyl, C18 alkynyl, C19 alkynyl or C20 alkynyl; The C _1-6 alkyl in each R may be C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl; the C _1-6 alkylene in each R may be independently selected from C1 alkylene, C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene, or C6 alkylene; the C _2-6 alkenyl groups in each R may each be independently selected from C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, or C6 alkenyl; The C _2-6 alkynyl in each R may be independently selected from C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl or C6 alkynyl, respectively;

in the compound C, each Z is independently selected from carbon-carbon single bond, -O-CH ₂-O-、-O-CH₂(CH₃)-O-、-OC(O)-、-NR⁴ C (O) -, -C (O) O-;

In the compound C, each Z ¹ is independently selected from-O-, -NR ⁴ -;

In the compound C, each R ⁴ is independently selected from H or C _1-6 alkyl;

in the compound D, R ⁶ is selected from hydrogen or unsubstituted C _1-3 alkyl; n is selected from any positive integer from 1 to 10 (i.e., 1, 2, 3, 4,5, 6, 7, 8, 9, or 10), and m is selected from any positive integer from 1 to 20 (i.e., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); R is selected from hydrogen, -Z ¹-C_1-20 alkyl, -Z ¹-C_2-20 alkenyl, -Z ¹-C_1-6 alkylene-Z-C _1-20 alkyl, -Z ¹-C_1-6 alkylene-Z-C _2-20 alkenyl, -Z ¹-C_2-6 alkenyl-Z-C _1-20 alkyl, -Z ¹-C_2-6 alkenyl-Z-C _2-20 alkenyl (the C _1-20 alkyl groups in each R may each be independently selected from C1 alkyl groups, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl or C20 alkyl; The C _2-20 alkenyl in each R is independently selected from C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C10 alkenyl, C11 alkenyl, C12 alkenyl, C13 alkenyl, C14 alkenyl, C15 alkenyl, C16 alkenyl, C17 alkenyl, C18 alkenyl, C19 alkenyl, or C20 alkenyl; the C _2-20 alkynyl group in each R may be C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl, C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C10 alkynyl, C11 alkynyl, C12 alkynyl, C13 alkynyl, C14 alkynyl, C15 alkynyl, C16 alkynyl, C17 alkynyl, C18 alkynyl, C19 alkynyl or C20 alkynyl; The C _1-6 alkyl in each R may be C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl; the C _1-6 alkylene in each R may be independently selected from C1 alkylene, C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene, or C6 alkylene; the C _2-6 alkenyl groups in each R may each be independently selected from C2 alkenyl, C3 alkenyl, C4 alkenyl, C5 alkenyl, or C6 alkenyl; The C _2-6 alkynyl in each R may be independently selected from C2 alkynyl, C3 alkynyl, C4 alkynyl, C5 alkynyl or C6 alkynyl, respectively;

In the compound D, each Z is independently selected from carbon-carbon single bond, -O-CH ₂-O-、-O-CH₂(CH₃)-O-、-OC(O)-、-NR⁴ C (O) -, -C (O) O-;

in the compound D, each Z ¹ is independently selected from-O-, -NR ⁴ -;

In the compound D, each R ⁴ is independently selected from H or C _1-6 alkyl.

In some embodiments, the halogen comprises at least one of F, cl, br, I.

(7) The lipid compound according to any one of (1) to (6), wherein the compound C is selected from the group consisting of compound L0461, compound L0464, compound L0465, compound L0467, compound L0468, compound L0469, compound L0470, compound L0471, compound L0472, compound L0475, compound L0476, compound L0477, compound L0478, compound L0479, compound L0491, compound L0492, compound L0497, and compound L0498;

The compound D is selected from the group consisting of compounds L0462、L0463、L0473、L0474、L0480、L0481、L0482、L0483、L0484、L0485、L0486、L0487、L0488、L0489、L0490、L0493、L0494、L0495、L0496、L0499、L0500、L0501、L0502、L0503、L0504、L0505、L0506;

In a second aspect, the present invention provides a lipid compound nanoparticle.

(8) The present invention provides a lipid compound nanoparticle as follows.

A lipid compound nanoparticle comprising the lipid compound of the first aspect and a helper material; or comprises the lipid compound of the first aspect, a nucleic acid and a helper material.

In some embodiments, the auxiliary material is selected from: at least one of PEG derivatives, lipids, alcohols, saccharides or inorganic salts.

In some embodiments, the PEG derivative is selected from at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, PEG-modified stearic acid, PEG-modified phosphatidylserine.

In some embodiments, the PEG derivative comprises 1, 2-dimyristoyl-sn-glycerogethoxy polyethylene glycol, 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [ amino (polyethylene glycol) ], dilauroyl phosphatidylethanolamine-polyethylene glycol, dimyristoyl phosphatidylethanolamine-polyethylene glycol, dipalmitoyl phosphatidylcholine polyethylene glycol, dipalmitoyl phosphatidylethanolamine-polyethylene glycol, PEG-distearoyl glycerol, PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceridem, PEG-dipalmitoyl phosphatidylethanolamine, or PEG-1, 2-dimyristol oxypropyl-3-amine.

In some embodiments, the PEG derivative comprises at least one of DMG-PEG2000, mPEG-DSPE, mPEG-STA, mPEG-PS, mPEG-DMPE, mPEG-DPPE, ALC-0159, mPEG2k-DMPE, DSPE-PEG 5000.

In some embodiments, the lipid comprises a lipid selected from phospholipids or sterols.

In some embodiments, the phospholipid comprises at least one selected from the group consisting of lecithin, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylethanolamine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dimyristoyl-sn-glycero-phosphorylcholine, 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine, 1, 2-bisundecoyl-sn-glycero-phosphorylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine.

In some embodiments, the sterols include at least one of cholesterol, lanosterol, 5α -cholestan-3β -ol, stigmasterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, lycorine, ursolic acid, or α -tocopherol.

(9) In the lipid compound nanoparticle described in (8), the lipid compound nanoparticle further includes the following components.

In some embodiments, in the lipid compound nanoparticle of (8), the lipid compound nanoparticle comprises the lipid compound of the first aspect and a helper material; or comprises the lipid compound of the first aspect, a nucleic acid and a helper material;

the auxiliary materials are PEG derivatives and lipids; the lipid comprises a member selected from the group consisting of phospholipids and sterols.

In some embodiments, the phospholipid comprises at least one selected from the group consisting of lecithin (PC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphocholine, 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine, 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-bisundecoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

(10) The lipid compound nanoparticle according to any one of (8) to (9), further comprising:

In some embodiments, in the lipid compound nanoparticle described in any one of (8) - (9), the lipid compound is present in an amount of 20.0mol% to 75.0mol% based on the total molar amount of each component in the lipid compound nanoparticle.

In some embodiments, the PEG derivative is present in an amount of 0.5mol% to 10mol% based on the total molar amount of each component in the lipid compound nanoparticle.

In some embodiments, the phospholipid is present in an amount of 4.0mol% to 20.0mol% based on the total molar amount of each component in the lipid compound nanoparticle.

In some embodiments, the sterol is present in an amount of 18.0mol% to 68.5mol% based on the total molar amount of each component in the lipid compound nanoparticle. In some embodiments, the sterol is present in an amount of 20.0mol% to 68.5mol% based on the total molar amount of each component in the lipid compound nanoparticle.

In some embodiments, the PEG derivative in the lipid compound nanoparticle: phospholipid: sterols: the lipid compound in the first aspect has a molar ratio of (0.5-10.0): (4.0-20.0): (18.0-68.5): (20.0-75.0).

In some embodiments, the PEG derivative in the lipid compound nanoparticle: phospholipid: sterols: the lipid compound in the first aspect has a molar ratio of (0.5-10.0): (4.0-20.0): (20.0-68.5): (20.0-75.0).

In some embodiments, the PEG derivative in the lipid compound nanoparticle: phospholipid: sterols: the lipid compound according to the first aspect has a molar ratio of 2.50:16.00:33.00:48.50、6.00:16.00:29.50:48.50、2.50:4.00:45.00:48.50、0.95:7.58:26.47:65.00、1.50:11.50:38.50:48.50、1.58:16.84:18.42:63.16、10.00:4.00:56.00:30.00、2.32:6.20:45.00:46.48、0.50:4.00:65.50:30.00、2.50:11.50:56.00:30.00、2.00:5.00:68.00:25.00、3.00:4.00:18.00:75.00、2.00:20.00:43.00:35.00、1.50:10.00:68.50:20.00、2.50:4.00:63.50:30.00、1.50:16.00:22.50:60.00、1.50:4.00:64.50:30.00 or 1.40:11.15:38.95:48.50.

In some embodiments, the molar ratio of PEG derivative to phospholipid to sterol of the lipid compound of the first aspect is (0.5-2.5): (7.58-16.0): (29.5-56.0): (35.0-48.5).

In some embodiments, the molar ratio of PEG derivative to phospholipid to sterol of the lipid compound nanoparticle of the first aspect is 0.50:4.00:65.50:30.00, 1.50:11.50:38.50:48.50, or 1.40:11.15:38.95:48.50.

In some embodiments, the PEG derivative is DMG-PEG2000 (i.e., DMG-PEG2 k), mPEG-DMPE, mPEG-DSPE, ALC-0159, mPEG-DPPE, mPEG-STA, mPEG-PS, mPEG2k-DMPE, DSPE-PEG5000.

In some embodiments, the phospholipid comprises at least one of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dimyristoyl-sn-glycero-phosphorylcholine (DMPC), lecithin (PC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

In some embodiments, the sterols include at least one of cholesterol (Chol), lanosterol (Lanosterol).

In a third aspect, the invention provides a nucleic acid nanoparticle complex.

(11) A nucleic acid nanoparticle complex comprising a nucleic acid and a lipid compound nanoparticle of the second aspect.

In some embodiments, the ratio of the molar amount of ionizable nitrogen atoms in the compound to the molar amount of phosphorus atoms of the nucleic acid (i.e., nitrogen to phosphorus ratio) in the lipid compound nanoparticle of the second aspect of the nucleic acid nanoparticle complex is from 6 to 65.

In some embodiments, the ratio of the molar amount of ionizable nitrogen atoms in the lipid compound to the molar amount of phosphorus atoms of the nucleic acid in the lipid compound nanoparticle of the second aspect of the nucleic acid nanoparticle complex is 6、7、8、9、10、11、12、13、13.5、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64 or 65.

In some embodiments, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

In some embodiments, the ribonucleic acid (RNA) comprises at least one of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), self-replicating RNA (saRNA), small hairpin RNA (shRNA), circular RNA (circRNA), messenger RNA (mRNA), or a combination thereof.

In some embodiments, the lipid compound complexes have an average particle size of 70nm to 300nm. In some embodiments, the lipid compound complexes have an average particle size of 70nm to 270nm. In some embodiments, the lipid compound complexes have an average particle size of 70nm to 268nm. In some embodiments, the lipid compound complexes have an average particle size of 70nm, 75nm, 80nm, 85nm

、90nm、95nm、100nm、105nm、110nm、115nm、120nm、125nm、130nm、135nm、140nm、145nm、150nm、155nm、160nm、165nm、170nm、175nm、180nm、185nm、190nm、195nm、200nm、205nm、210nm、215nm、220nm、225nm、230nm、235nm、240nm、245nm、250nm、255nm、260nm、265nm、268nm、270nm、275nm、280nm、285nm、290nm、295nm Or 300nm.

In some embodiments, the lipid compound complex has a polydispersity of 0.30 or less.

In some embodiments, the nucleic acid nanoparticle complex is a numbered nucleic acid nanoparticle complex selected from any one of:

In a fourth aspect, the present invention provides a pharmaceutical composition.

(12) A pharmaceutical composition.

In some embodiments, a pharmaceutical composition comprising a lipid compound nanoparticle according to the second aspect or a nucleic acid nanoparticle complex according to the third aspect, and a pharmaceutically acceptable adjuvant.

In some embodiments, the dosage form of the pharmaceutical composition includes an injection, a suppository, an eye drop, a tablet, a capsule, a suspension, or an inhalant.

In a fifth aspect, the invention provides a use.

(13) Use of a lipid compound according to the first aspect, a lipid compound nanoparticle according to the second aspect, a nucleic acid nanoparticle complex according to the third aspect or a pharmaceutical composition according to the fourth aspect for the preparation of a product for in vivo delivery of a nucleic acid.

Advantageous effects

Compared with the prior art, the invention has at least one of the following technical effects:

The lipid compound, the lipid compound nanoparticle or the nucleic acid nanoparticle compound provided by the invention has the advantages of good biocompatibility, high transfection efficiency, good immunocompetence, good in vivo delivery effect, low toxicity and the like, and has excellent technical effect.

Drawings

FIG. 1 is a statistical plot of serum IgG antibody levels of mice immunized with the nucleic acid nanocomposite of novel S-mRNA-loaded crowns of example 8;

FIG. 2 shows a statistical plot of serum IgG antibody titers of mice immunized with the nucleic acid nanocomposite of novel corona S-mRNA of example 8.

Definition of terms

Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings:

In the present invention, the expressions "L0461", "compound L0461" and "compound represented by formula L0461" mean the same compound.

"V/V" or "V/V" means the volume ratio.

The terms "plurality", "several" mean at least 2, such as 2, 3, 4, 5, etc.

The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

"Any positive integer selected from 1-20" means 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

"Any positive integer selected from 1 to 10" means 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

The term "mol%" means mole percent.

The terms "optional," "optional," or "optionally" mean that the subsequently described event or circumstance may, but need not, occur. For example, the case where "optionally, the heteroatom in the heterocycle includes at least one selected from nitrogen, oxygen, and sulfur" means "the heteroatom in the heterocycle includes at least one selected from nitrogen, oxygen, and sulfur" may be present or absent.

"Room temperature" in the present invention refers to an ambient temperature, which is from about 10 ℃ to about 40 ℃. In some embodiments, "room temperature" refers to a temperature from about 20 ℃ to about 30 ℃; in other embodiments, "room temperature" refers to a temperature from about 25 ℃ to about 30 ℃; in still other embodiments, "room temperature" refers to 10 ℃, 15 ℃,20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, and the like.

"Alkyl" is a hydrocarbon containing a normal, secondary, tertiary, or cyclic carbon atom. For example, an alkyl group may have 1 to-20 carbon atoms (i.e., a C ₁-C₂₀ alkyl group), 1 to-8 carbon atoms (i.e., a C ₁-C₈ alkyl group), or 1 to-6 carbon atoms (i.e., a C ₁-C₆ alkyl group). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, -CH ₃), ethyl (Et, -CH ₂CH₃), 1-propyl (i-Pr, i-propyl, -CH ₂CH₂CH₃), 2-propyl (i-Pr, i-propyl), -CH (CH ₃)₂), 1-butyl (n-Bu, n-butyl, -CH ₂CH₂CH₂CH₃), 2-methyl-1-propyl (i-Bu, i-butyl, -CH ₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, -CH (CH ₃)CH₂CH₃), a catalyst for the preparation of a pharmaceutical composition, 2-methyl-2-propyl (t-Bu, t-butyl, -C (CH ₃)₃), 1-pentyl (n-pentyl, -CH ₂CH₂CH₂CH₂CH₃), 2-pentyl (-CH (CH ₃)CH₂CH₂CH₃), 3-pentyl (-CH (CH ₂CH₃)₂)), a catalyst for the preparation of a pharmaceutical composition, 2-methyl-2-butyl (-C (CH ₃)₂CH₂CH₃), 3-methyl-2-butyl (-CH (CH ₃)CH(CH₃)₂), 3-methyl-1-butyl (-CH ₂CH₂CH(CH₃)₂), 2-methyl-1-butyl (-CH ₂CH(CH₃)CH₂CH₃), 1-hexyl (-CH ₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl (-CH (CH ₃)CH₂CH₂CH₂CH₃), 3-hexyl (-CH (CH ₂CH₃)(CH₂CH₂CH₃)), 2-methyl-2-pentyl (-C (CH ₃)₂CH₂CH₂CH₃)), a catalyst, 3-methyl-2-pentyl (-CH (CH ₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (-CH (CH ₃)CH₂CH(CH₃)₂), 3-methyl-3-pentyl (-C (CH ₃)(CH₂CH₃)₂), 2-methyl-3-pentyl (-CH (CH ₂CH₃)CH(CH₃)₂)), a catalyst for the preparation of a pharmaceutical composition, 2, 3-dimethyl-2-butyl (-C (CH ₃)₂CH(CH₃)₂), 3-dimethyl-2-butyl (-CH (CH ₃)C(CH₃)₃) or octyl (- (CH ₂)₇CH₃)).

The term "alkylene" means a saturated divalent hydrocarbon group resulting from the removal of two hydrogen atoms from a saturated straight or branched hydrocarbon group. Unless otherwise specified, alkylene groups contain 1 to 12 carbon atoms. In one embodiment, the alkylene group contains 1 to 6 carbon atoms; in another embodiment, the alkylene group contains 1 to 4 carbon atoms; in yet another embodiment, the alkylene group contains 1 to 3 carbon atoms; in yet another embodiment, the alkylene group contains 1 to 2 carbon atoms. Examples include methylene (-CH ₂ -), ethylene (-CH ₂CH₂ -), isopropylidene (-CH (CH ₃)CH₂ -), etc.. The alkylene group is optionally substituted with one or more substituents as described herein.

The term "carbocyclyl" or "carbocycle" means a monovalent or polyvalent, non-aromatic, saturated or partially unsaturated, monocyclic, bicyclic or tricyclic ring system containing 3 to 12 carbon atoms. Bicyclic or tricyclic ring systems may include fused rings, bridged rings, and spiro rings. Carbobicyclo groups include spirocarbobicyclo groups and fused carbobicyclo groups, and suitable carbocyclyl groups include, but are not limited to, cycloalkyl, cycloalkenyl, and cycloalkynyl groups. In one embodiment, the carbocyclyl group comprises 3 to 10 carbon atoms, such as a C _3-C₁₀ carbocyclyl group; in another embodiment, the carbocyclyl group comprises 3 to 8 carbon atoms, such as a C _3-C₈ carbocyclyl group; in yet another embodiment, the carbocyclyl group contains 3 to 6 carbon atoms, such as a C _3-C₆ carbocyclyl group. In another embodiment, a monocyclic carbocyclyl group contains 4 to 8 carbon atoms, such as a C _4-C₈ monocyclic carbocyclyl group; in yet another embodiment, a monocyclic carbocyclyl group contains 4 to 6 carbon atoms, such as a C _4-C₆ monocyclic carbocyclyl group. Examples of carbocyclyl groups further include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopentyl-1-enyl, 1-cyclopentyl-2-enyl, 1-cyclopentyl-3-enyl, cyclohexyl, 1-cyclohexyl-1-enyl, 1-cyclohexyl-2-enyl, 1-cyclohexyl-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and the like. The carbocyclyl group is optionally substituted with one or more substituents described herein. Examples of carbocyclyl groups further include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopentyl-1-enyl, 1-cyclopentyl-2-enyl, 1-cyclopentyl-3-enyl, cyclohexyl, 1-cyclohexyl-1-enyl, 1-cyclohexyl-2-enyl, 1-cyclohexyl-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and the like. When the structure clearly requires a linking group, the markush variables recited for that group are understood to be linking groups. For example, if the structure requires a linking group and the markush group definition for that variable enumerates a "carbocyclyl" it is to be understood that the "carbocyclyl" represents a linked carbocyclylene group.

The term "cycloalkyl" denotes a monovalent or polyvalent saturated monocyclic, bicyclic or tricyclic ring system containing 3 to 12 carbon atoms. Bicyclic or tricyclic ring systems may include fused rings, bridged rings, and spiro rings. In one embodiment, cycloalkyl contains 3 to 10 carbon atoms, such as C _3-C₁₀ cycloalkyl; in another embodiment, cycloalkyl contains 3 to 8 carbon atoms, such as C _3-C₈ cycloalkyl; in yet another embodiment, cycloalkyl contains 3 to 6 carbon atoms, such as C _3-C₆ cycloalkyl. The cycloalkyl groups may independently be unsubstituted or substituted with one or more substituents described herein.

The term "alkoxy" means that the alkyl group is attached to the remainder of the molecule through an oxygen atom, wherein the alkyl group has the meaning as described herein. Unless otherwise specified, the alkoxy groups contain 1 to 12 carbon atoms. In one embodiment, the alkoxy group contains 1 to 6 carbon atoms; in another embodiment, the alkoxy group contains 1 to 4 carbon atoms; in yet another embodiment, the alkoxy group contains 1 to 3 carbon atoms. The alkoxy group may be optionally substituted with one or more substituents described herein.

Examples of alkoxy groups include, but are not limited to, methoxy (MeO, -OCH ₃), ethoxy (EtO, -OCH ₂CH₃), 1-propoxy (n-PrO, n-propoxy, -OCH ₂CH₂CH₃), 2-propoxy (i-PrO, i-propoxy, -OCH (CH ₃)₂), 1-butoxy (n-BuO, n-butoxy, -OCH ₂CH₂CH₂CH₃), 2-methyl-l-propoxy (i-BuO, i-butoxy, -OCH ₂CH(CH₃)₂), 2-butoxy (s-BuO, s-butoxy, -OCH (CH ₃)CH₂CH₃), 2-methyl-2-propoxy (t-BuO, t-butoxy, -OC (CH ₃)₃), and the like.

"Alkenyl" is a hydrocarbon containing a normal carbon atom, a secondary carbon atom, a tertiary carbon atom, or a cyclic carbon atom with at least one unsaturated site, i.e., a carbon-carbon sp ² double bond. For example, alkenyl groups may have 2 to 10 carbon atoms (C ₂-C₁₀ alkenyl), 2 to 12 carbon atoms (C ₂-C₁₂ alkenyl), or 2 to 6 carbon atoms (C ₂-C₆ alkenyl). Wherein the alkenyl group may be optionally substituted with one or more substituents described herein, including the positioning of "cis" and "trans", or the positioning of "E" and "Z". Examples of suitable alkenyl groups include, but are not limited to, ethylene or vinyl (-ch=ch ₂), allyl (-CH ₂CH＝CH₂), cyclopentenyl (-C ₅H₇) 5-hexenyl (-CH ₂CH₂CH₂CH₂CH＝CH₂).

"Alkynyl" is a hydrocarbon containing a normal carbon atom, a secondary carbon atom, a tertiary carbon atom, or a cyclic carbon atom with at least one site of unsaturation, i.e., a carbon-carbon sp triple bond. Wherein the alkynyl group may be optionally substituted with one or more substituents described herein. For example, an alkynyl group may have 2 to 10 carbon atoms (C ₂-C₁₀ alkynyl), 2 to 12 carbon atoms (C ₂-C₁₂ alkynyl), or 2 to 6 carbon atoms (C ₂-C₆ alkynyl). Examples of suitable alkynyl groups include, but are not limited to, ethynyl (-c=ch), propargyl (-CH ₂ c=ch), or analogs thereof.

The term "aryl" means a monocyclic, bicyclic and tricyclic carbocyclic ring system containing 6 to 20 ring atoms, or 6 to 14 ring atoms, or 6 to 12 ring atoms, or 6 to 10 ring atoms, wherein at least one ring system is aromatic, wherein each ring system contains rings of 3 to 7 atoms. The aryl group is typically, but not necessarily, attached to the parent molecule through an aromatic ring of the aryl group. The term "aryl" may be used interchangeably with the term "aromatic ring" or "aromatic ring". Examples of aryl groups may include benzene (e.g., phenyl), substituted benzene, naphthalene, anthracene, biphenyl, and the like derived groups, and the like. The aryl group is optionally substituted with one or more substituents described herein.

"Arylalkyl" refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom (typically a terminal or sp ³ carbon atom) is replaced with an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like. Arylalkyl groups can include from 7 to 20 carbon atoms, for example, the alkyl moiety is from I to 6 carbon atoms, and the aryl moiety is from 6 to 14 carbon atoms.

The terms "substituted" such as "substituted C ₁-C₁₀ alkyl", "substituted C ₆-C₂₀ aryl", "substituted arylalkyl", "substituted C ₁-C₂₀ heterocycle" and "substituted carbocyclyl" in relation to alkyl, aryl, arylalkyl, heterocyclyl, heteroaryl, carbocyclyl and the like, mean C ₁-C₁₀ alkyl, C ₆-C₂₀ aryl, arylalkyl, C ₁-C₂₀ heterocycle, alkylamino, carbocyclyl, respectively, wherein one or more hydrogen atoms are each independently replaced by a non-hydrogen substituent. Unless otherwise indicated, when the term "substituted" is used in conjunction with a group having two or more moieties capable of substitution, such as arylalkyl, a substituent may be attached to the aryl moiety, the alkyl moiety, or both.

The term "alkylamino" or "alkylamino" includes "N-alkylamino" and "N, N-dialkylamino" in which the amino groups are each independently substituted with one or two alkyl groups, where the alkyl groups have the meaning as described herein. Suitable alkylamino groups may be mono-or di-alkylamino, examples of which include, but are not limited to, N-methylamino, N-ethylamino, N, N-dimethylamino, N, N-diethylamino, and the like. The alkylamino group is optionally substituted with one or more substituents described herein.

The term "hydroxyalkyl" means an alkyl group substituted with one or more hydroxyl groups, wherein the alkyl group has the meaning as described herein; examples include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxy-1-propyl, 3-hydroxy-1-propyl, 2, 3-dihydroxypropyl, and the like.

The term "cyanoalkyl" means that an alkyl group is substituted with one or more cyano groups, wherein the alkyl group has the meaning as described herein; examples include, but are not limited to, cyanomethyl, 2-cyanoethyl, 2-cyano-1-propyl, 3-cyano-1-propyl, 2, 3-dicyanopropyl, and the like.

The term "aminoalkyl" means an alkyl group substituted with one or more amino groups, wherein the alkyl group has the meaning as described herein; examples include, but are not limited to, aminomethyl, 2-aminoethyl, 2-amino-1-propyl, 3-amino-1-propyl, 2, 3-diaminopropyl, and the like.

The term "haloalkyl" means an alkyl group substituted with one or more halogen atoms, wherein the alkyl group has the meaning as described herein, examples of which include, but are not limited to ,-CHF₂、-CF₃、-CHFCH₂F、-CF₂CHF₂、-CH₂CF₃、-CHFCH₃、-CH₂CH₂F、-CF₂CH₃、-CH₂CF₂CHF₂ and the like. In one embodiment, the C ₁-C₆ haloalkyl comprises a fluoro-substituted C ₁-C₆ alkyl; in another embodiment, the C ₁-C₄ haloalkyl comprises a fluoro-substituted C ₁-C₄ alkyl; in yet another embodiment, the C ₁-C₂ haloalkyl comprises a fluoro-substituted C ₁-C₂ alkyl.

The term "haloalkoxy" means that the alkoxy group is substituted with one or more halogen atoms, wherein the alkoxy group has the meaning as described herein, examples of which include, but are not limited to ,-OCHF₂、-OCF₃、-OCHFCH₂F、-OCF₂CHF₂、-OCH₂CF₃、-OCHFCH₃、-OCH₂CH₂F、-OCF₂CH₃、-OCH₂CF₂CHF₂ and the like. In one embodiment, the C ₁-C₆ haloalkoxy group comprises a fluoro-substituted C ₁-C₆ alkoxy group; in another embodiment, the C ₁-C₄ haloalkoxy group comprises a fluoro-substituted C ₁-C₄ alkoxy group; in yet another embodiment, the C ₁-C₂ haloalkoxy group comprises a fluoro-substituted C ₁-C₂ alkoxy group.

The term "j-k atoms" or "j-k elements" means that the cyclic group consists of j-k ring atoms including carbon atoms and/or O, N, S, P or other heteroatoms; each of j and k is independently any non-zero natural number, and k > j; the term "j-k" includes j, k and any natural number therebetween. For example, "3-8 atom" or "3-8 membered", "3-6 atom" or "3-6 membered", "5-10 atom" or "5-10 membered", "5-6 atom" or "5-6 membered" means that the cyclic group is composed of 3-8 (i.e., 3,4, 5, 6, 7 or 8), 3-6 (i.e., 3,4, 5 or 6), 5-10 (i.e., 5, 6, 7, 8, 9 or 10), or 5-6 (i.e., 5 or 6) ring atoms, including heteroatoms such as carbon atoms and/or O, N, S, P. For another example, a piperidinyl group is a 6-atom-containing heterocyclic group or a 6-membered heterocyclic group, and a pyridinyl group is a 6-atom-containing heteroaryl group or a 6-membered heteroaryl group.

The terms "j-k", "j-k-element" or "C _j-C_k" are each independently any natural number other than zero, and k > j; for example, "1-4" means 1,2, 3 or 4, and "4-6" means 4-, 5-or 6-membered; "C ₃-C₆" means C ₃、C₄、C₅ or C ₆. And so on.

The terms "(alkoxy) -alkylene", "(alkylamino) -alkylene", "(cycloalkyl) -alkylene", "(heterocyclyl) -alkylene", "(aryl) -alkylene", "(heteroaryl) -alkylene" mean that the alkoxy, alkylamino, cycloalkyl, heterocyclyl, aryl or heteroaryl groups are each independently attached to the remainder of the molecule through an alkylene group, wherein the alkoxy, alkylamino, cycloalkyl, heterocyclyl, aryl, heteroaryl and alkylene groups have the meaning described herein. For example, examples of (cycloalkyl) -alkylene include, but are not limited to, cyclopropylmethylene, cyclobutylmethylene, cyclopentylmethylene, cyclohexylmethylene, and the like. Also for example, examples of (aryl) -alkylene groups include, but are not limited to, phenylmethylene, phenylethylene, phenylpropylene, and the like. The (alkoxy) -alkylene, (alkylamino) -alkylene, (cycloalkyl) -alkylene, (heterocyclyl) -alkylene, (aryl) -alkylene, (heteroaryl) -alkylene are each independently optionally substituted with one or more substituents described herein.

The term "prodrug" refers to any compound that when administered to a biological system produces a drug, i.e., an active ingredient, as a result of spontaneous chemical reactions, enzyme-catalyzed chemical reactions, photolysis, and/or metabolic chemical reactions. Prodrugs are thus covalently modified analogues or potential forms of the therapeutically active compound.

"Heterocycle" or "heterocyclyl" includes by way of example and not limitation those heterocycles described in the following: paquette, leo A.; PRINCIPLES OF MODERN HETEROCYCLICCHEMISTRY (w.a. benjamin, new York, 1968), in particular chapters 1, 3,4, 6, 7 and 9; THE CHEMISTRY of Heterocyclic Compounds, A Series of Monographs (John Wiley & Sons, new York,1950 to now), in particular volumes 13, 14, 16, 19 and 28 and J.Am.chem.Soc. (1960) 82:5566. In a particular embodiment of the invention, "heterocycle" includes "carbocycle" as defined herein in which one or more (e.g., 1, 2, 3, or 4) carbon atoms have been replaced with a heteroatom (e.g., O, N or S). The term "heterocycle" or "heterocyclyl" includes saturated rings, partially unsaturated rings, and aromatic rings (i.e., heteroaromatic rings). Substituted heterocyclyl groups include, for example, heterocycles substituted with any substituent disclosed herein including carbonyl.

The terms "heterocyclyl" and "heterocycle" are used interchangeably herein to refer to a non-aromatic, saturated or partially unsaturated, monocyclic, bicyclic or tricyclic ring system containing 3 to 12 ring atoms, wherein the bicyclic or tricyclic ring system may include fused rings, bridged rings and spiro rings. Wherein one or more atoms in the ring are independently replaced by heteroatoms having the meaning as described herein. In one embodiment, the heterocyclyl is a monocyclic heterocyclyl consisting of 3 to 8 ring atoms (1 to 7 carbon atoms and 1 to 4 heteroatoms selected from N, O, P or S, where S or P are optionally substituted with one or more oxygen atoms to give a group like SO, SO ₂,PO,PO₂); in yet another embodiment, the heterocyclyl is a monocyclic heterocyclyl consisting of 3 to 6 ring atoms (1 to 5 carbon atoms and 1 to 4 heteroatoms selected from N, O, P or S, where S or P are optionally substituted with one or more oxygen atoms to give a group like SO, SO ₂,PO,PO₂); in another embodiment, the heterocyclyl is a bicyclic heterocyclyl consisting of 7-12 ring atoms (1-11 carbon atoms and 1-4 heteroatoms selected from N, O, P or S where S or P is optionally substituted with one or more oxygen atoms to give a group like SO, SO ₂,PO,PO₂). The heterocyclyl group is optionally substituted with one or more substituents described herein.

The ring atoms of the heterocyclic groups may be carbon groups or heteroatom groups. Wherein the-CH ₂ -group of the ring is optionally replaced by-C (=O) -and the sulfur atom of the ring is optionally oxidized to S-oxide and the nitrogen atom of the ring is optionally oxidized to N-oxide. Examples of heterocyclyl groups include, but are not limited to, oxiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, 1, 3-dioxacyclopentyl, dithiocyclopentyl, tetrahydropyranyl, dihydropyranyl, 2H-pyranyl, 4H-pyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, dioxanyl, dithianyl, thiazalkyl, homopiperazinyl, homopiperidinyl, oxacycloheptyl, thietanyl, oxazanyl, and the likeRadical, diazaRadical, thiazasA group, 2-oxa-5-azabicyclo [2, 1] hept-5-yl group, and the like. Examples of the substitution of the-CH ₂ -group in the heterocyclyl group by-C (=o) -include, but are not limited to, 2-oxo-pyrrolidinyl, oxo-1, 3-thiazolidinyl, 2-piperidonyl, 3, 5-dioxopiperidyl, pyrimidinedionyl, and the like. Examples of sulfur atoms in the heterocyclyl group that are oxidized include, but are not limited to, sulfolane, thiomorpholino 1, 1-dioxide, and the like. The heterocyclyl group is optionally substituted with one or more substituents described herein.

Examples of heterocycles include by way of example and not by way of limitation, pyridyl, dihydropyridinyl, tetrahydropyridinyl (piperidinyl), thiazolyl, tetrahydrothienyl, thioxotetrahydrothienyl, pyrimidinyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuryl, thianaphtyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidinonyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azacinyl (azacyclooctanyl), triazinyl, 6H-1,2, 5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chroenyl, xanthenyl, phenoflavinyl 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4 aH-carbazolyl, beta-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, and pharmaceutical compositions containing the same phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochroman, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazole, benzisoxazolyl, oxindolyl, benzoxazolinyl, indolinyl, isatinyl and bis-tetrahydrofuranyl.

The term "heteroaryl" means monocyclic, bicyclic and tricyclic ring systems containing 5 to 12 ring atoms, or 5 to 10 ring atoms, or 5 to 6 ring atoms, wherein at least one ring system is aromatic and at least one ring system contains one or more heteroatoms, wherein each ring system contains a ring of 5 to 7 atoms. Heteroaryl groups are typically, but not necessarily, attached to the parent molecule through an aromatic ring of the heteroaryl group. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring", "aromatic heterocycle" or "heteroaromatic. The heteroaryl group is optionally substituted with one or more substituents described herein. Non-limiting examples of suitable heteroatoms that may be included on the aromatic ring include oxygen, sulfur, and nitrogen. Non-limiting examples of heteroaryl rings include all those aromatic rings listed in the definition of "heterocyclyl" including pyridyl, pyrrolyl, oxazolyl, indolyl, isoindolyl, purinyl, furanyl, thienyl, benzofuranyl, benzothienyl, carbazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, quinolinyl, isoquinolinyl, pyridazinyl, pyrimidinyl, pyrazolyl, and the like. In one embodiment, the heteroaryl group of 5 to 10 ring atoms comprises 1,2,3 or 4 heteroatoms independently selected from O, S or N.

Examples of heteroaryl groups include, but are not limited to, 2-furyl, 3-furyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g., 3-pyridazinyl), 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl), triazolyl (e.g., 2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl, pyrazolyl (e.g., 2-pyrazolyl), isothiazolyl, 1,2, 3-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,2, 4-oxadiazolyl, 1,2, 3-triazolyl, 1, 3-dithiotriazinyl, 1, 3-dithio, 3-triazolyl, 1, 3-triazolyl; the following bicyclic rings are also included, but are in no way limited to: benzimidazolyl, benzofuranyl, benzothienyl, indolyl (e.g., 2-indolyl), purinyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl, 4-quinolinyl), isoquinolinyl (e.g., 1-isoquinolinyl, 3-isoquinolinyl, or 4-isoquinolinyl), imidazo [1,2-a ] pyridinyl, pyrazolo [1,5-a ] pyrimidinyl, imidazo [1,2-b ] pyridazinyl, [1,2,4] triazolo [4,3-b ] pyridazinyl, [1,2,4] triazolo [1,5-a ] pyrimidinyl, [1,2,4] triazolo [1,5-a ] pyridinyl, and the like.

The term "treating" as used herein, unless indicated otherwise, means reversing, alleviating, inhibiting the progression of, or preventing a disorder or condition for which the term is applicable or one or more symptoms of such disorder or condition. The term "treatment" as used herein refers to a therapeutic action, as "treatment" is defined immediately above.

The compounds of the invention also include reference to physiologically acceptable salts thereof, examples including salts derived from suitable bases such as alkali or alkaline earth metals (e.g., na ⁺、Li⁺、K⁺、Ca⁺² and Mg ⁺²), ammonium and NR ₄ ⁺ (where R is as defined herein). Physiologically acceptable salts of nitrogen atoms or amino groups include: (a) Acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; (b) Salts with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, isethionic acid, lactobionic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolic acid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid, phthalic acid, mandelic acid, lactic acid, ethanesulfonic acid, lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, leucine and the like; and (c) salts with elemental anions such as chlorine, bromine, and iodine. Physiologically acceptable salts of hydroxy compounds include combinations of anions of the compounds with suitable cations such as Na ⁺ and NR ₄ ⁺.

Whenever a compound described herein is substituted with more than one of the same designated groups (e.g., "R" or "R ¹"), it is understood that these groups may be the same or different, i.e., each group is independently selected.

Pharmaceutical preparation

The compounds of the present invention are formulated with conventional carriers and excipients which will be selected in accordance with conventional practices. Although the active ingredients can be administered alone, they are preferably formulated into pharmaceutical formulations. The formulations of the invention, whether for veterinary or human use, comprise at least one active ingredient as defined above together with one or more acceptable carriers therefor, and optionally further therapeutic ingredients, particularly those further therapeutic ingredients as disclosed herein. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and physiologically harmless to its recipient.

Formulations include those suitable for the above routes of administration. The formulations may be conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations can generally be found in Remington's Pharmaceutical Sciences (Mack Publishing co., easton, PA.). Such methods include the step of mixing the active ingredient with a carrier that constitutes one or more accessory ingredients. In general, the formulation is prepared as follows: the product is shaped by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary.

Route of administration:

one or more compounds of the invention (referred to herein as active ingredients) are administered by any route suitable for the condition being treated. Suitable routes include oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural), and the like. It will be appreciated that the preferred route may vary with the condition of the recipient, for example.

Detailed Description

In order to better understand the technical solution of the present invention, some non-limiting examples are further disclosed below to further describe the present invention in detail.

The reagents used in the present invention are all commercially available or can be prepared by the methods described herein.

In the present invention, nM means nanomole per liter; mu M represents micromoles per liter; mmol represents millimoles; equiv represents equivalent.

The term "x g" means a centrifugal acceleration of how many times the gravitational acceleration, for example "5000 x g" means a centrifugal acceleration of 5000 times the gravitational acceleration.

Body represents a mouse living body; lung represents lung; liver denotes the liver; spleen denotes the spleen; muscle means muscle.

DMG-PEG2000 means 1, 2-dimyristoyl-sn-glycerylmethoxy polyethylene glycol 2000 (CAS: 160743-62-4); PEG-DMPE means dimyristoyl phosphatidylethanolamine-polyethylene glycol; PEG-DPPC represents dipalmitoyl phosphatidylcholine polyethylene glycol; mPEG-STA represents methoxypolyethylene glycol-monostearate; mPEG-PS represents methoxypolyethylene glycol-phosphatidylserine; mPEG-DPPE represents L-phosphatidylethanolamine PEG (purchased from https:// www.aladdin-e.com/zh_cn/d163583. Html); mPEG-DSPE represents methoxy-polyethylene glycol-phosphatidylethanolamine (CAS: 147867-65-0); DSPE-PEG5000 (mPEG-DSPE with PEG molecular weight of 5000); mPEG-DMPE represents 2-myristoyl-3-phosphatidylethanolamine PEG (purchased from:

https:// www.aladdin-e.com/zh_cn/d163570. Html); mPEG2k-DMPE (mPEG-DMPE with PEG molecular weight 2000); ALC-0159 (methoxypolyethylene glycol bitetradecylamide, CAS: 1849616-42-7); DOTAP represents (2, 3-dioleoyl-propyl) -trimethylamine sulfate; DOPE represents 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine; DSPC represents 1, 2-distearoyl-sn-glycero-3-phosphorylcholine; chol represents cholesterol; DMPC represents 1, 2-dimyristoyl-sn-glycero-phosphorylcholine; PC represents lecithin; Representing tween 20; DPPC represents 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine; Representing span 80.EDCI stands for 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. DMAP represents 4-dimethylaminopyridine. HCTU represents 6-chlorobenzotriazole-1, 3-tetramethylurea hexafluorophosphate. NMM represents N-methylmorpholine. Ultra means dichloromethane/methanol/ammonia = 75:22:3 (volume ratio). DCC means N, N' -dicyclohexylcarbodiimide.

L0461 represents an L0461 compound, L0462 represents an L0462 compound, L0463 represents an L0463 compound, and so on.

FLuc-mRNA represents messenger RNA encoding firefly luciferase; EGFP-pDNA represents a plasmid encoding a green fluorescent protein; EGFP-siRNA represents a small interfering RNA for silencing enhanced green fluorescent protein gene expression; S-mRNA or Spike-mRNA represents messenger RNA encoding S protein.

"IH" means subcutaneous injection; "IM" means intramuscular injection; "IV" intravenous injection; "IP" means intraperitoneal injection.

FLuc-mRNA manufacturer: shanghai megadimension technology development Co., ltd (Hongene Biotech Corporation).

Specific information for the FLuc-mRNA stock solution is:

product name: FLuc-mRNA (N1-Me-pseudo U);

description of the product: 1939 nucleotides in length;

Modifications (modification): fully substituted with N1-Me-pseudo UTP; (all replaced with N1-Me-pseudo UTP);

concentration: 1.0mg/mL;

storage environment: 1mM sodium citrate, pH 6.4;

storage requirements are: -40 ℃ or less.

Example 1: synthesis of hydrophobic chains of ionizable lipid compounds

The ionizable lipid compounds of the invention are produced by any previously known synthetic method known to those of ordinary skill in the art. The raw materials of the preparation method, namely compound 1, compound 2, compound 4, compound 6, epoxypropionic acid, 1-decanol, 6AF6, SS10C, 2-hexyl octanoic acid, 2-hexyl decanoic acid, 1, 4-butanediol, 1, 6-hexanediol, 4AFV8, 6AFV6, 6AE6, oleylamine, 8MN, 10MN, 12MN, 14MN, boc-2A, boc-4A, boc-6A, boc-2DA, boc anhydride, GABA, 8-pentadecanol, 1-decanol and the like, can be purchased commercially or can be synthesized by a conventional method.

The simple synthesis method and specific process for synthesizing the hydrophobic chain of the ionizable lipid compound are described as follows:

synthesis of Compound 3:

HCTU (6.83 g) was added in portions to a solution of dodecylamine (2.78 g), epoxypropionic acid (1.32 g) and NMM (1.82 mL) in DMF (N, N-dimethylformamide, 60 mL) and stirred for 12h. The reaction was diluted with 150mL of ethyl acetate and the resulting solution was washed with saturated sodium chloride solution (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=5:1 (V/V)) to give a pale yellow powdery compound 3(3.1g).¹H NMR(400MHz,Chloroform-d)δ6.15(s,1H),3.40(dd,J＝4.8,2.8Hz,1H),3.20(dp,J＝16.8,6.4Hz,2H),2.95(t,J＝5.2Hz,1H),2.71(dd,J＝5.6,2.8Hz,1H),1.45(p,J＝6.8Hz,2H),1.34–1.16(m,18H),0.85(t,J＝6.8Hz,3H);HRMS(ESI,m/z)[M+H]⁺calcd for C₁₅H₃₀NO₂:256.22711;found:256.22806.

Synthesis of Compound 5:

HCTU (9.10 g) was added in portions to a solution of decylamine (3.46 g), epoxypropionic acid (1.76 g) and NMM (2.42 mL) in 60mL DMF and stirred for 12h. The reaction was diluted with 150mL of ethyl acetate and the resulting solution was washed with saturated sodium chloride solution (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=5:1 (V/V)) to give a pale yellow powdery compound 5(3.2g).¹H NMR(400MHz,Chloroform-d)δ6.10(s,1H),3.43(dd,J＝4.8,2.8Hz,1H),3.23(dp,J＝19.6,6.4Hz,2H),2.98(t,J＝5.2Hz,1H),2.73(dd,J＝5.6,2.4Hz,1H),1.47(p,J＝6.8Hz,2H),1.33–1.19(m,14H),0.87(t,J＝6.8Hz,3H);HRMS(ESI,m/z)[M+H]⁺calcd for C₁₃H₂₆NO₂:228.19581;found:228.19937.

Synthesis of Compound 7:

DCC (4.47 g) was added to a mixed solution of dodecanol (4.10 g), epoxypropionic acid (1.76 g) and DMAP (244.4 mg) in 60mL DCM under ice-bath conditions. The resulting mixture was stirred in an ice bath for half an hour and then moved to room temperature for stirring for 12h. The reaction was quenched with 100mL saturated ammonium chloride and extracted with dichloromethane (50 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=20:1 (V/V)) to give the compound as pale yellow powder 7(3.0g).¹H NMR(400MHz,Chloroform-d)δ4.16(qt,J＝10.8,6.8Hz,2H),3.41(dd,J＝4.0,2.4Hz,1H),2.93(qd,J＝6.4,3.2Hz,2H),1.64(p,J＝6.8Hz,2H),1.34–1.17(m,18H),0.86(t,J＝6.8Hz,3H);HRMS(ESI,m/z)[M+H]⁺ calcd for C₁₅H₂₉O₃:257.21112;found:257.21190.

Synthesis of Compound GA-14C:

DCC (1.36 g) was added to a mixed solution of tetradecanol (1.42 g), epoxypropionic acid (0.53 g) and DMAP (73.0 mg) in 60mL DCM under ice-bath conditions. The resulting mixture was stirred in an ice bath for half an hour and then moved to room temperature for stirring for 12h. The reaction was quenched with 50mL saturated ammonium chloride and extracted with dichloromethane (50 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=20:1 (V/V)) to give the compound as a white powder GA-14C(0.96g).¹H NMR(400MHz,Chloroform-d)δ4.27–4.08(m,2H),3.43(dd,J＝4.0,2.4Hz,1H),3.02–2.86(m,2H),1.66(p,J＝6.8Hz,2H),1.38–1.20(m,22H),0.88(t,J＝6.8Hz,3H).

Synthesis of Compound GA-10C:

Epoxiconazole (0.50 g) and 1-decanol (1.20 mL) were dissolved in 15mL DCM. DMAP (71.0 mg) and DCC (1.29 g) were added thereto at 0℃and stirred at that temperature for 15min, after which the reaction system was moved to room temperature and stirred for a further 12h. The reaction was quenched with 5mL of saturated ammonium chloride solution, filtered through celite, washed with 60mL of DCM, the filtrate was washed twice with 20mL of saturated aqueous ammonium chloride solution and 2 times with 20mL of saturated aqueous sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by column chromatography (PE: EA (i.e. petroleum ether: ethyl acetate) =15:1, v/v). Obtaining a colorless oily liquid GA-10C(0.699g).¹H NMR(400MHz,Chloroform-d)δ4.22–4.08(m,2H),3.41(dd,J＝4.0,2.8Hz,1H),2.98–2.88(m,2H),1.70–1.60(m,2H),1.38–1.17(m,14H),0.86(t,J＝6.8Hz,3H).

Synthesis of Compound GA-6AF 6:

Epoxiconacid (1.77 g) was mixed with 80mL DCM, and 6AF6 (5.13 g) was added to the reaction system. DMAP (250.0 mg) and DCC (4.49 g) were added at 0deg.C, reacted for 15min, followed by reaction at room temperature for 12h. The reaction was quenched with saturated ammonium chloride solution, filtered through celite, the filtrate was washed twice with saturated ammonium chloride solution, 2 times with sodium chloride solution, dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by column chromatography (PE: ea=15:1 (V/V)). Obtaining the liquid compound as colorless oil GA-6AF6(3.00g).¹H NMR(400MHz,Chloroform-d)δ4.65(s,2H),4.28–4.10(m,2H),3.51(td,J＝6.8,2.8Hz,4H),3.42(dd,J＝4.0,2.8Hz,1H),3.01–2.87(m,2H),1.71–1.65(m,2H),1.64–1.51(m,4H),1.44–1.24(m,10H),0.88(t,J＝6.8Hz,3H);HRMS(ESI,m/z)[M+Na]⁺calcd for C₁₆H₃₀NaO₅:325.19855;found:325.19388.

Synthesis of Compound GA-4E8B 6:

2-hexyl octanoic acid (10.53 g) and 1, 4-butanediol (15.52 mL) were mixed and 290mL DCM was added. DMAP (505.3 mg) and EDCI (12.58 g) were added at 0℃and reacted for 30min, followed by a reaction at room temperature for 24h. After the reaction was completed, it was diluted with DCM, washed 2 times with saturated ammonium chloride, then 2 times with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by column chromatography (PE: ea=3:1 (V/V)) to give the compound as a pale yellow oily liquid 4E8B6(9.890g).HRMS(ESI,m/z):[M+H]⁺ calcd.For:C₁₈H₃₆O₃,301.27372;found:301.27568.

Epoxiconacid (1.20 g) was mixed with 4E8B6 (4.10 g), followed by 48mL of DCM, DMAP (165.0 mg) and EDCI (5.22 g) were added at 0deg.C and reacted for 30min. Then reacted at room temperature for 12 hours. The reaction solution was washed 2 times with saturated ammonium chloride solution, 2 times with saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate=7/1 (V/V)) to give a colorless oily compound GA-4E8B6(3.09g).¹H NMR(400MHz,Chloroform-d)δ4.21–4.07(m,2H),4.02(t,J＝6.0Hz,2H),3.34(t,J＝3.3Hz,1H),2.93–2.80(m,2H),2.30–2.18(m,1H),1.75–1.59(m,4H),1.57–1.44(m,2H),1.41–1.30(m,2H),1.29–1.05(m,16H),0.79(t,J＝6.8Hz,6H).HRMS(ESI,m/z):[M+H]+ calcd.For:C₂₁H₃₈O₅,371.27920;found:371.28013.

Synthesis of Compound GA-6E8B 6:

2-hexyl octanoic acid (10.53 g) and 1, 6-hexanediol (20.70 g) were mixed and 300mL DCM was added. DMAP (505.3 mg) and EDCI (12.58 g) were added at 0℃and reacted for 30min, followed by stirring at room temperature for 12h. After the reaction was completed, it was diluted with DCM, washed 2 times with saturated ammonium chloride, then 2 times with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by column chromatography (PE: ea=3:1 (V/V)) to give compound 6E8B6 (9.70 g) as a pale yellow oil.

Epoxy propionic acid (0.83 g) and 6E8B6 (3.41 g) were mixed, followed by addition of 25mL DCM to the reaction system. DMAP (114 mg) and EDCI (2.13 g) were added at 0℃and reacted for 30min, followed by reaction at room temperature for 12h. The reaction was quenched with saturated ammonium chloride solution, filtered through celite (7 μm), the filtrate was washed 2 times with saturated ammonium chloride solution, then 2 times with saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=7/1 (V/V)) to give the compound as a pale yellow oily liquid compound GA-6E8B6(2.09g).¹H NMR(400MHz,Chloroform-d)δ4.19–4.06(m,2H),4.01(t,J＝6.8Hz,2H),3.36(t,J＝3.2Hz,1H),2.93–2.83(m,2H),2.31–2.20(m,1H),1.69–1.45(m,6H),1.44–1.30(m,6H),1.30–1.04(m,16H),0.81(t,J＝6.8Hz,6H).HRMS(ESI,m/z):[M+H]+ calcd.For:C₂₃H₄₂O₅,399.31050;found:399.31127.

Synthesis of Compound GA-6E10B 6:

2-hexyl decanoic acid (10.01 g) and 1, 6-hexanediol (18.42 g) were mixed and 250mL DCM was added. DMAP (473.0 mg) and EDCI (11.21 g) were added at 0deg.C, reacted for 30min, followed by reaction at room temperature for 24h. After the reaction, the reaction system was ice-bathed at 0℃for 15min, followed by suction filtration through 7 μm celite, washing the filtrate with saturated ammonium chloride 2 times, washing with saturated sodium chloride 2 times, drying over anhydrous sodium sulfate, and vacuum concentrating. The crude product was purified by column chromatography (PE: ea=3/1 (V/V)) to give the product as a pale yellow oily liquid compound 6E10B6(9.05g).HRMS(ESI,m/z):[M+H]⁺ calcd.For:C₂₂H₄₄O₃,357.33632;found:357.33778.

Epoxy propionic acid (0.83 g) and 6E10B6 (3.70 g) were mixed, followed by addition of 25mL DCM to the reaction system. DMAP (144.0 mg) and EDCI (2.13 g) were added at 0℃and reacted for 30min, followed by reaction at room temperature for 12h. The reaction was quenched with saturated ammonium chloride solution, filtered through celite (7 μm), the filtrate was washed 2 times with saturated ammonium chloride solution, then 2 times with saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by column chromatography (petroleum ether: ethyl acetate=7/1 (V/V)) to give the final product as a pale yellow oily liquid compound GA-6E10B6(2.09g).¹H NMR(400MHz,Chloroform-d)δ4.23–4.09(m,2H),4.04(t,J＝6.8Hz,2H),3.40(t,J＝3.2Hz,1H),2.98–2.87(m,2H),2.34–2.23(m,1H),1.71–1.49(m,6H),1.45–1.33(m,6H),1.32–1.14(m,20H),0.84(t,J＝6.8Hz,6H).HRMS(ESI,m/z):[M+H]+ calcd.For:C₂₅H₄₆O₅,427.34180;found:427.34297.

Synthesis of Compound GA-4AFV 8:

Epoxiconamic acid (1.21 g) was mixed with 30mL DCM, and 4AFV8 (4.86 g) was added to the reaction system. DMAP (165.0 mg) and DCC (3.92 g) were added and mixed at 0deg.C, and the resulting mixture was stirred in an ice bath for half an hour, then moved to room temperature and stirred for 12h. The reaction was quenched with 5mL of saturated aqueous NH ₄ Cl, filtered and washed with 10mL of saturated aqueous NaCl. The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate=7:1 (V/V)) to give the compound as a colorless oil GA-4AFV8(1.22g).¹H NMR(400MHz,Chloroform-d)δ5.50–5.39(m,1H),5.39–5.27(m,1H),4.64(s,2H),4.22–4.10(m,2H),3.51(td,J＝6.8,2.0Hz,4H),3.40(dd,J＝4.0,2.8Hz,1H),2.98–2.87(m,2H),2.31(q,J＝7.2Hz,2H),2.04(p,J＝7.6Hz,2H),1.71–1.62(m,2H),1.62–1.51(m,2H),1.43–1.33(m,4H),0.94(t,J＝7.6Hz,3H).HRMS(ESI,m/z):[M+NH4]⁺ calcd.For:C₁₆H₂₈O₅,318.22750;found:318.22821.

Synthesis of the Compound GA-6AFV 6:

Epoxiconamic acid (1.95 g) was mixed with 37mL DCM, and 6AFV6 (2.45 g) was added to the reaction system. DMAP (133.0 mg) and DCC (2.41 g) were added at 0deg.C and mixed, and the resulting mixture was stirred in an ice bath for a further half an hour, then moved to room temperature and stirred for 12h. The reaction was quenched with 5mL of saturated aqueous NH ₄ Cl, filtered and washed with 10mL of saturated aqueous NaCl. The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate=7:1 (V/V)) to give the compound as a colorless oil GA-6AFV6(0.97g).¹H NMR(400MHz,Chloroform-d)δ5.51–5.41(m,1H),5.41–5.30(m,1H),4.65(s,2H),4.28–4.13(m,2H),3.53(dt,J＝12.0,6.8Hz,4H),3.41(dd,J＝4.0,2.8Hz,1H),2.99–2.87(m,2H),2.32(q,J＝7.2Hz,2H),2.03(q,J＝6.8Hz,2H),1.81–1.70(m,2H),1.70–1.59(m,2H),1.37–1.25(m,4H),0.88(t,J＝6.8Hz,3H).HRMS(ESI,m/z):[M+H]⁺ calcd.For:C₁₆H₂₈O₅,301.20095;found:301.20201.

Synthesis of Compound GA-6AE 6:

Epoxy propionic acid (1.63 g) and 6AE6 (4.58 g) were mixed, followed by addition of 65mL DCM to the reaction system. DMAP (224.0 mg) and EDCI (5.35 g) were added at 0deg.C and the resulting mixture was stirred in an ice bath for 30min, then moved to room temperature and stirred for 12h. The reaction solution was diluted with 150mL DCM and washed with saturated aqueous NaCl (80 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure, and the crude product was purified by column chromatography (eluent: petroleum ether: ethyl acetate=6:1 (V/V)) to give a colorless oily compound GA-6AE6(3.20g).HRMS(ESI,m/z):[M+Na]+ calcd.For:C₁₇H₃₂O₅,339.21420;found:339.21558.

Synthesis of Compound GA-18 OleN:

Epoxy propionic acid (1.30 g) was mixed with oleylamine (4.73 g), and 50mL of DMF was added, followed by NMM (1.83 mL) and HCTU (6.72 g) to the reaction system and the reaction at room temperature for 12h. The reaction solution was concentrated under reduced pressure, then diluted with EA, and washed 3 times with saturated sodium chloride (if floc is precipitated and dissolved in an organic phase, diatomaceous earth of 7 μm was required). The organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by silica gel column chromatography (dry loading, petroleum ether: ethyl acetate=2/1 (V/V)) to give a pale yellow solid compound GA-18OleN(2.093g).¹H NMR(400MHz,Chloroform-d)δ6.12(s,1H),5.48–5.20(m,2H),3.45–3.38(m,1H),3.31–3.11(m,2H),2.96(t,J＝5.2Hz,1H),2.72(dd,J＝6.0,2.8Hz,1H),2.15–1.78(m,4H),1.56–1.38(m,2H),1.38–1.17(m,22H),0.86(t,J＝6.8Hz,3H).

Synthesis of Compound GA-8 MN:

EDCI (4.89 g) and DMAP (0.19 g) were added to a mixed solution of epoxypropionic acid (2.15 g) and 100mM LDMF under ice bath, and stirring was continued for 30min. 8MN (2.15 g) was added to the above system, and the reaction system was moved to room temperature and stirred for 12 hours. DMF was removed under reduced pressure, the residue was dissolved in 300mL of ethyl acetate, the resulting solution was washed twice with 100mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated and purified by silica gel column chromatography to give the objective GA-8MN (2.19 g). GA-8MN HRMS (ESI, M/z): [ M+H ] ⁺

calcd.For C₁₂H₂₄NO₂,214.18015；found:214.18013.

Synthesis of Compound GA-10 MN:

EDCI (3.50 g) and DMAP (0.10 g) were added to a mixed solution of epoxypropionic acid (1.46 g) and 80mL DMF under ice bath and stirring was continued for 30min. 10MN (1.42 g) was added to the above system, and the reaction system was moved to room temperature and stirred for 12 hours. Removing DMF under reduced pressure, dissolving the residue with 300mL of ethyl acetate, washing the obtained solution with 100mL of saturated saline twice, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography to obtain the target product GA-10MN(1.04g).GA-10MN:HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₄H₂₈NO₂,242.21145;found:242.21238.

Synthesis of Compound GA-12 MN:

EDCI (4.14 g) and DMAP (0.22 g) were added to a mixed solution of epoxypropionic acid (1.90 g) and 100mL DMF under ice bath and stirring was continued for 30min. 12MN (3.59 g) was added to the above system, and the reaction system was moved to room temperature and stirred for 12 hours. Removing DMF under reduced pressure, dissolving the residue with 300mL of ethyl acetate, washing the obtained solution with 100mL of saturated saline twice, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography to obtain the target product GA-12MN(3.84g).GA-12MN:HRMS(ESI,m/z):[M+H]⁺calcd.For C₁₆H₃₂NO₂,270.24275;found:270.24537.

Synthesis of Compound GA-14 MN:

EDCI (4.22 g) and DMAP (0.19 g) were added to a mixed solution of epoxypropionic acid (1.76 g) and 120mL DMF under ice bath and stirring was continued for 30min. 14MN (2.27 g) was added to the above system, and the reaction system was moved to room temperature and stirred for 12 hours. Removing DMF under reduced pressure, dissolving the residue with 300mL of ethyl acetate, washing the obtained solution with 100mL of saturated saline twice, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography to obtain the target product GA-14MN(1.79g).GA-14MN:HRMS(ESI,m/z):[M+H]⁺calcd.For C₁₈H₃₆NO₂,298.27405;found:298.27392.

Synthesis of Compound GA-2A8B 6:

EDCI (3.62 g) and DMAP (0.15 g) were added to a mixed solution of 2-hexyl octanoic acid 8B6 (3.58 g) and 100mL DCM under ice bath, and stirring was continued under ice bath for 30 min; n- (tert-Butoxycarbonyl) ethanolamine Boc-2A (2.10 g) was added to the above system and reacted at room temperature for 12 hours. The reaction was diluted with DCM, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified with petroleum ether: ethyl acetate=4:1 (v/v) as eluent was purified by flash medium pressure preparative chromatography to give compound Boc-2A8B6 (4.37 g).

Boc-2A8B6 obtained in the previous step was dissolved in TFA/DCM (27 mL,1:2, v/v) and reacted at room temperature for 2h. After TLC monitoring showed that the reaction was complete, TFA (trifluoroacetic acid) and DCM were removed by concentration under reduced pressure, the residue was diluted with 200mL of DCM, washed twice with 100mL of saturated sodium bicarbonate solution, and the organic phase was collected and dried over anhydrous sodium sulfate; DCM was removed by concentration under reduced pressure and the residue was purified by flash medium pressure preparative chromatography using DCM/ultra=2:1 (v/v) as eluent to give compound 2A8B6 (2.65 g).

EDCI (4.20 g) and DMAP (0.12 g) were added to a mixed solution of epoxypropionic acid (1.73 g) and 100mL DMF under ice bath and stirring was continued for 30min. 2A8B6 (2.66 g) obtained in the previous step was added to the above system, and the reaction system was allowed to move to room temperature and stirred for 12 hours. Removing DMF under reduced pressure, dissolving the residue with 300mL of ethyl acetate, washing the obtained solution with 100mL of saturated saline twice, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography to obtain the target product GA-2A8B6(0.75g).¹H NMR(400MHz,Chloroform-d)δ6.38(s,1H),4.23–4.05(m,2H),3.59–3.47(m,2H),3.44(dd,J＝4.8,2.4Hz,1H),3.02–2.94(m,1H),2.74(dd,J＝5.6,2.4Hz,1H),2.41–2.25(m,1H),1.63–1.52(m,2H),1.49–1.37(m,2H),1.31–1.18(m,16H),0.94–0.80(m,6H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₉H₃₆NO₄,342.26388;found:342.26362.

Synthesis of Compound GA-4A8B 6:

EDCI (1.55 g) and DMAP (65.0 mg) were added to a mixed solution of 2-hexyl octanoic acid 8B6 (1.53 g) and 50mL DCM under ice, and stirring was continued under ice for 30 min; 4- (N-Boc-amino) -1-butanol Boc-4A (1.03 g) was added to the above system and reacted at room temperature for 12 hours. The reaction was diluted with DCM, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified with petroleum ether: ethyl acetate=4:1 (v/v) as eluent by flash medium pressure preparative chromatography gave compound Boc-4A8B6 (1.75 g).

Boc-4A8B6 obtained in the previous step was dissolved in TFA/DCM (10.5 mL,1:2, v/v) and reacted at room temperature for 2h. After TLC monitoring showed that the reaction was complete, TFA and DCM were removed by concentration under reduced pressure, the residue was diluted with 200mL DCM, washed twice with 100mL saturated sodium bicarbonate solution, and the organic phase was collected and dried over anhydrous sodium sulfate; DCM was removed by concentration under reduced pressure and the residue was purified by flash medium pressure preparative chromatography using DCM/ultra=2:1 (v/v) as eluent to give compound 4A8B6 (1.10 g).

EDCI (1.59 g) and DMAP (46.0 mg) were added to a mixed solution of epoxypropionic acid (0.65 g) and 50mL DMF under ice bath, and stirring was continued for 30min. 4A8B6 (1.11 g) obtained in the previous step was added to the above system, and the reaction system was allowed to stand at room temperature for stirring for 12 hours. Removing DMF under reduced pressure, dissolving the residue with 300mL of ethyl acetate, washing the obtained solution with 100mL of saturated saline twice, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography to obtain the target product GA-4A8B6(0.83g).¹H NMR(400MHz,Chloroform-d)δ6.16(s,1H),4.07(t,J＝6.4Hz,2H),3.44(dd,J＝4.8,2.8Hz,1H),3.37–3.18(m,2H),2.99(t,J＝5.2Hz,1H),2.74(dd,J＝5.6,2.8Hz,1H),2.37–2.24(m,1H),1.63–1.51(m,6H),1.47–1.37(m,2H),1.32–1.20(m,16H),0.87(t,J＝6.8Hz,6H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₂₁H₄₀NO₄,370.29518;found:370.29600.

Synthesis of Compound GA-6A8B 6:

EDCI (1.34 g) and DMAP (56 mg) were added to a mixed solution of 2-hexyl octanoic acid 8B6 (1.33 g) and 100mL DCM under ice bath, and stirring was continued under ice bath for 30 min; 4- (N-Boc-amino) -1-hexanol (Boc-6A) (2.69 g) was added to the above system, and reacted at room temperature for 12 hours. The reaction was diluted with DCM, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified with petroleum ether: ethyl acetate=4:1 (v/v) as eluent was purified by flash medium pressure preparative chromatography to give compound Boc-6A8B6 (1.45 g).

Boc-6A8B6 obtained in the previous step was dissolved in TFA/DCM mixture (27 mL,1:2, v/v) and reacted at room temperature for 2h. After TLC monitoring showed that the reaction was complete, TFA and DCM were removed by concentration under reduced pressure, the residue was diluted with 200mL DCM, washed twice with 100mL saturated sodium bicarbonate solution, and the organic phase was collected and dried over anhydrous sodium sulfate; DCM was removed by concentration under reduced pressure and the residue was purified by flash medium pressure preparative chromatography using DCM/ultra=2:1 (v/v) as eluent to give compound 6A8B6 (0.90 g).

EDCI (1.16 g) and DMAP (33.0 mg) were added to a mixed solution of epoxypropionic acid (0.48 g) and 100mL DMF under ice bath and stirring was continued for 30min. 6A8B6 (0.88 g) obtained in the previous step was added to the above system, and the reaction system was allowed to stand at room temperature for stirring for 12 hours. Removing DMF under reduced pressure, dissolving the residue with 300mL of ethyl acetate, washing the obtained solution with 100mL of saturated saline twice, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography to obtain the target product GA-6A8B6(0.74g).¹H NMR(400MHz,Chloroform-d)δ6.12(s,1H),4.05(t,J＝6.8Hz,2H),3.43(dd,J＝4.8,2.8Hz,1H),3.33–3.14(m,2H),2.98(t,J＝5.2Hz,1H),2.73(dd,J＝5.6,2.8Hz,1H),2.36–2.23(m,1H),1.64–1.32(m,12H),1.29–1.20(m,16H),0.86(t,J＝6.8Hz,6H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₂₃H₄₄NO₄,398.32648;found:398.32640.

Synthesis of Compound GA-2A10B 6:

EDCI (3.62 g) and DMAP (0.15 g) were added to a mixed solution of 2-hexyldecanoic acid 10B6 (3.82 g) and 100mL DCM under ice bath, and stirring was continued under ice bath for 30 minutes; 2- (N-Boc-amino) -1-ethanol Boc-2A (2.10 g) was added to the above system, and reacted at room temperature for 12 hours. The reaction was diluted with DCM, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified with petroleum ether: ethyl acetate=4:1 (v/v) as eluent was purified by flash medium pressure preparative chromatography to give compound Boc-2a10B6 (4.86 g).

Boc-2A10B6 obtained in the previous step was dissolved in TFA/DCM (28.5 mL,1:2, v/v) and reacted at room temperature for 2h. After TLC monitoring showed that the reaction was complete, TFA and DCM were removed by concentration under reduced pressure, the residue was diluted with 200mL DCM, washed twice with 100mL saturated sodium bicarbonate solution, and the organic phase was collected and dried over anhydrous sodium sulfate; DCM was removed by concentration under reduced pressure and the residue was purified by flash medium pressure preparative chromatography using DCM/ultra=2:1 (v/v) as eluent to give compound 2a10B6 (2.74 g).

EDCI (3.92 g) and DMAP (0.11 g) were added to a mixed solution of epoxypropionic acid (1.73 g) and 100mL DMF under ice bath and stirring was continued for 30min. The 2A10B6 (2.73 g) obtained in the previous step was added to the above system, and the reaction system was allowed to move to room temperature and stirred for 12 hours. Removing DMF under reduced pressure, dissolving the residue with 300mL of ethyl acetate, washing the obtained solution with 100mL of saturated saline twice, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography to obtain the product GA-2A10B6(2.34g).¹H NMR(400MHz,Chloroform-d)δ6.38(s,1H),4.24–4.05(m,2H),3.62–3.47(m,2H),3.44(dd,J＝4.8,2.8Hz,1H),2.98(t,J＝5.2Hz,1H),2.74(dd,J＝5.6,2.8Hz,1H),2.43–2.23(m,1H),1.62–1.52(m,2H),1.49–1.37(m,2H),1.33–1.19(m,20H),0.87(t,J＝6.8Hz,6H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₂₁H₄₀NO₄,370.29518;found:370.29420.

Synthesis of Compound GA-4A10B 6:

EDCI (1.53 g) and DMAP (0.06 g) were added to a mixed solution of 2-hexyldecanoic acid (10B 6) (1.63 g) and 50mL DCM under ice bath, and stirring was continued under ice bath for 30 minutes; 4- (N-Boc-amino) -1-butanol Boc-4A (1.01 g) was added to the above system and reacted at room temperature for 12 hours. The reaction was diluted with DCM, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified with petroleum ether: ethyl acetate=4:1 (v/v) as eluent was purified by flash medium pressure preparative chromatography to give compound Boc-4a10B6 (2.05 g).

Boc-4A10B6 obtained in the previous step was dissolved in TFA/DCM (18.0 mL,1:2, v/v) and reacted at room temperature for 2h. After TLC monitoring showed that the reaction was complete, TFA and DCM were removed by concentration under reduced pressure, the residue was diluted with 200mL DCM, washed twice with 100mL saturated sodium bicarbonate solution, and the organic phase was collected and dried over anhydrous sodium sulfate; DCM was removed by concentration under reduced pressure and the residue was purified by flash medium pressure preparative chromatography using DCM/ultra=2:1 (v/v) as eluent to give compound 4a10B6 (1.97 g).

EDCI (2.58 g) and DMAP (0.07 g) were added to a mixed solution of epoxypropionic acid (1.06 g) and 100mL DMF under ice bath and stirring was continued for 30min. 4A10B6 (1.97 g) obtained in the previous step was added to the above system, and the reaction system was allowed to stand at room temperature for stirring for 12 hours. Removing DMF under reduced pressure, dissolving the residue with 300mL of ethyl acetate, washing the obtained solution with 100mL of saturated saline twice, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography to obtain the product GA-4A10B6(1.87g).¹H NMR(400MHz,Chloroform-d)δ6.16(s,1H),4.06(t,J＝6.4Hz,2H),3.53–3.39(m,1H),3.38–3.18(m,2H),2.98(t,J＝5.2Hz,1H),2.73(dd,J＝5.6,2.8Hz,1H),2.35–2.24(m,1H),1.68–1.50(m,6H),1.48–1.36(m,2H),1.33–1.17(m,20H),0.86(t,J＝6.8Hz,6H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₂₃H₄₄NO₄,398.32648;found:398.32794.

Synthesis of Compound GA-6A10B 6:

EDCI (1.32 g) and DMAP (0.06 g) were added to a mixed solution of 2-hexyldecanoic acid 10B6 (1.41 g) and 50mL DCM under ice bath, and stirring was continued under ice bath for 30 minutes; 6- (N-Boc-amino) -1-hexanol Boc-6A (1.0 g) was added to the above system and reacted at room temperature for 12 hours. The reaction was diluted with DCM, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified with petroleum ether: ethyl acetate=4:1 (v/v) as eluent was purified by flash medium pressure preparative chromatography to give compound Boc-6a10B6 (2.04 g).

Boc-6A10B6 obtained in the previous step was dissolved in TFA/DCM (10.5 mL,1:2, v/v) and reacted at room temperature for 2h. After TLC monitoring showed that the reaction was complete, TFA and DCM were removed by concentration under reduced pressure, the residue was diluted with 200mL DCM, washed twice with 100mL saturated sodium bicarbonate solution, and the organic phase was collected and dried over anhydrous sodium sulfate; DCM was removed by concentration under reduced pressure and the residue was purified by flash medium pressure preparative chromatography using DCM/ultra=2:1 (v/v) as eluent to give compound 6a10B6 (1.07 g).

EDCI (1.29 g) and DMAP (0.04 g) were added to a mixed solution of epoxypropionic acid (0.53 g) and 100mL DMF under ice bath and stirring was continued for 30min. 6A10B6 (1.07 g) obtained in the previous step was added to the above-mentioned system, and the reaction system was allowed to stand at room temperature for stirring for 12 hours. Removing DMF under reduced pressure, dissolving the residue with 300mL of ethyl acetate, washing the obtained solution with 100mL of saturated saline twice, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating, and purifying by silica gel column chromatography to obtain the product GA-6A10B6(0.70g).¹H NMR(400MHz,Chloroform-d)δ6.11(s,1H),4.05(t,J＝6.8Hz,2H),3.43(dd,J＝4.8,2.6Hz,1H),3.35–3.13(m,2H),2.98(t,J＝5.2Hz,1H),2.73(dd,J＝5.6,2.8Hz,1H),2.36–2.23(m,1H),1.65–1.20(m,32H),0.87(t,J＝6.8Hz,6H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₂₅H₄₈NO₄,426.35778;found:426.35713.

Synthesis of Compound GA-2DA8B 6:

EDCI (1.82 g) and DMAP (77.0 mg) were added to a mixed solution of alcohol Boc-2DA (1.01 g), 2-hexyl octanoic acid 8B6 (1.79 g) and 30mL DCM under ice. The resulting mixture was stirred under ice bath for an additional 30 minutes, then warmed to room temperature and stirred for 12h. The reaction solution was diluted with 100mL DCM and washed with saturated aqueous NaCl (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=4:1 (V/V)) to give Boc-2DA8B6 (1.90 g) as a colorless oil.

Boc-2DA8B6 obtained in the above synthesis step was dissolved in a mixture of TFA and DCM (18.0 mL,1:2, v/v) and stirred at room temperature for 2 hours. TFA and DCM were removed under reduced pressure, the residue was dissolved in 100mL DCM and washed three times with 50mL saturated aqueous NaHCO ₃. The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: DCM: ultra=2:1 (V/V)) to give 2DA8B6 (1.25 g) as a colorless oil.

EDCI (1.98 g) and DMAP (57.0 mg) were added to a mixed solution of alcohol 2DA8B6 (1.07 g), epoxypropionic acid (0.81 g) and 30mL DMF under ice bath conditions. The resulting mixture was kept stirring under ice bath conditions for 30 minutes, then warmed to room temperature and stirred for 12h. The reaction solution was diluted with 100mL of ethyl acetate and washed with saturated aqueous NaCl (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=2:1) to give the compound as a colorless oil GA-2DA8B6(1.30g).¹H NMR(400MHz,Chloroform-d)δ6.70(s,1H),5.99(t,J＝5.2Hz,1H),3.55–3.27(m,5H),2.99–2.95(m,1H),2.77(dd,J＝5.6,2.5Hz,1H),2.04–1.95(m,1H),1.62–1.50(m,2H),1.44–1.35(m,2H),1.32–1.18(m,16H),0.86(t,J＝6.8Hz,6H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₉H₃₇N₂O₃,341.27987;found:341.27937.

Synthesis of the Compound GA-GABA8B 7:

gamma-aminobutyric acid GABA (1.04 g) was dissolved in 2mL of water and diluted with 50mL of methanol, and after cooling with an ice water bath for 5 minutes, boc anhydride (2.34 g) was added, the resulting mixture was brought to room temperature and stirred for 30 minutes, and then the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography to give Boc-GABA (1.82 g) as a white solid.

EDCI (1.70 g) and DMAP (77.0 mg) were added to a mixed solution of Boc-GABA (1.42 g), 8-pentadecanol (1.40 g) and 50mL DCM under ice. The resulting mixture was stirred under ice bath for an additional 30 minutes, then warmed to room temperature and stirred for 12h. The reaction solution was diluted with 100mL DCM and washed with saturated aqueous NaCl (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=4:1 (V/V)) to give Boc-GABA8B7 (1.82 g) as a colorless oil.

Boc-GABA8B7 obtained in the above synthesis step was dissolved in a mixture of TFA and DCM (10.5 mL,1:2, v/v) and stirred at room temperature for 2 hours. TFA and DCM were removed under reduced pressure, the residue was dissolved in 100mL DCM and washed three times with 50mL saturated aqueous NaHCO ₃. The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: DCM: ultra=2:1 (V/V)) to give GABA8B7 (1.08 g) as a colorless oil.

EDCI (1.21 g) and DMAP (35.0 mg) were added to a mixed solution of alcohol GABA8B7 (1.07 g), epoxypropionic acid (0.49 g) and 20mL DMF under ice bath conditions. The resulting mixture was kept stirring under ice bath conditions for 30 minutes, then warmed to room temperature and stirred for 12h. The reaction solution was diluted with 150mL of ethyl acetate and washed with saturated aqueous NaCl (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=2:1) to give the compound as a colorless oil GA-GABA8B7(0.73g).¹H NMR(400MHz,Chloroform-d)δ6.29(s,1H),4.86(p,J＝6.4Hz,1H),3.42(dd,J＝4.8,2.8Hz,1H),3.28(th,J＝13.6,6.8Hz,2H),2.97(t,J＝5.2Hz,1H),2.74(dd,J＝6.0,2.8Hz,1H),2.31(t,J＝7.2Hz,2H),1.82(p,J＝7.2Hz,2H),1.50(q,J＝6.4Hz,4H),1.36–1.18(m,20H),0.87(t,J＝6.8Hz,6H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₂₂H₄₂NO₄,384.31083;found:384.30914.

Synthesis of the Compound GA-GABA 10C:

EDCI (4.06 g) and DMAP (242.0 mg) were added to a mixed solution of alcohol Boc-GABA (3.98 g), 1-decanol (3.80 g) and 100mL DCM under ice-bath. The resulting mixture was stirred under ice bath for an additional 30 minutes, then warmed to room temperature and stirred for 12h. The reaction solution was diluted with 200mL DCM and washed with saturated aqueous NaCl (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=4:1 (V/V)) to give Boc-GABA10C (5.13 g) as a colorless oil.

Boc-GABA10C obtained in the above synthesis step was dissolved in a mixture of TFA and DCM (10.5 mL,1:2, v/v) and stirred at room temperature for 2 hours. TFA and DCM were removed under reduced pressure, the residue was dissolved in 100mL DCM and washed three times with 50mL saturated aqueous NaHCO ₃. The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent removed under reduced pressure, and the residue purified by silica gel column chromatography (eluent: DCM: ultra=2:1 (V/V)) to give gaba10c·hcl (2.30 g, quantitative) as a colorless oil.

EDCI (2.82 g) and DMAP (148.0 mg) were added to a mixed solution of epoxypropionic acid (1.06 g) and 40mL DMF under ice bath conditions. The resulting mixture was kept stirring at 0℃for 30 minutes, and GABA10C (2.24 g) and TEA (1.12 mL) were added, and the resulting reaction mixture was warmed to room temperature and stirred for 12 hours. The reaction solution was diluted with 200mL of ethyl acetate and washed with saturated aqueous NaCl (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate=1:1 (V/V)) to give a colorless oily compound GABA10C(0.82g).¹H NMR(400MHz,Chloroform-d)δ6.28(s,1H),4.06(t,J＝6.8Hz,2H),3.43(dd,J＝4.8,2.8Hz,1H),3.38–3.21(m,2H),2.98(dd,J＝5.6,4.8Hz,1H),2.75(dd,J＝5.6,2.8Hz,1H),2.33(t,J＝7.6Hz,2H),1.83(p,J＝7.2Hz,2H),1.66–1.56(m,3H),1.37–1.21(m,14H),0.88(t,J＝6.8Hz,3H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₇H₃₂NO₄,314.23258;found:314.23084.

Example 2: preparation of lipid compounds

Preparation of compound L0463:

0.1mmol of Amine E (Amine E) and 0.78mmol of compound 3 were dissolved in 2.0mL of isopropanol (Isopropanol) and the resulting solution was stirred at 80℃for 24h. The isopropanol is removed by decompression concentration, and the residue is purified by silica gel column chromatography to obtain the target product compound L0463.

Preparation of compound L0474:

0.1mmol of Amine F (Amine F) and 0.78mmol of compound 3 were dissolved in 2.0mL of isopropanol and the resulting solution was stirred at 80℃for 24h. The isopropanol is removed by decompression concentration, and the residue is purified by silica gel column chromatography to obtain the target product compound L0474.

The hydrogen spectrum and mass spectrum of the obtained compounds L0463 and L0474 were measured by taking appropriate amounts of the compounds, respectively, and the results were as follows:

Hydrogen spectrum of compound L0463 ：¹H NMR(400MHz,Chloroform-d)δ7.13(t,J＝6.0Hz,6H),4.14(dd,J＝8.0,3.2Hz,6H),3.33–3.09(m,12H),3.05–2.55(m,24H),1.49(p,J＝6.8Hz,12H),1.41–1.11(m,108H),0.86(t,J＝6.8Hz,18H).

Compound L0463 mass spectrum ：HRMS(ESI,m/z):[M+H]⁺ calcd.For C₉₆H₁₉₃N₁₀O₁₂,1679.48275;found1679.48310.

Hydrogen spectrum of compound L0474 ：¹H NMR(400MHz,Chloroform-d)δ7.08(t,J＝6.0Hz,6H),4.08(dd,J＝8.8,3.6Hz,6H),3.20(q,J＝6.8Hz,12H),2.90(dd,J＝13.6,4.0Hz,6H),2.76–2.57(m,12H),2.39–2.23(m,6H),1.61–1.41(m,18H),1.33–1.20(m,108H),0.86(t,J＝6.8Hz,18H).

Compound L0474 mass spectrum ：HRMS(ESI,m/z):[M+H]⁺ calcd.For C₉₉H₁₉₉N₁₀O₁₂,1721.52970;found:1721.53481.

Example 3: compound L0461, compound L0462, compound L0464, compound L0465, compound L0467, compound L0468, compound L0469, compound L0470, compound L0471, compound L0472, compound L0473, compound L0475, compound L0476, compound L0477, compound L0478, compound L0479, compound L0480, compound L0481, compound L0482, compound L0483, compound L0484, compound L0485, compound L0486, compound L0487, compound L0488, compound L0489, compound L0490, compound L0491, compound L0492, compound L0493, compound L0494, compound L0495, compound L0496, compound L0497, compound L0498, compound L0499, compound L0500, compound L0501, compound L0502, compound L0503, compound 0506 and method for preparing 0506

The compounds L0461, L0462, L0464, L0465, L0467, L0468, L0469, L0470, L0471, L0472, L0473, L0475, L0476, L0477, L0478, L0479, L0480, L0481, L0482, L0483, L0484, L0485, L0486, L0487, L0488, L0489, L0490, L0491, L0492, L0493, L0494, L0495, L0496, L0497, L0498, L0499, L0500, L0501, L2, L0502, L0503 and the epoxy amide compounds 0506 are commercially available, according to the structural characteristics of the target product, 0.1mmol of amine E or amine F and 0.78mmol of epoxy propionate/amide compound (the epoxy propionate/amide compound has a hydrophobic chain structure synthesized as shown in example 1 or is prepared according to the similar preparation method of example 1), corresponding epoxy propionate/amide compound is selected according to the structural characteristics of the target product, the substrate is dissolved in 2.0mL of isopropanol, and the obtained solution is stirred for 24h at 80 ℃. The isopropanol was removed by concentration under reduced pressure, and the residue was purified by silica gel column chromatography to give the objective products L0461-L0506, respectively.

The hydrogen spectrum and mass spectrum of the obtained compound L0461-L0506 were measured by taking a proper amount of the compound, respectively, and the results are as follows:

Compounds of formula (I) L0461：¹H NMR(400MHz,Chloroform-d)δ4.27(dd,J＝8.0,3.2Hz,6H),4.23–4.01(m,12H),3.25–2.52(m,24H),1.64(p,J＝7.2Hz,12H),1.39–1.20(m,108H),0.87(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₉₆H₁₈₇N₄O₁₈,1685.38685;found 1685.38831.

Compounds of formula (I) L0462：¹H NMR(400MHz,Chloroform-d)δ7.14(t,J＝6.0Hz,6H),4.12(dd,J＝8.0,3.2Hz,6H),3.30–3.10(m,12H),3.03–2.80(m,10H),2.78–2.53(m,14H),1.58–1.40(m,12H),1.36–1.13(m,84H),0.86(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]+ calcd.For C₈₄H₁₆₉N₁₀O₁₂,1510.29160;found 1510.29342.

Compounds of formula (I) L0464：¹H NMR(400MHz,Chloroform-d)δ4.64(s,12H),4.27(dd,J＝8.0,3.2Hz,6H),4.21–4.05(m,12H),3.50(t,J＝6.8Hz,24H),3.14–2.61(m,24H),1.76–1.47(m,36H),1.44–1.27(m,60H),0.88(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₀₂H₁₉₉N₄O₃₀,1961.41972;found 1961.41685.

Compounds of formula (I) L0465：¹H NMR(400MHz,Chloroform-d)δ5.53–5.42(m,6H),5.39–5.31(m,6H),4.65(s,12H),4.27(dd,J＝8.0,3.2Hz,6H),4.21–4.08(m,12H),3.52(td,J＝6.8,3.6Hz,24H),3.09–2.63(m,24H),2.32(q,J＝7.2Hz,12H),2.05(p,J＝7.6Hz,12H),1.69–1.62(m,12H),1.61–1.54(m,12H),1.41–1.34(m,24H),0.96(t,J＝7.6Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₀₂H₁₈₇N₄O₃₀,1949.32582;found 1949.31849.

Compounds of formula (I) L0467：¹H NMR(400MHz,Chloroform-d)δ5.59–5.39(m,6H),5.39–5.23(m,6H),4.65(s,12H),4.26(dd,J＝8.0,3.2Hz,6H),4.21–4.03(m,12H),3.51(td,J＝6.8,3.2Hz,24H),3.38–3.06(m,6H),2.94–2.60(m,18H),2.32(q,J＝7.0Hz,12H),2.11–1.85(m,18H),1.71–1.52(m,24H),1.43–1.32(m,24H),0.95(t,J＝7.6Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₀₅H₁₉₃N₄O₃₀,1991.37277;found 1991.37367.

Compounds of formula (I) L0468：¹H NMR(400MHz,Chloroform-d)δ5.54–5.41(m,6H),5.41–5.28(m,6H),4.64(s,12H),4.36–4.06(m,18H),3.52(dt,J＝9.6,6.8Hz,24H),3.08–2.50(m,24H),2.32(q,J＝7.2Hz,12H),2.03(q,J＝6.8Hz,12H),1.73(p,J＝6.8Hz,12H),1.68–1.55(m,12H),1.40–1.28(m,24H),0.88(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₀₂H₁₈₇N₄O₃₀,1949.32582;found:1949.33204.

Compounds of formula (I) L0469：¹H NMR(400MHz,Chloroform-d)δ4.26(dd,J＝8.0,3.6Hz,6H),4.21–4.06(m,12H),4.04(t,J＝6.8Hz,12H),3.15–2.40(m,24H),2.33–2.24(m,6H),1.74–1.48(m,36H),1.47–1.33(m,36H),1.31–1.20(m,96H),0.85(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₄₄H₂₇₁N₄O₃₀,2537.98312;found:2537.98943.

Compounds of formula (I) L0470：¹H NMR(400MHz,Chloroform-d)δ5.56–5.41(m,6H),5.41–5.30(m,6H),4.65(s,12H),4.33–4.07(m,18H),3.53(dt,J＝10.0,6.8Hz,24H),3.10–2.47(m,24H),2.32(q,J＝7.2Hz,12H),2.04(q,J＝6.8Hz,12H),1.93–1.67(m,18H),1.67–1.58(m,12H),1.38–1.28(m,24H),0.93–0.81(m,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₀₅H₁₉₃N₄O₃₀,1991.37277;found:1991.38136.

Compounds of formula (I) L0471：¹H NMR(400MHz,Chloroform-d)δ4.23(dd,J＝8.0,3.2Hz,6H),4.19–4.04(m,12H),3.06–2.50(m,24H),1.93–1.41(m,18H),1.34–1.19(m,84H),0.86(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₈₇H₁₆₉N₄O₁₈,1558.24264;found:1558.24973.

Compounds of formula (I) L0472：¹H NMR(400MHz,Chloroform-d)δ4.26(dd,J＝8.0,3.6Hz,6H),4.20–3.99(m,12H),3.41–3.08(m,6H),2.91–2.59(m,18H),2.03–1.81(m,6H),1.62(p,J＝6.8Hz,12H),1.35–1.20(m,108H),0.86(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺calcd.For C₉₉H₁₉₃N₄O₁₈,1727.43380;found:1727.43652.

Compounds of formula (I) L0473：¹H NMR(400MHz,Chloroform-d)δ7.08(t,J＝6.0Hz,6H),4.08(dd,J＝8.8,3.6Hz,6H),3.19(q,J＝6.8Hz,12H),2.90(dd,J＝13.6,4.0Hz,6H),2.77–2.56(m,12H),2.44–2.23(m,6H),1.50(dp,J＝14.4,7.2,6.8Hz,18H),1.32–1.20(m,84H),0.85(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+3H]³⁺ calcd.For C₈₇H₁₇₇N₁₀O₁₂,518.11770;found:518.12216.

Compounds of formula (I) L0475：¹H NMR(400MHz,Chloroform-d)δ4.63(s,12H),4.24(dd,J＝8.0,3.6Hz,6H),4.20–3.99(m,12H),3.49(t,J＝6.8Hz,24H),3.31–2.99(m,6H),2.89–2.69(m,14H),2.68–2.56(m,4H),1.99–1.76(m,6H),1.75–1.44(m,36H),1.43–1.24(m,60H),0.86(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₀₅H₂₀₅N₄O₃₀,2003.46667;found:2003.47735.

Compounds of formula (I) L0476：¹H NMR(400MHz,Chloroform-d)δ4.27(dd,J＝8.4,3.2Hz,6H),4.22–3.99(m,24H),3.45–3.01(m,6H),2.93–2.72(m,14H),2.72–2.57(m,4H),2.34–2.25(m,6H),2.01–1.86(m,6H),1.71–1.51(m,36H),1.48–1.34(m,36H),1.32–1.22(m,96H),0.86(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+2H]²⁺ calcd.For C₁₄₇H₂₇₈N₄O₃₀,1290.51868;found:1290.52785.

Compounds of formula (I) L0477：¹H NMR(400MHz,Chloroform-d)δ4.49–3.96(m,24H),3.44–2.73(m,24H),1.69–1.51(m,12H),1.25(d,J＝11.2Hz,84H),0.86(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺calcd.For C₈₄H₁₆₃N₄O₁₈,1516.19569;found:1516.20294.

Compounds of formula (I) L0478：¹H NMR(400MHz,Chloroform-d)δ4.40–4.23(m,6H),4.20–4.03(m,12H),3.36–2.62(m,24H),1.70–1.51(m,12H),1.42–1.10(m,132H),0.86(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₀₈H₂₁₁N₄O₁₈,1853.57465;found:1853.57832.

Compounds of formula (I) L0479：¹H NMR(400MHz,Chloroform-d)δ4.63(q,J＝5.2Hz,6H),4.25(dd,J＝8.0,3.2Hz,6H),4.20–4.04(m,12H),3.59–3.50(m,12H),3.37(dt,J＝9.2,6.8Hz,12H),3.08–2.53(m,24H),1.71–1.48(m,36H),1.39–1.23(m,78H),0.86(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₀₈H₂₁₁N₄O₃₀,2045.51362;found:2045.51478.

Compounds of formula (I) L0480：¹H NMR(400MHz,Chloroform-d)δ4.52–4.37(m,6H),3.42–3.22(m,12H),3.11–2.85(m,20H),2.84–2.53(m,22H),1.73–1.45(m,18H),1.34–1.18(m,108H),0.86(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₀₅H₂₁₁N₁₀O₁₂,1805.62360;found:1805.62826.

Compounds of formula (I) L0481：¹H NMR(400MHz,Chloroform-d)δ4.56–4.31(m,5H),3.39–3.19(m,10H),3.07–2.93(m,9H),2.92–2.85(m,6H),2.82–2.35(m,22H),1.72–1.41(m,16H),1.32–1.14(m,90H),0.85(t,J＝6.8Hz,15H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₈₉H₁₈₀N₉O₁₀,m/z:1535.38477;found:1535.39418.

Compounds of formula (I) L0482：¹H NMR(400MHz,Chloroform-d)δ4.47(t,J＝8.8Hz,6H),3.40–3.22(m,12H),3.10–2.94(m,14H),2.94–2.85(m,10H),2.85–2.56(m,18H),1.60–1.43(m,12H),1.33–1.16(m,108H),0.86(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₀₂H₂₀₅N₁₀O₁₂,1763.57665;found:1763.58230.

Compounds of formula (I) L0483：¹H NMR(400MHz,Chloroform-d)δ4.50–4.39(m,6H),3.42–3.20(m,12H),3.00(s,11H),2.97–2.47(m,31H),1.59–1.42(m,12H),1.32–1.16(m,84H),0.85(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₉₀H₁₈₁N₁₀O₁₂,1594.38550;found:1594.39622.

Compounds of formula (I) L0484：¹H NMR(400MHz,Chloroform-d)δ4.50–4.31(m,6H),3.47–3.13(m,12H),3.00(s,10H),2.89(s,8H),2.79–2.31(m,24H),1.87–0.91(m,102H),0.91–0.59(m,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₉₃H₁₈₇N₁₀O₁₂,1637.43580;found:1637.43913.

Compounds of formula (I) L0485：¹H NMR(400MHz,Chloroform-d)δ4.53–4.38(m,6H),3.40–3.21(m,12H),3.01(s,11H),2.96–2.47(m,31H),1.62–1.41(m,12H),1.34–1.14(m,60H),0.85(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₇₈H₁₅₇N₁₀O₁₂,1426.19770;found:1426.20547.

Compounds of formula (I) L0486：¹H NMR(400MHz,Chloroform-d)δ4.48–4.36(m,6H),3.45–3.17(m,12H),3.09–2.95(m,11H),2.94–2.85(m,7H),2.84–2.36(m,24H),1.73–1.38(m,18H),1.34–1.13(m,60H),0.85(t,J＝6.4Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₈₁H₁₆₃N₁₀O₁₂,1468.24465;found:1468.25309.

Compounds of formula (I) L0487：¹H NMR(400MHz,Chloroform-d)δ4.50–4.39(m,6H),3.42–3.21(m,12H),3.06–2.97(m,11H),2.94–2.83(m,11H),2.81–2.46(m,20H),1.61–1.43(m,12H),1.36–1.10(m,132H),0.86(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+3H]³⁺ calcd.For C₁₁₄H₂₃₁N₁₀O₁₂,644.59300;found:644.59279.

Compounds of formula (I) L0488：¹H NMR(400MHz,Chloroform-d)δ4.51–4.35(m,6H),3.40–3.23(m,12H),3.05–2.96(m,11H),2.94–2.86(m,7H),2.83–2.40(m,24H),1.71–1.44(m,18H),1.36–1.10(m,132H),0.85(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₁₇H₂₃₅N₁₀O₁₂,1973.81140;found:1973.82239.

Compounds of formula (I) L0489：¹H NMR(400MHz,Chloroform-d)δ7.25–6.95(m,6H),4.16(d,J＝7.6Hz,6H),4.05(t,J＝6.8Hz,12H),3.34–3.14(m,12H),3.10–2.47(m,24H),2.34–2.25(m,6H),1.70–1.46(m,36H),1.46–1.19(m,132H),0.86(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₄₄H₂₇₇N₁₀O₂₄,2532.07903;found:2532.09648.

Compounds of formula (I) L0490：¹H NMR(400MHz,Chloroform-d)δ7.18(t,J＝6.0Hz,6H),4.16(dd,J＝8.8,3.2Hz,6H),4.03(t,J＝6.8Hz,12H),3.28–3.15(m,J＝6.8Hz,14H),3.04–2.87(m,8H),2.86–2.58(m,14H),2.29(tt,J＝9.2,5.6Hz,6H),1.93–1.70(m,6H),1.68–1.46(m,36H),1.44–1.17(m,132H),0.85(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₄₇H₂₈₃N₁₀O₂₄,2574.12598;found:2574.14153.

Compounds of formula (I) L0491：¹H NMR(400MHz,Chloroform-d)δ7.59–7.27(m,6H),4.34–4.04(m,18H),3.61–3.43(m,J＝7.6,6.8Hz,12H),3.03–2.51(m,24H),2.38–2.28(m,6H),1.63–1.50(m,12H),1.48–1.37(m,12H),1.32–1.16(m,96H),0.85(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₂₀H₂₂₉N₁₀O₂₄,2195.70343;found:2195.71164.

Compounds of formula (I) L0492：¹H NMR(400MHz,Chloroform-d)δ7.57–7.29(m,6H),4.25–4.05(m,18H),3.48(q,J＝6.0Hz,12H),3.09–2.60(m,24H),2.36–2.26(m,6H),1.93–1.67(m,6H),1.61–1.50(m,12H),1.47–1.37(m,12H),1.30–1.16(m,96H),0.85(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+3H]³⁺calcd.For C₁₂₃H₂₃₇N₁₀O₂₄,746.58831;found:746.58730.

Compounds of formula (I) L0493：¹H NMR(400MHz,Chloroform-d)δ7.26–7.08(m,6H),4.26–4.10(m,6H),4.06(t,J＝6.4Hz,12H),3.36–3.19(m,12H),3.18–2.39(m,24H),2.34–2.24(m,6H),1.71–1.49(m,36H),1.47–1.37(m,12H),1.33–1.21(m,96H),0.86(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺calcd.For C₁₃₂H₂₅₃N₁₀O₂₄,2363.89123;found:2363.90057.

Compounds of formula (I) L0494：¹H NMR(400MHz,Chloroform-d)δ7.12(t,J＝6.4Hz,6H),4.22–3.97(m,18H),3.26(q,J＝6.8Hz,12H),3.01–2.87(m,5H),2.86–2.48(m,13H),2.38–2.15(m,12H),1.71–1.45(m,42H),1.44–1.37(m,12H),1.32–1.20(m,96H),0.85(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺calcd.For C₁₃₅H₂₅₉N₁₀O₂₄,2405.93818;found:2405.95028.

Compounds of formula (I) L0495：¹H NMR(400MHz,Chloroform-d)δ7.23–6.97(m,6H),4.25–4.09(m,6H),4.04(t,J＝6.8Hz,12H),3.35–3.13(m,12H),3.06–2.45(m,24H),2.36–2.24(m,6H),1.68–1.47(m,36H),1.46–1.16(m,156H),0.86(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺calcd.For C₁₅₆H₃₀₁N₁₀O₂₄,2700.26683;found:2700.29074.

Compounds of formula (I) L0496：¹H NMR(400MHz,Chloroform-d)δ7.20–6.91(m,6H),4.26–3.91(m,18H),3.33–3.14(m,12H),3.00–2.86(m,5H),2.84–2.47(m,13H),2.41–2.17(m,12H),1.69–1.45(m,42H),1.44–1.16(m,156H),0.85(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺calcd.For C₁₅₉H₃₀₇N₁₀O₂₄,2742.31378;found:2742.33817.

Compounds of formula (I) L0497：¹H NMR(400MHz,Chloroform-d)δ7.60–7.28(m,6H),4.45–3.95(m,18H),3.66–3.41(m,12H),3.20–2.43(m,24H),2.41–2.28(m,6H),1.65–1.52(m,12H),1.50–1.39(m,12H),1.35–1.18(m,120H),0.87(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺calcd.For C₁₃₂H₂₅₃N₁₀O₂₄,2363.89123;found:2363.88239.

Compounds of formula (I) L0498：¹H NMR(400MHz,Chloroform-d)δ7.49–7.26(m,6H),4.31–4.00(m,18H),3.62–3.40(m,12H),3.07–2.55(m,20H),2.44–2.24(m,10H),1.66–1.37(m,30H),1.34–1.16(m,120H),0.86(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₃₅H₂₅₉N₁₀O₂₄,2405.93818;found:2405.92726.

Compounds of formula (I) L0499：¹H NMR(400MHz,Chloroform-d)δ7.25–7.05(m,6H),4.25–4.09(m,6H),4.06(t,J＝6.4Hz,12H),3.38–3.17(m,12H),3.04–2.45(m,24H),2.34–2.24(m,6H),1.74–1.48(m,36H),1.47–1.37(m,12H),1.33–1.15(m,120H),0.86(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺calcd.For C₁₄₄H₂₇₇N₁₀O₂₄,2532.07903;found:2532.07588.

Compounds of formula (I) L0500：¹H NMR(400MHz,Chloroform-d)δ7.11(t,J＝6.0Hz,6H),4.27–3.96(m,18H),3.28(q,J＝6.8Hz,12H),3.12–2.38(m,19H),2.37–2.22(m,11H),1.70–1.47(m,40H),1.47–1.36(m,14H),1.34–1.18(m,120H),0.86(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₄₇H₂₈₃N₁₀O₂₄,2574.12598;found:2574.12599.

Compounds of formula (I) L0501：¹H NMR(400MHz,Chloroform-d)δ7.90–7.59(m,6H),7.21–6.89(m,6H),5.84(s,6H),4.24–4.02(m,6H),3.51–3.24(m,24H),3.04–2.87(m,8H),2.81–2.46(m,16H),2.11–1.99(m,6H),1.59–1.45(m,12H),1.42–1.33(m,12H),1.30–1.15(m,96H),0.85(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]+ calcd.For C₁₂₀H₂₃₅N₁₆O₁₈,2189.79933;found:2189.79622.

Compounds of formula (I) L0502：¹H NMR(400MHz,Chloroform-d)δ7.80–7.61(m,6H),7.17–6.89(m,6H),5.81–5.38(m,5H),4.17–3.98(m,6H),3.51–3.22(m,24H),2.94–2.79(m,6H),2.76–2.39(m,16H),2.33–2.23(m,2H),2.10–1.98(m,6H),1.65–1.42(m,18H),1.41–1.32(m,12H),1.31–1.14(m,96H),0.84(t,J＝6.4Hz,36H).HRMS(ESI,m/z):[M+H]+ calcd.For C₁₂₃H₂₄₁N₁₆O₁₈,2231.84628;found:2231.84191.

Compounds of formula (I) L0503：¹H NMR(400MHz,Chloroform-d)δ7.26–7.16(m,6H),4.84(p,J＝6.4Hz,6H),4.25–4.05(m,6H),3.36–3.20(m,12H),3.11–2.47(m,24H),2.32(t,J＝7.6Hz,12H),1.83(p,J＝7.2Hz,12H),1.56–1.43(m,24H),1.34–1.18(m,120H),0.86(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₃₈H₂₆₅N₁₀O₂₄,2447.98513;found:2447.98335.

Compounds of formula (I) L0504：¹H NMR(400MHz,Chloroform-d)δ7.26–7.20(m,6H),4.84(p,J＝6.4Hz,6H),4.21–4.03(m,6H),3.35–3.21(m,12H),3.03–2.41(m,20H),2.32(t,J＝7.6Hz,16H),1.87–1.78(m,12H),1.67–1.35(m,30H),1.34–1.17(m,120H),0.86(t,J＝6.8Hz,36H).HRMS(ESI,m/z):[M+H]⁺calcd.For C₁₄₁H₂₇₁N₁₀O₂₄,2489.02873;found:2489.04173.

Compounds of formula (I) L0505：¹H NMR(400MHz,Chloroform-d)δ7.26–7.21(m,6H),6.17–5.37(m,6H),4.15(dd,J＝8.4,3.0Hz,6H),4.04(t,J＝6.8Hz,12H),3.39–3.20(m,12H),3.05–2.42(m,24H),2.34(t,J＝7.6Hz,12H),1.84(p,J＝7.2Hz,12H),1.60(p,J＝6.8Hz,12H),1.36–1.20(m,84H),0.87(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₀₈H₂₀₅N₁₀O₂₄,2027.51563;found:2027.51361.

Compounds of formula (I) L0506：¹H NMR(400MHz,Chloroform-d)δ7.24(d,J＝6.4Hz,6H),4.09(dd,J＝8.8,3.6Hz,6H),4.04(t,J＝6.8Hz,12H),3.37–3.17(m,J＝6.8Hz,12H),3.01–2.57(m,18H),2.33(t,J＝7.6Hz,18H),1.83(p,J＝7.2Hz,12H),1.67–1.44(m,18H),1.34–1.19(m,84H),0.86(t,J＝6.8Hz,18H).HRMS(ESI,m/z):[M+H]⁺ calcd.For C₁₁₁H₂₁₁N₁₀O₂₄,2069.56258;found:2069.55766.

Example 4: nucleic acid nanoparticle complex preparation

Nucleic acid drug stock solution (wherein nucleic acid can be FLuc mRNA, EGFP pDNA, EGFP siRNA, spike mRNA) is diluted with buffer solution to a nucleic acid concentration of 0.04mg/mL (buffer solution can be: pH 3-6, 1-200 mM PBS buffer, citric acid-sodium citrate buffer, citric acid-disodium hydrogen phosphate buffer, HEPES buffer, sodium acetate buffer, tris buffer, preferably ph=4, 10mM, 20mM, 50mM PBS buffer, citric acid-sodium citrate buffer; ph=4, 50mM citric acid-sodium citrate buffer is used in this example) as aqueous phase.

Nucleic acid nanoparticle complexes were prepared according to the formulations described in the examples (Table 1 of example 5, table 2 of example 6, table 4 of example 7, table 5 of example 8, etc.), the desired PEG derivatives, phospholipids, cholesterol analogs and lipid compounds were equilibrated from the refrigerator to room temperature, and the PEG derivatives, phospholipids, cholesterol analogs and lipid compounds were weighed and dissolved with ethanol (the PEG derivatives were dissolved with ethanol at a concentration ranging from 1mg/mL to 10mg/mL; dissolving phospholipid with ethanol in the concentration range of 1 mg/mL-20 mg/mL; dissolving cholesterol analog with ethanol in the concentration range of 1 mg/mL-20 mg/mL, dissolving lipid compound with ethanol in the concentration range of 1 mg/mL-30 mg/mL), ultrasonic dispersing to aid dissolution, mixing the dissolved PEG derivative, phospholipid, cholesterol analog and lipid compound according to the ratio of each of the formulations (Table 1 of example 5, table 2 of example 6, table 4 of example 7, table 5 of example 8, etc.) according to each of the examples, adding appropriate amount of ethanol, mixing (the addition amount of ethanol is adaptively adjusted according to the component ratio of each of the formulations of nucleic acid nanoparticle complexes, the ratio of aqueous phase to organic phase in the subsequent operation), preparing ethanol solution containing PEG derivative, phospholipid, cholesterol analog as organic phase, mixing the two phases rapidly according to the volume ratio of aqueous phase to organic phase of 3:1, removing ethanol and water in the mixed solution by dialysis or ultrafiltration method with a nanoparticle preparation instrument according to the ratio of 3:1, adding nucleic acid in the mixed solution to make the concentration of nucleic acid template to be 0.04mg/mL, the nucleic acid nanoparticle complex formulations described in each example (Table 1 of example 5, table 2 of example 6, table 4 of example 7, table 5 of example 8, etc.) were finally obtained and stored at 2-8℃for use.

Wherein a prescription prepared by commercially available ionizable lipid molecules SM-102 (CAS: 2089251-47-6), ALC-0315 (CAS: 2036272-55-4) was used as a control.

Example 5: characterization of nucleic acid nanoparticle complexes according to the invention

Nucleic acid nanoparticle complexes of Table 1 were prepared as described in example 4 using FLuc-mRNA as model mRNA, and the dynamic light scattering particle size (size), surface Potential (Zeta Potential) and Polydispersity (PDI) of the nucleic acid nanoparticle complexes were tested at 25℃using a Markov nanoparticle sizer (Malvern Zetasizer Nano ZSE).

Table 1: characterization of nucleic acid nanoparticle complexes

Remarks: the nitrogen-to-phosphorus ratio described in Table 1 represents the ratio of the molar amount of ionizable nitrogen atoms to the molar amount of phosphorus atoms of the nucleic acid in the lipid compound of the present invention.

The nitrogen-to-phosphorus ratio of the lipid compounds to the nucleic acids described in Table 1 represents the ratio of the molar amount of ionizable nitrogen atoms in the lipid compounds (i.e., the compounds provided herein, such as at least one of compounds L0461-L0506, or ALC-0315 or SM-102) to the molar amount of phosphorus atoms in the nucleic acids in the nucleic acid nanoparticle complexes.

The results are shown in Table 1, and the results show that the average particle size of the nucleic acid nanoparticle composite provided by the invention is in the range of 70nm to 268mm, PDI is small, the particle size uniformity is good, and the surface charge of the nanoparticle composite is in the range of-9 mV to +17 mV.

Example 6: in vitro cell transfection experiments of nucleic acid nanoparticle complexes

In vitro silencing gene expression experiments of nucleic acid nanoparticle complexes with EGFP-siRNA (EGFP-siRNA as model siRNA) transfected into HeLa-EGFP cells (polyclonal cell lines stably expressing EGFP fluorescent proteins):

The cell suspension in logarithmic growth phase was packed in 24-well plates at a density of 1X 10 ⁵ cells per well, and placed in a 5% CO ₂ incubator for stationary culture at 37 ℃. After 24 hours, EGFP-siRNA was taken and the nucleic acid nanoparticle complexes shown in Table 2 were prepared according to the preparation method described in example 4, and the nucleic acid nanoparticle complex mixture was diluted with opti-MEM, respectively, and after standing, 160ng of EGFP-siRNA-containing nucleic acid nanoparticle complex was added to each well, and 3 wells were repeated for each sample. After 4h of administration, the medium aspirated into the 96-well plate was replaced with complete medium. Culturing is continued for 18 hours, cells are digested and collected, the fluorescence intensity of the FITC channel of each well living cell is detected by a flow cytometer, and the geometric mean of the fluorescence intensities of the compound well EGFP positive cells is calculated. The siRNA silencing efficiency (i.e., percent decrease in mean fluorescence intensity of the cells) was calculated as follows:

siRNA silencing efficiency (%) = (geometric mean of fluorescence intensity of cells not transfected nucleic acid nanoparticle complex-geometric mean of fluorescence intensity of cells transfected nucleic acid nanoparticle complex)/geometric mean of fluorescence intensity of cells not transfected nucleic acid nanoparticle complex x 100%

Table 2: silencing efficiency of EGFP-siRNA-loaded nucleic acid nanoparticle complexes delivery to Hela-EGFP cells

Remarks: the nitrogen-to-phosphorus ratio shown in Table 2 represents the ratio of the molar amount of ionizable nitrogen atoms in the lipid compound of the present invention to the molar amount of phosphorus atoms in the nucleic acid.

The results are shown in Table 2. Conclusion: the results show that the nucleic acid-lipid compound with the EGFP-siRNA entrapped therein shows better gene silencing effect in cell transfection, wherein Rp.74-Rp.83 deliver siRNA in cells with silencing efficiency exceeding 70 percent, which is superior to that of control Rp.84 and Lipo2000.

Example 7: detection of transfection of nucleic acid nanoparticle complexes in mice by fluorescence imaging of small animals

7.1 Investigation of the Effect of nucleic acid lipid nanoparticle formulations on the in vivo delivery of FLuc-mRNA in mice

Three BALB/c mice per group, nucleic acid nanoparticle complexes containing FLuc-mRNA in Table 3 were prepared as described in example 4, using FLuc-mRNA as model mRNA. After completion of the formulation, the formulation was taken for Intramuscular (IM) or Intravenous (IV) or Intraperitoneal (IP) or subcutaneous (IH) injections, and about 75 μl of a nucleic acid nanoparticle complex formulation containing 3 μg FLuc-mRNA was injected into each mouse using an insulin needle for a total of 3 mice. When the administration mode is intravenous injection, the injection site is the tail vein of the mice. When the administration mode is intraperitoneal injection, the injection site is the abdominal cavity of the mouse. When the administration mode is intramuscular injection, the injection site is thigh muscle of the mouse. In the case of subcutaneous injection, the injection site is subcutaneous on the back of the mouse.

Living imaging (IVIS Lumina III, perkin Elmer Co., ltd.) was performed after a certain period of time from the injection, and intravenous injection was usually performed 3 hours later, intraperitoneal injection was usually performed 6 hours later, intramuscular injection was performed 24 hours later, and subcutaneous injection was performed 24 hours later.

A proper amount of substrate D-Luciferin (D-fluorescein) is taken, diluted by PBS to prepare a solution with the concentration of 15mg/mL, the solution is kept away from light for later use, 200 mu L of 15mg/mL of D-Luciferin working solution is injected into each mouse intraperitoneally 10-15 minutes before imaging, and a biological imaging system (PERKINELMER, IVIS Lumina Series III) for animals is used for carrying out whole-body in-vivo imaging bioluminescence image detection on the mice. The bioluminescence image of the mice was taken and the luminescence signal was counted.

Table 3: nucleic acid nanoparticle complexes carrying FLuc-mRNA for in vivo delivery in mice

Remarks:

(1) The nitrogen-to-phosphorus ratio shown in Table 3 represents the ratio of the molar amount of ionizable nitrogen atoms in the lipid compound of the present invention to the molar amount of phosphorus atoms in the nucleic acid.

(2) IV represents intravenous administration; IM means intramuscular administration.

Results: the results are shown in table 3, and the experimental group nucleic acid nanoparticle complex formulations all showed expression of luciferase in whole body in vivo imaging, and the greater the fluorescence intensity, the more luciferase was expressed.

Conclusion: the nucleic acid nanoparticle complexes of each experimental group with the FLuc-mRNA has better luciferase expression in the body of mice.

As shown in table 3, in the formulations of the intramuscular administration experimental group, all formulations were expressed efficiently in mice after administration. In the prescription of the intravenous injection administration experimental group, all prescriptions are effectively expressed in mice after administration. The expression of luciferases of formulas Rp.02, rp.07, rp.13, rp.15, rp.18, rp.23, rp.24, rp.53, rp.54, rp.65, etc. is significantly higher than that of formulas Rp.72 and Rp.73 prepared from commercially available ionizable lipids SM-102 and ALC-0315.

7.2 Investigation of the in vivo delivery Effect of nucleic acid lipid nanoparticle formulations on FLuc-circRNA in mice

Three BALB/c mice per group were prepared as nucleic acid nanoparticle complexes containing FLuc-circRNA in Table 4 using FLuc-circRNA (manufacturer: shanghai megadimension technologies development Co., ltd. (Hongene Biotech Corporation)) as model RNA according to the preparation method described in example 4. After the preparation was completed, the preparation was taken for intramuscular injection (rat thigh rectus muscle) or intravenous injection (rat tail vein) or intraperitoneal injection or subcutaneous injection (back subcutaneous), and about 75 μl was injected per mouse. Living imaging (IVIS Lumina III, perkin Elmer Co., ltd.) was performed after a certain period of injection, 200. Mu.L of 15mg/mL of a D-Luciferin (D-fluorescein) working solution was intraperitoneally injected 10-15 minutes before imaging, and imaging analysis was performed using a living imager.

Table 4: nucleic acid nanoparticle complexes carrying FLuc-circRNA for in vivo delivery in mice

Remarks: the nitrogen-to-phosphorus ratio shown in Table 4 represents the ratio of the molar amount of ionizable nitrogen atoms in the lipid compound of the present invention to the molar amount of phosphorus atoms in the nucleic acid.

Results: the results are shown in table 4, where the experimental set of nucleic acid nanoparticle complex formulations all showed expression of luciferase in whole body in vivo imaging, the greater the fluorescence intensity, the more luciferase expression.

Conclusion: the nucleic acid nanoparticle complexes of each experimental group, which are coated with FLuc-circRNA, have better luciferase expression in mice.

As shown in table 4, in the formulations of the intramuscular administration experimental group, all formulations were expressed effectively in mice after administration. In the prescription of the intravenous injection administration experimental group, all prescriptions are effectively expressed in mice after administration. The expression of the luciferases of the prescriptions Rp.85, rp.86 and Rp.87, etc. is significantly higher than the prescriptions Rp.89 prepared by the commercial ionizable lipid SM-102, and the expression of the luciferases of the prescriptions Rp.85 and Rp.86, etc. is significantly higher than the prescriptions Rp.88 prepared by the commercial ionizable lipid ALC-0315.

Example 8: evaluation of humoral immune Effect of nucleic acid nanoparticle Complex in mice

The novel crown S-mRNA is used as model mRNA, and provided by Shanghai megadimension technology development Co., ltd (Hongene Biotech Corporation), and the nucleotide sequence of the novel crown S-mRNA (cap 1 structure, N1-me-pseudo U modified) is shown in the S-mRNA in the sequence table.

Specific information for S-mRNA stock solution is:

Product name: COVID-19Spike Protein,Full Length-mRNA;

description of the product: a length of 4088 nucleotides;

concentration: 1.0mg/mL;

storage environment: 1mM sodium citrate, pH 6.4;

storage requirements are: -40 ℃ or less.

The experimental process comprises the following steps:

table 5: nucleic acid nanoparticle complexes carrying S-mRNA for in vivo immunization of mice

8.1, In vivo immunization experiments:

Lipid nanoparticles encapsulating nucleic acid drugs were prepared as shown in Table 5 according to the method shown in example 4, and then administered after DELTA SPIKE full LENGTH MRNA (Shanghai megasciences development Co., ltd.) formulation, each mouse was dosed at 5. Mu.g, an equal volume of PBS buffer was used as a blank control group, and 5. Mu.g of naked S-mRNA was used as a negative control group. The whole blood of the mouse is taken by taking blood from the outer canthus on the 14 th day after the first immunization, and the whole blood of the mouse is taken by taking blood from the outer canthus on the 14 th day after the second immunization by repeated administration. Centrifuging whole blood with 5000rcf for 5min, collecting supernatant, centrifuging with 10000rcf for 5min to obtain serum, packaging into a row of tubes, and storing in a refrigerator at-20deg.C.

8.2, Elisa or Elisa titer experiments to detect specific antibody levels:

8.2.1, coating: mu.L of 0.2. Mu.g/100. Mu.L of Delta-S protein (Suzhou offshore protein technologies Co., ltd.) dilution was added to each well and placed in a refrigerator at 4℃for 12 hours.

8.2.2, Cleaning: mu.L of PBST (PBS buffer at pH 7.4 containing 0.05% Tween-20) was added to each well and washed three times.

8.2.3, Closing: mu.L of 5% (w/v) BSA-PBS solution (BSA bovine serum albumin, beijing Boaosen Biotechnology Co., ltd.) was added to each well and shaken at room temperature for 2h using a shaker at 50 rcf.

8.2.4, Cleaning: 200. Mu.L of PBST was added to each well and washed once.

8.2.5, Add sample: 100. Mu.L of serum (i.e., 0.5. Mu.L of serum) diluted 200-fold with PBS was added to each well (ELISA titer test serum diluted 1000-fold, 10000-fold, 100000-fold, 1000000-fold) and 50rcf was shaken at room temperature for 2 hours using a shaker.

8.2.6, Cleaning: 200. Mu.L of PBST was added to each well and washed three times.

8.2.7, Adding detection antibody: 100. Mu.L of HRP-labeled goat anti-mouse IgG detection antibody (i.e., 0.1. Mu.L of antibody stock solution) diluted 1000-fold was added to each well (manufactured by Biotechnology (Shanghai) Co., ltd.) and shaken for 1h at room temperature using a shaker.

8.2.8, Cleaning: 200. Mu.L of PBST was added to each well and washed three times.

8.2.9, TMB (beijing solebao biotechnology limited): adding 50 mu L of TMB mixed solution with the volume ratio of solution A to solution B of 1:1 into each hole, and reacting for about 5 min.

8.2.10 Phosphoric acid (ala Ding Shiji (Shanghai) limited) was added: the reaction was terminated by adding 50. Mu.L of 1mol/L phosphoric acid per well.

8.2.11, Detection: and (5) detecting by using a multifunctional enzyme-labeled instrument.

Serum IgG antibody level results: as shown in fig. 1.

Conclusion: the results show that the OD values of the prescriptions Rp.91, rp.92, rp.93 and Rp.94 after the second immunization are obviously higher than those of the blank control group and the naked mRNA negative control group, and obvious immune response is caused, so that the prescriptions nucleic acid nanoparticle complex has stronger serum conversion efficiency and humoral immune activation function.

Serum IgG antibody titer results: as shown in fig. 2.

Conclusion: the results show that the OD values of the serum after the second immunization are obviously higher than that of the blank control group and the naked mRNA negative control group after the serum is diluted by 1X 10 ⁶ times, the OD values are lower than that of the commercial ionizable cationic lipid molecule ALC-0315 prescription Rp.90, the immune effects of the prescriptions Rp.91 and Rp.93 are weaker than those of the commercial ionizable cationic lipid molecule ALC-0315 prescription Rp.90, and the prescriptions nucleic acid nanoparticle complex has better functions in terms of mRNA delivery and expression although the immune response is weaker.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. A lipid compound, the structure of which is shown in formula A,

in,

Each n is independently selected from any positive integer from 1 to 20, and each m is independently selected from any positive integer from 1 to 20;

R ¹ , R ² and R ³ are each independently selected from hydrogen, substituted or unsubstituted C _1-20 alkyl, substituted or unsubstituted C _2-20 alkenyl, substituted or unsubstituted C _2-20 alkynyl, substituted or unsubstituted C _2-20 heteroalkyl,

wherein the substitution in the substituted or unsubstituted C _1-20 alkyl, substituted or unsubstituted C _2-20 alkenyl, substituted or unsubstituted C _2-20 alkynyl or substituted or unsubstituted C _2-20 heteroalkyl independently represents that at least one hydrogen atom is replaced by 1-5 halogen, cyano, -OR ⁴ , -SR ⁴ , N(R ⁴ ) ₂ , -N(R ⁴ ) ₃ ⁺ , oxo, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl or C _1-6 haloalkyl;

Each R ⁴ is independently selected from hydrogen, substituted or unsubstituted C _1-12 alkyl, substituted or unsubstituted C _2-12 alkenyl or substituted or unsubstituted C _2-12 alkynyl; wherein the substitution in each substituted or unsubstituted C _1-12 alkyl, substituted or unsubstituted C _2-12 alkenyl or substituted or unsubstituted C _2-12 alkynyl in each R ⁴ independently represents that at least one hydrogen atom is replaced by 1-5 halogens, cyano, -OH, -SR ⁵ , N(R ⁵ ) ₂ or -N(R ⁵ ) ₃ ⁺ , or at least 2 hydrogen atoms are replaced by =O;

Each R ⁵ is independently selected from hydrogen, substituted or unsubstituted C _1-12 alkyl, substituted or unsubstituted C _2-12 alkenyl or substituted or unsubstituted C _2-12 alkynyl; wherein the substitution in each substituted or unsubstituted C _1-12 alkyl, substituted or unsubstituted C _2-12 alkenyl or substituted or unsubstituted C _2-12 alkynyl in each R ⁵ independently represents that at least one hydrogen atom is replaced by 1-5 halogens, cyano, -OH, -SH, NH ₂ , -NH(C _1-6 alkyl), -N(C _1-6 alkyl) ₂ , -N(C _1-6 alkyl) ₃ ⁺ , or at least an even number of hydrogen atoms are replaced by =O;

Each L is independently selected from C _1-10 alkylene or C _3-10 heteroalkylene;

Each Y is independently selected from -O- or -NR ⁶ -;

Each R ⁶ is independently selected from hydrogen, substituted or unsubstituted C _1-6 alkyl, substituted or unsubstituted C _2-6 alkenyl, substituted or unsubstituted C _2-6 alkynyl or substituted or unsubstituted C _1-6 haloalkyl; wherein the substitution in each substituted or unsubstituted C _1-6 alkyl, substituted or unsubstituted C _2-6 alkenyl, substituted or unsubstituted C _2-6 alkynyl or substituted or unsubstituted C _1-6 haloalkyl in each ^R 6 independently represents that at least one hydrogen atom is replaced by 1-5 halogens, cyano, -OH, -NH ₂ , -NH(C _1-6 alkyl), -N(C _1-6 alkyl) ₂ , -N(C _1-6 alkyl) ₃ ⁺ , C _1-6 alkoxy or C _1-6 haloalkoxy, or at least 2 hydrogen atoms are replaced by =O;

Each R is independently selected from hydrogen, -Z ¹ -C _1-20 alkyl, -Z ¹ -C _2-20 alkenyl, -Z ¹ -C _2-20 alkynyl, -Z ¹ -heterocyclyl, -Z ¹ -C _1-6 alkylene-ZC _1-20 alkyl, -Z ¹ -C _1-6 alkylene-ZC _2-20 alkenyl, -Z ¹ -C _1-6 alkylene-ZC _2-20 alkynyl, -Z ¹ -C _1-6 alkylene-Z-heterocyclyl, -Z ¹ -C _2-6 alkenyl-ZC _1-20 alkyl _, _-Z ¹ -C _2-6 alkenyl-ZC _2-20 alkenyl, -Z ¹ -C 2-6 alkenyl-ZC 2-20 alkynyl, -Z ¹ -C _2-6 alkynyl-ZC _1-20 alkyl, -Z ¹ -C _{-Z 1 -C 2-6} alkynyl-ZC _2-20 alkenyl or -Z ¹ -C _2-6 alkynyl-ZC _2-20 alkynyl;

each Z is independently selected from a carbon-carbon single bond, -O-, -O-CH ₂ -O-, -O-CH ₂ (CH ₃ )-O-, -OC(O)-, -NR ⁴ C(O)-, -C(O)O-, -NR ⁴ -, -S-, -SS-, -C(O)-, -C(O)NR ⁴ -, -S(O)-, -S(O) ₂ -, -NR ⁴ C(O)O-, -NR ⁴ C(O)NR ⁴ -, -NR ⁴ S(O)-, or -S(O) ₂ NR ⁴ -;

each Z ¹ is independently selected from a carbon-carbon single bond, -O-, -NR ⁴ -, -S-, -SS-, -C(O)-, -C(O)O-, -C(O)NR ⁴ -, -S(O)-, -S(O) ₂ -, -NR ⁴ C(O)-, -NR ⁴ C(O)O-, -NR ⁴ C(O)NR ⁴ -, -NR ⁴ S(O)-, or -S(O) ₂ NR ⁴ -;

Each R ⁴ is independently selected from H or C _1-6 alkyl;

In the compound represented by formula A, each The linear moiety contains at least 6 linear atoms or contains 6-20 linear atoms.

2. The lipid compound according to claim 1, wherein R ¹ , R ² and R ³ are independently selected from hydrogen, substituted or unsubstituted C _1-20 alkyl,

Each R is independently selected from hydrogen, -Z ¹ -C _1-20 alkyl, -Z ¹ -C _2-20 alkenyl, -Z ¹ -C _1-6 alkylene-ZC _1-20 alkyl, -Z ¹ -C _1-6 alkylene-ZC _2-20 alkenyl, -Z ¹ -C _2-6 alkenyl-ZC _1-20 alkyl, -Z ¹ -C _2-6 alkenyl-ZC _2-20 alkenyl.

3. The lipid compound according to any one of claims 1-2, wherein n is selected from any positive integer in the range of 1-20, and m is selected from any positive integer in the range of 1-20;

R ¹ , R ² and R ³ are independently selected from hydrogen, unsubstituted C _1-20 alkyl or

^R6 is selected from hydrogen, substituted or unsubstituted _C1-6 alkyl;

Each Y is independently selected from -O- or -NR ⁶ -;

R is selected from hydrogen, -Z ¹ -C _1-20 alkyl, -Z ¹ -C _2-20 alkenyl, -Z ¹ -C _1-6 alkylene-ZC _1-20 alkyl, -Z ¹ -C _1-6 alkylene-ZC _2-20 alkenyl, -Z ¹ -C _2-6 alkenyl-ZC _1-20 alkyl, -Z ¹ -C _2-6 alkenyl-ZC _2-20 alkenyl;

Each Z is independently selected from a carbon-carbon single bond, -O-, -O-CH ₂ -O-, -O-CH ₂ (CH ₃ )-O-, -OC(O)-, -NR ⁴ C(O)-, -C(O)O-;

Each Z ¹ is independently selected from -O-, -NR ⁴ -;

Each R ⁴ is independently selected from H or C _1-6 alkyl.

4. The lipid compound according to any one of claims 1 to 3, wherein the compound represented by formula A comprises compound B,

5. The lipid compound according to any one of claims 1 to 4, wherein the compound represented by formula B comprises: compound C, compound D,

6. The lipid compound according to claim 5, wherein in the compound C, n is selected from any positive integer of 1-10, m is selected from any positive integer of 1-20; R is selected from hydrogen, -Z ¹ -C _1-20 alkyl, -Z ¹ -C _2-20 alkenyl, -Z ¹ -C _1-6 alkylene-ZC _1-20 alkyl, -Z ¹ -C _1-6 alkylene-ZC _2-20 alkenyl, ^-Z 1 -C _2-6 alkenyl-ZC _1-20 alkyl, -Z ¹ -C _2-6 alkenyl-ZC _2-20 alkenyl;

In the compound C, each Z is independently selected from a carbon-carbon single bond, -O-, -O-CH ₂ -O-, -O-CH ₂ (CH ₃ )-O-, -OC(O)-, -NR ⁴ C(O)-, -C(O)O-;

In the compound C, each Z ¹ is independently selected from -O-, -NR ⁴ -;

In the compound C, each R ⁴ is independently selected from H or C _1-6 alkyl;

In the compound D, ^R6 is selected from hydrogen or unsubstituted _C1-3 alkyl; n is selected from any positive integer from 1 to 10, and m is selected from any positive integer from 1 to 20; R is selected from hydrogen, ^-Z1 - _C1-20 alkyl, ^-Z1 - _C2-20 alkenyl, ^-Z1 - _C1-6 alkylene- _ZC1-20 alkyl, ^-Z1 - _C1-6 alkylene- _ZC2-20 ^alkenyl , -Z1- _C2-6 alkenyl- _ZC1-20 alkyl, -Z1 ^- _C2-6 alkenyl- _ZC2-20 alkenyl;

In the compound D, each Z is independently selected from a carbon-carbon single bond, -O-, -O-CH ₂ -O-, -O-CH ₂ (CH ₃ )-O-, -OC(O)-, -NR ⁴ C(O)-, -C(O)O-;

In the compound D, each Z ¹ is independently selected from -O-, -NR ⁴ -;

In the compound D, each R ⁴ is independently selected from H or C _1-6 alkyl.

7. The lipid compound according to claim 6, wherein the compound C is selected from the group consisting of compound L0461, compound L0464, compound L0465, compound L0467, compound L0468, compound L0469, compound L0470, compound L0471, compound L0472, compound L0475, compound L0476, compound L0477, compound L0478, compound L0479, compound L0491, compound L0492, compound L0497, and compound L0498;

The compound D is selected from the group consisting of compounds L0462, L0463, L0473, L0474, L0480, L0481, L0482, L0483, L0484, L0485, L0486, L0487, L0488, L0489, L0490, L0493, L0494, L0495, L0496, L0499, L0500, L0501, L0502, L0503, L0504, L0505, and L0506;

8. A lipid compound nanoparticle, characterized in that it comprises the lipid compound according to any one of claims 1 to 7 and an auxiliary material; or comprises the lipid compound according to any one of claims 1 to 7, a nucleic acid and an auxiliary material;

Optionally, the auxiliary material is selected from at least one of: PEG derivatives, lipids, lipidoids, alcohols, sugars or inorganic salts;

Optionally, the PEG derivative is selected from at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, PEG-modified stearic acid, and PEG-modified phosphatidylserine;

Optionally, the PEG derivative comprises 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)], dilauroylphosphatidylethanolamine-polyethylene glycol, dimyristoylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylcholine polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol, PEG-distearylglycerol, PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide, PEG-dipalmitoylphosphatidylethanolamine or PEG-1,2-dimyristyloxypropyl-3-amine;

Optionally, the PEG derivative includes at least one of DMG-PEG2000, mPEG-DSPE, mPEG-STA, mPEG-PS, mPEG-DMPE, mPEG-DPPE, ALC-0159, mPEG2k-DMPE, and DSPE-PEG5000;

Optionally, the lipid comprises a phospholipid or a sterol;

Optionally, the phospholipids include at least one selected from lecithin, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine;

Optionally, the sterol comprises at least one of cholesterol, lanosterol, 5α-cholestane-3β-ol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid or α-tocopherol.

9. The lipid compound nanoparticle according to claim 8, wherein the lipid compound nanoparticle comprises the lipid compound according to any one of claims 1 to 7 and an auxiliary material; or comprises the lipid compound according to any one of claims 1 to 7, a nucleic acid and an auxiliary material;

The auxiliary materials are PEG derivatives and lipids; the lipids are selected from phospholipids or sterols;

Optionally, the phospholipids include at least one selected from phosphatidylcholine (PC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine;

10. The lipid compound nanoparticles according to any one of claims 8 to 9, wherein the content of the lipid compound according to any one of claims 1 to 7 is 20.0 mol% to 75.0 mol% calculated based on the total molar amount of each component in the lipid compound nanoparticles; and/or

The content of the PEG derivative is 0.5 mol%-10 mol% calculated based on the total molar amount of each component in the lipid compound nanoparticles; and/or

Calculated based on the total molar amount of each component in the lipid compound nanoparticles, the content of the phospholipid is 4.0 mol%-20.0 mol%; and/or

Calculated based on the total molar amount of each component in the lipid compound nanoparticles, the content of the sterol is 18.0 mol%-68.5 mol%; or the content of the sterol is 20.0 mol%-68.5 mol%; and/or

The molar ratio of PEG derivative: phospholipid: sterol: lipid compound according to any one of claims 1 to 7 in the lipid compound nanoparticles is (0.5-10.0): (4.0-20.0): (18.0-68.5): (20.0-75.0); and/or

The molar ratio of PEG derivative: phospholipid: sterol: lipid compound according to any one of claims 1 to 7 in the lipid compound nanoparticles is (0.5-10.0): (4.0-20.0): (20.0-68.5): (20.0-75.0); and/or

The molar ratio of PEG derivative: phospholipid: sterol: lipid compound according to any one of claims 1 to 7 in the lipid compound nanoparticles is 2.50:16.00:33.00:48.50, 6.00:16.00:29.50:48.50, 2.50:4.00:45.00:48.50, 0.95:7.58:26.47:65.00, 1.50:11.50:38.50:48.50, 1.58:16.84:18.42:63.16, 10.00:4.00:56.00:30.00, 2.32:6.20:45.00:46.48:0 .50:4.00:65.50:30.00, 2.50:11.50:56.00:30.00, 2.00:5.00:68.00:25.00, 3.00:4.00:18.00:75.00, 2.00:20.00:43.00:35.00, 1.50:10.00:68.50:20.00, 2.50:4.00:63.50:30.00, 1.50:16.00:22.50:60.00, 1.50:4.00:64.50:30.00 or 1.40:11.15:38.95:48.50; and/or

The molar ratio of PEG derivative: phospholipid: sterol: lipid compound according to any one of claims 1 to 7 in the lipid compound nanoparticles is (0.5-2.5): (7.58-16.0): (29.5-56.0): (35.0-48.5); and/or

The molar ratio of PEG derivative: phospholipid: sterol: lipid compound according to any one of claims 1 to 7 in the lipid compound nanoparticles is 0.50:4.00:65.50:30.00, 1.50:11.50:38.50:48.50 or 1.40:11.15:38.95:48.50; and/or

The PEG derivative is selected from DMG-PEG2000, mPEG-DMPE, mPEG-DSPE, ALC-0159, mPEG-DPPE, mPEG-STA, mPEG-PS, mPEG2k-DMPE, DSPE-PEG5000; and/or

The phospholipids include at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), phosphatidylcholine (PC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); and/or

The sterol includes at least one of cholesterol (Chol) and lanosterol (Lanosterol).

11. A nucleic acid nanoparticle complex, characterized in that it comprises a nucleic acid and the lipid compound nanoparticles according to any one of claims 8 to 10;

Optionally, the ratio of the molar amount of ionizable nitrogen atoms in the compound in the lipid compound nanoparticles according to any one of claims 8 to 10 to the molar amount of phosphorus atoms in the nucleic acid in the nucleic acid nanoparticle complex is 6 to 65;

Optionally, the ratio of the molar amount of ionizable nitrogen atoms in the lipid compound to the molar amount of phosphorus atoms in the nucleic acid in the lipid compound nanoparticles of any one of claims 8 to 10 in the nucleic acid-nanoparticle complex is 6, 7, 8, 9, 10, 11, 12, 13, 13.5, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65;

Optionally, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA);

Optionally, the ribonucleic acid (RNA) includes at least one of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), self-replicating RNA (saRNA), small hairpin RNA (shRNA), circular RNA (circRNA), messenger RNA (mRNA), or a combination thereof;

Optionally, the average particle size of the lipid compound complex is 70nm-300nm; and/or

Optionally, the lipid compound complex has a polydispersity coefficient of ≤ 0.30.

12. A pharmaceutical composition, characterized in that it comprises the lipid compound nanoparticles according to claims 8-10 or the nucleic acid nanoparticle complex according to claim 11, and a pharmaceutically acceptable excipient;

Optionally, the dosage form of the pharmaceutical composition includes injection, suppository, eye drop, tablet, capsule, suspension or inhalant.

13. Use of the lipid compound according to any one of claims 1 to 7, the lipid compound nanoparticles according to claims 8 to 10, the nucleic acid nanoparticle complex according to claim 11 or the pharmaceutical composition according to claim 12 in the preparation of a product for in vivo delivery of nucleic acids.