Ionizable lipid molecules containing a tocopherol structure, lipid nanoparticles comprising the same and uses thereof

Abstract

The present application relates to ionizable lipid molecules containing a tocopherol structure, lipid nanoparticles comprising the same and uses thereof. Specifically, an ionizable lipid molecule containing a tocopherol and derivatives thereof structure represented by formula (1), lipid nanoparticles comprising the same, methods of preparation and uses thereof are provided. Compared with ionizable lipid molecules conventionally used in the art, the lipid nanoparticles prepared from the ionizable lipid molecules represented by formula (1) of the present application can significantly improve the delivery efficiency and expression of nucleic acids.

Description

Technical Field

[0001] This disclosure pertains to the field of pharmaceuticals and biotechnology, specifically relating to ionizable lipid molecules capable of delivering nucleic acids and containing tocopherol structures, lipid nanoparticles containing such molecules, and their uses. Background Technology

[0002] The rise of nucleic acid therapy undoubtedly marks a significant milestone in the history of modern medicine. The success of mRNA vaccines has not only provided a powerful tool for controlling epidemics but has also greatly promoted the research and application of nucleic acid drugs in the treatment of genetic diseases, tumors, and viral infections. Nucleic acid drugs, through regulation at the post-transcriptional and pre-translational stages of genes, achieve precise control over the expression of disease-related proteins, demonstrating significant advantages such as simple design, short development cycle, high targeting specificity, broad therapeutic range, and long-lasting effects.

[0003] However, the development and application of nucleic acid drugs face a core challenge—how to effectively deliver nucleic acid molecules to target cells while ensuring their stability during delivery and their functionality within the cell. An ideal delivery carrier needs to possess characteristics such as safety, stability, and high efficiency. The emergence of lipid nanoparticle (LNP) technology provides an innovative solution for nucleic acid drug delivery. LNPs, composed of ionizable lipids, steroidal lipids, accessory lipids, and polyethylene glycol lipids, can encapsulate mRNA, protecting it from enzymatic degradation and clearance by the immune system during delivery, promoting its transmembrane transport, and releasing mRNA into the cytoplasm for protein translation, thereby stimulating the body's immune response. The successful application of LNPs not only solves the problems of RNA protection and delivery but, due to their flexibility, safety, and scalable production methods, has become a key technology for mRNA vaccines and therapeutics.

[0004] Tocopherol, or vitamin E, is an important fat-soluble vitamin widely distributed in the human body and plays an indispensable role in various physiological processes. Tocopherol not only possesses strong antioxidant capabilities, protecting cells from damage by free radicals and reactive oxygen species, but also exhibits a variety of biological functions, including anti-inflammatory effects, regulation of lipid metabolism, and radiation protection. Furthermore, tocopherol and its derivatives have shown potential therapeutic value in the treatment of various diseases, including cardiovascular diseases, neurodegenerative diseases, and tumors. Importantly, tocopherol's high fat solubility allows it to mix with phospholipids and other lipid molecules to form stable lipid bilayer structures. Summary of the Invention

[0005] Based on these properties of tocopherol and its derivatives, the inventors developed a novel class of ionizable lipid molecules. These molecules incorporate the structural features of tocopherol and its derivatives and have been applied to the preparation of nucleic acid delivery carriers. These novel lipid molecules have demonstrated significant advantages in improving nucleic acid delivery efficiency, enhancing nucleic acid expression in vivo, and improving therapeutic efficacy. Particularly in the field of tumor therapy, the application of these novel lipid molecules offers new possibilities for achieving efficient and safe tumor suppression.

[0006] The main objective of this invention is to provide an ionizable lipid molecule containing tocopherol and its derivatives as shown in formula (1), lipid nanoparticles containing the same, a method for preparing the same, and its uses. Compared with conventionally used ionizable lipid molecules in the art, the lipid nanoparticles prepared from the ionizable lipid molecule shown in formula (1) of this invention can achieve highly efficient delivery and expression of nucleic acids.

[0007] [Ionizable lipid molecules - compounds of formula (1) or pharmaceutically acceptable salts thereof]

[0008] This invention provides a compound of formula (1) or a pharmaceutically acceptable salt thereof.

[0009]

[0010] in,

[0011] X is selected from N, CH, or

[0012] Selected from

[0013] L 1 L 2 and L 3 Independently selected from single bonds, C 1-10 Alkylene, C 6-10 aryl or -C 1-10 Alkylene-NR d -C 1-10 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens;

[0014] R d Selected from C 1-10 Alkyl, -C 1-10 Alkylene-NHC(=O)-C 1-30 Alkyl, -C 1-10 Alkylene-NHC(=O)-C 2-30 alkenyl or -C 1-10 Alkylene-OC(=O)-C1-6 Alkylene-SC 1-6 Alkylene-C(=O)OC 1-30 alkyl;

[0015] L 4 L 5 and L 6 Independently selected from single bonds, C 1-10 Alkylene, -C 1-6 Alkylene-SC 1-6 alkylene-, -C 1-6 Alkylene-SSC 1-6 alkylene- or -C 1-10 Alkylene-NR e -C 1-10 Alkylene -, wherein the alkylene is unsubstituted or converted by one or more OH, NH2, halogen, -NHC(=O)OC 1-30 Alkyl group, -NHC(=O)OC 2-30 alkenyl, -NHC(=O)-C 1-30 Alkyl group, -NHC(=O)-C 2-30 alkenyl, -C 1-10 Alkylene-C(=O)NH-C 1-10 Alkylene-NR e -C 1-10 Alkylene-OC(=O)-C 2-30 alkenyl, -C(=O)OC 1-30 Alkyl group, -C(=O)OC 2-30 alkenyl, -OC(=O)-C 1-30 Alkyl or -OC(=O)-C 2-30 Alkenyl substitution;

[0016] R e Selected from -C 1-10 Alkylene-OC(=O)-C(=O)-R f -C 1-10 Alkylene-C(=O)OC 1-30 Alkyl, -C 1-10 Alkylene-OC(=O)-C 1-30 Alkyl, -C 1-10 Alkylene-C(=O)OC 2-30 alkenyl, -C 1-10 Alkylene-OC(=O)-C 2-30 alkenyl, -C 1-10 Alkylene-OC(=O)-L b -C(=O)OC 1-30 Alkyl, -C 1-10 Alkylene-OC(=O)-C 1-20Alkylene-C(=O)-R f -C 1-10 Alkylene-OC(=O)-C 1-10 alkylene-(5-8-membered heteroaryl)-C 1-10 Alkyl, -C 1-10 Alkylene-OC(=O)NH-C 1-30 Alkyl, -C 1-10 Alkylene-NHC(=O)-C 1-30 Alkyl or -C 1-10 Alkylene-OC 1-30 alkyl;

[0017] R f Selected from

[0018] L b Selected from or -C 1-10 Alkylene-SSC 1-10 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens;

[0019] n is selected from 1, 2, 3, 4, 5, 6, 7 or 8;

[0020] G 1 G 2 and G 3 Independently selected from single bonds, -NR 3 -, -O-, -S-, -C(=O)-, -OC(=O)-, -OC(=O)O-, -C(=O)C(=O)O-, -NR 3 C(=O)-、-NR 3 C(=O)O-, 5 to 10 membered heterocyclic groups, -OC(=O)NR 3 -L a -C(=O)O-、-OC(=O)NR 3 -L a -NR 3 -C(=O)O-、-OC(=O)-L a -OC(=O)-NR 3 -、-NR 3 C(=O)-L a -C(=O)-NR 3 -、-NR 3 C(=O)OL a -C(=O)-NR 3 -or-C(=O)OL a -OC(=O)-NR 3-;

[0021] G 4 G 5 and G 6 Independently selected from single bonds, -NR 3 -, -O-, -C(=O)-, -OC(=O)-, -OC(=O)O-, -C(=O)C(=O)O-, -NR 3 C(=O)-、-NR 3 C(=O)O-、-C(=O)-L a -C(=O)-、-OC(=O)-L a -C(=O)-、-C(=O)OL a -C(=O)NR 3 -、-C(=O)OL a -OC(=O)NR 3 -、-C(=O)-L a -C(=O)NR 3 -, 5-8 quinone heteroaryl or -C(=O)NR 3 -L a -OC(=O)C(=O)-;

[0022] Each R 3 Independently selected from H and C 1-30 Alkyl, -C 1-10 Alkylene-OC(=O)-C 1-30 Alkyl, -C 1-10 Alkylene-OC(=O)-C 2-30 alkenyl, -C 1-10 Alkylene-C(=O)OC 1-30 Alkyl, -C 1-10 Alkylene-C(=O)OC 2-30 alkenyl or -C 1-10 Alkylene-OC(=O)-C 1-8 Alkylene-SC 1-8 Alkylene-C(=O)OC 1-30 alkyl;

[0023] Each L a Independently selected from single bonds, C 1-20 Alkylene, -[OCH2CH2] n -、-C 1-10 Alkylene-SSC 1-10 alkylene-, -C 1-10 Alkylene-SC 1-10 alkylene-, -C 1-10 Alkylene-C(=O)-C 1-10 alkylene- or -C 1-10Alkylene-NR e -C 1-10 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens;

[0024] R 1 and R 2 Selected independently

[0025] C 1-30 Alkyl or C 2-30 Alkenyl group, wherein the alkyl or alkenyl group is unsubstituted or substituted by one or more OH, NH2 or halogens.

[0026] In some implementations, L 1 L 2 and L 3 Independently selected from single bonds, C 1-10 Alkylene, C 6-10 aryl or -C 1-10 Alkylene-NR d -C 1-10 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens;

[0027] R d Selected from C 1-10 Alkyl, -C 1-10 Alkylene-NHC(=O)-C 1-30 Alkyl, -C 1-10 Alkylene-NHC(=O)-C 2-30 alkenyl or -C 1-10 Alkylene-OC(=O)-C 1-6 Alkylene-SC 1-6 Alkylene-C(=O)OC 1-30 alkyl.

[0028] In some implementations, L 1 L 2 and L 3 Independently selected from single bonds, C 1-8 Alkylene, C 6-10 aryl or -C 1-8 Alkylene-NR d -C 1-8 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens;

[0029] R d Selected from C 1-8 Alkyl, -C 1-8 Alkylene-NHC(=O)-C8-26 Alkyl, -C 1-8 Alkylene-NHC(=O)-C 8-26 alkenyl or -C 1-8 Alkylene-OC(=O)-C 1-4 Alkylene-SC 1-4 Alkylene-C(=O)OC 8-26 alkyl.

[0030] In some implementations, L 1 L 2 and L 3 Independently selected from single bonds, C 1-6 alkylene, phenylene or -C 1-6 Alkylene-NR d -C 1-6 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens;

[0031] R d Selected from C 1-6 Alkyl, -C 1-6 Alkylene-NHC(=O)-C 10-20 Alkyl or -C 1-6 Alkylene-OC(=O)-C 1-4 Alkylene-SC 1-4 Alkylene-C(=O)OC 10-20 alkyl.

[0032] In some implementations, L 1 L 2 and L 3 Independently selected from single bonds, -CH2-, -CH2CH2-,

[0033] In some implementations, L 4 L 5 and L 6 Independently selected from single bonds, C 1-10 Alkylene, -C 1-6 Alkylene-SC 1-6 alkylene-, -C 1-6 Alkylene-SSC 1-6 alkylene- or -C 1-10 Alkylene-NR e -C 1-10 Alkylene -, wherein the alkylene is unsubstituted or converted by one or more OH, NH2, halogen, -NHC(=O)OC 1-30 Alkyl group, -NHC(=O)OC 2-30 alkenyl, -NHC(=O)-C1-30 Alkyl group, -NHC(=O)-C 2-30 alkenyl, -C 1-10 Alkylene-C(=O)NH-C 1-10 Alkylene-NR e -C 1-10 Alkylene-OC(=O)-C 2-30 alkenyl, -C(=O)OC 1-30 Alkyl group, -C(=O)OC 2-30 alkenyl, -OC(=O)-C 1-30 Alkyl or -OC(=O)-C 2-30 Alkenyl substitution;

[0034] R e Selected from -C 1-10 Alkylene-OC(=O)-C(=O)-R f -C 1-10 Alkylene-C(=O)OC 1-30 Alkyl, -C 1-10 Alkylene-OC(=O)-C 1-30 Alkyl, -C 1-10 Alkylene-C(=O)OC 2-30 alkenyl, -C 1-10 Alkylene-OC(=O)-C 2-30 alkenyl, -C 1-10 Alkylene-OC(=O)-L b -C(=O)OC 1-30 Alkyl, -C 1-10 Alkylene-OC(=O)-C 1-20 Alkylene-C(=O)-R f -C 1-10 Alkylene-OC(=O)-C 1-10 alkylene-(5-8-membered heteroaryl)-C 1-10 Alkyl, -C 1-10 Alkylene-OC(=O)NH-C 1-30 Alkyl, -C 1-10 Alkylene-NHC(=O)-C 1-30 Alkyl or -C 1-10 Alkylene-OC 1-30 alkyl;

[0035] R f Selected from

[0036] L b Selected from or -C 1-10 Alkylene-SSC 1-10Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens;

[0037] n is selected from 1, 2, 3, 4, 5, 6, 7 or 8.

[0038] In some implementations, L 4 L 5 and L 6 Independently selected from single bonds, C 1-8 Alkylene, -C 1-4 Alkylene-SC 1-4 alkylene-, -C 1-4 Alkylene-SSC 1-4 alkylene- or -C 1-8 Alkylene-NR e -C 1-8 Alkylene -, wherein the alkylene is unsubstituted or converted by one or more OH, NH2, halogen, -NHC(=O)OC 8-26 Alkyl group, -NHC(=O)OC 8-26 alkenyl, -NHC(=O)-C 8-26 Alkyl group, -NHC(=O)-C 8-26 alkenyl, -C 1-8 Alkylene-C(=O)NH-C 1-8 Alkylene-NR e -C 1-8 Alkylene-OC(=O)-C 8-26 alkenyl, -C(=O)OC 8-26 Alkyl group, -C(=O)OC 8-26 alkenyl, -OC(=O)-C 8-26 Alkyl or -OC(=O)-C 8-26 Alkenyl substitution;

[0039] R e Selected from -C 1-8 Alkylene-OC(=O)-C(=O)-R f -C 1-8 Alkylene-C(=O)OC 8-26 Alkyl, -C 1-8 Alkylene-OC(=O)-C 8-26 Alkyl, -C 1-8 Alkylene-C(=O)OC 8-26 alkenyl, -C 1-8 Alkylene-OC(=O)-C 8-26 alkenyl, -C 1-8 Alkylene-OC(=O)-L b -C(=O)OC 8-26 Alkyl, -C 1-8Alkylene-OC(=O)-C 1-16 Alkylene-C(=O)-R f -C 1-8 Alkylene-OC(=O)-C 1-8 alkylene-(5-6-membered heteroaryl)-C 4-10 Alkyl, -C 1-8 Alkylene-OC(=O)NH-C 8-26 Alkyl, -C 1-8 Alkylene-NHC(=O)-C 8-26 Alkyl or -C 1-8 Alkylene-OC 8-26 alkyl;

[0040] R f Selected from

[0041] L b Selected from or -C 1-8 Alkylene-SSC 1-8 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens;

[0042] n is selected from 1, 2, 3, 4, 5, or 6.

[0043] In some implementations, L 4 L 5 and L 6 Independently selected from single bonds, C 1-6 Alkylene, -C 1-4 Alkylene-SC 1-4 alkylene-, -C 1-4 Alkylene-SSC 1-4 alkylene- or -C 1-6 Alkylene-NR e -C 1-6 Alkylene -, wherein the alkylene is unsubstituted or converted by one or more OH, NH2, halogen, -NHC(=O)OC 10-24 Alkyl group, -NHC(=O)OC 10-24 alkenyl, -NHC(=O)-C 10-24 Alkyl group, -NHC(=O)-C 10-24 alkenyl, -C 1-6 Alkylene-C(=O)NH-C 1-6 Alkylene-NR e -C 1-6 Alkylene-OC(=O)-C 10-24 alkenyl, -C(=O)OC 10-24 Alkyl group, -C(=O)OC10-24 alkenyl, -OC(=O)-C 10-24 Alkyl or -OC(=O)-C 10-24 Alkenyl substitution;

[0044] R e Selected from -C 1-6 Alkylene-OC(=O)-C(=O)-R f -C 1-6 Alkylene-C(=O)OC 10-24 Alkyl, -C 1-6 Alkylene-OC(=O)-C 10-24 Alkyl, -C 1-6 Alkylene-C(=O)OC 10-24 alkenyl, -C 1-6 Alkylene-OC(=O)-C 10-24 alkenyl, -C 1-6 Alkylene-OC(=O)-L b -C(=O)OC 10-24 Alkyl, -C 1-6 Alkylene-OC(=O)-C 6-12 Alkylene-C(=O)-R f -C 1-6 Alkylene-OC(=O)-C 1-6 alkylene-(5-membered heteroaryl)-C 4-10 Alkyl, -C 1-6 Alkylene-OC(=O)NH-C 10-24 Alkyl, -C 1-6 Alkylene-NHC(=O)-C 10-24 Alkyl or -C 1-6 Alkylene-OC 8-16 alkyl;

[0045] R f Selected from

[0046] L b Selected from or -C 1-6 Alkylene-SSC 1-6 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens;

[0047] n is selected from 1, 2, 3 or 4.

[0048] In some implementations, L 4 L 5 and L 6 Independently selected from single bonds, -CH2-, -CH2-CH2-,

[0049] In some implementations, G 1 G 2 and G 3 Independently selected from single bonds, -NR 3 -, -O-, -S-, -C(=O)-, -OC(=O)-, -OC(=O)O-, -C(=O)C(=O)O-, -NR 3 C(=O)-、-NR 3 C(=O)O-, 5 to 10 membered heterocyclic groups, -OC(=O)NR 3 -L a -C(=O)O-、-OC(=O)NR 3 -L a -NR 3 -C(=O)O-、-OC(=O)-L a -OC(=O)-NR 3 -、-NR 3 C(=O)-L a -C(=O)-NR 3 -、-NR 3 C(=O)OL a -C(=O)-NR 3 -or-C(=O)OL a -OC(=O)-NR 3 -

[0050] In some implementations, G 1 G 2 and G 3 Independently selected from single bonds, -NR 3 -, -O-, -S-, -C(=O)-, -OC(=O)-, -NR 3 C(=O)-、-NR 3 C(=O)O-, 5- to 8-membered heterocyclic groups, -OC(=O)NR 3 -L a -C(=O)O-、-OC(=O)NR 3 -L a -NR 3 -C(=O)O-、-OC(=O)-L a -OC(=O)-NR 3 -、-NR 3 C(=O)-L a -C(=O)-NR 3 -、-NR 3 C(=O)OL a-C(=O)-NR 3 -or-C(=O)OL a -OC(=O)-NR 3 -

[0051] In some implementations, G 1 G 2 and G 3 Independently selected from single bonds, -NH-, -O-, -S-, -C(=O)-, -OC(=O)-, -NHC(=O)-, -OC(=O)NH-,

[0052] In some implementations, G 4 G 5 and G 6 Independently selected from single bonds, -NR 3 -, -O-, -C(=O)-, -OC(=O)-, -OC(=O)O-, -C(=O)C(=O)O-, -NR 3 C(=O)-、-NR 3 C(=O)O-、-C(=O)-L a -C(=O)-、-OC(=O)-L a -C(=O)-、-C(=O)OL a -C(=O)NR 3 -、-C(=O)OL a -OC(=O)NR 3 -、-C(=O)-L a -C(=O)NR 3 -, 5-8 quinone heteroaryl or -C(=O)NR 3 -L a -OC(=O)C(=O)-.

[0053] In some implementations, G 4 G 5 and G 6 Independently selected from single bonds, -NR 3 -, -O-, -C(=O)-, -OC(=O)-, -C(=O)C(=O)O-, -NR 3 C(=O)-、-NR 3 C(=O)O-、-C(=O)-L a -C(=O)-、-OC(=O)-L a -C(=O), -C(=O)-L a -C(=O)NR 3 -, 5-6 quinone heteroaryl, -C(=O)-NR3 -C 1-8 Alkylene-NR e -C 1-8 Alkylene -OC(=O)C(=O)-.

[0054] In some implementations, G 4 G 5 and G 6 Independently selected from -O-, -C(=O)-, -OC(=O)-, -NHC(=O)-, -NHC(=O)O-,

[0055] In some implementations, each R 3 Independently selected from H and C 1-30 Alkyl, -C 1-10 Alkylene-OC(=O)-C 1-30 Alkyl, -C 1-10 Alkylene-OC(=O)-C 2-30 alkenyl, -C 1-10 Alkylene-C(=O)OC 1-30 Alkyl, -C 1-10 Alkylene-C(=O)OC 2-30 alkenyl or -C 1-10 Alkylene-OC(=O)-C 1-8 Alkylene-SC 1-8 Alkylene-C(=O)OC 1-30 alkyl.

[0056] In some implementations, each R 3 Independently selected from H and C 8-26 Alkyl, -C 1-8 Alkylene-OC(=O)-C 8-26 Alkyl, -C 1-8 Alkylene-OC(=O)-C 8-26 alkenyl, -C 1-8 Alkylene-C(=O)OC 8-26 Alkyl, -C 1-8 Alkylene-C(=O)OC 8-26 alkenyl or -C 1-8 Alkylene-OC(=O)-C 1-6 Alkylene-SC 1-6 Alkylene-C(=O)OC 8-26 alkyl.

[0057] In some implementations, each R 3 Independently selected from H and C 10-22 Alkyl, -C 1-6Alkylene-OC(=O)-C 10-22 Alkyl, -C 1-6 Alkylene-OC(=O)-C 10-22 alkenyl, -C 1-6 Alkylene-C(=O)OC 10-22 Alkyl, -C 1-6 Alkylene-C(=O)OC 10-22 alkenyl or -C 1-6 Alkylene-OC(=O)-C 1-4 Alkylene-SC 1-4 Alkylene-C(=O)OC 10-22 alkyl.

[0058] In some implementations, each L a Independently selected from single bonds, C 1-20 Alkylene, -[OCH2CH2] n -、-C 1-10 Alkylene-SSC 1-10 alkylene-, -C 1-10 Alkylene-SC 1-10 alkylene-, -C 1-10 Alkylene-C(=O)-C 1-10 alkylene- or -C 1-10 Alkylene-NR e -C 1-10 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens.

[0059] In some implementations, each L a Independently selected from single bonds, C 1-16 Alkylene, -[OCH2CH2] n -、-C 1-8 Alkylene-SSC 1-8 alkylene-, -C 1-8 Alkylene-SC 1-8 alkylene-, -C 1-8 Alkylene-C(=O)-C 1-8 alkylene- or -C 1-8 Alkylene-NR e -C 1-8 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens.

[0060] In some implementations, each L a Independently selected from single bonds, C 1-12 Alkylene, -[OCH2CH2] n -、-C 1-6 Alkylene-SSC1-6 alkylene-, -C 1-6 Alkylene-SC 1-6 alkylene-, -C 1-6 Alkylene-C(=O)-C 1-6 alkylene- or -C 1-6 Alkylene-NR e -C 1-6 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens.

[0061] In some implementations, each L a Independently selected from single bonds, -CH2-, -CH2CH2-,

[0062] In some implementation schemes, Selected from

[0063] In some implementation schemes, Selected from

[0064] In some implementation schemes, R 1 and R 2 Selected independently

[0065] C 1-30 Alkyl or C 2-30 Alkenyl group, wherein the alkyl or alkenyl group is unsubstituted or substituted by one or more OH, NH2 or halogen;

[0066] In some implementation schemes, R 1 and R 2 Selected independently

[0067] C 1-26 Alkyl or C 6-26 Alkenyl group, wherein the alkyl or alkenyl group is unsubstituted or substituted by one or more OH, NH2 or halogens.

[0068] In some implementation schemes, R 1 and R 2 Selected independently

[0069] C 1-24 Alkyl or C 8-24 Alkenyl group, wherein the alkyl or alkenyl group is unsubstituted or substituted by one or more OH, NH2 or halogens.

[0070] In some implementation schemes, R 1 and R 2 Selected independently

[0071] In some embodiments, the present invention provides a compound of formula (1) or a pharmaceutically acceptable salt thereof, said compound being selected from the following:

[0072]

[0073]

[0074]

[0075]

[0076] [Lipid carrier]

[0077] The present invention provides a lipid carrier comprising ionizable lipid molecules, polyethylene glycol lipid molecules, steroidal lipid molecules and auxiliary lipid molecules, wherein the ionizable lipid molecules include compounds represented by formula (1) above or pharmaceutically acceptable salts thereof.

[0078] In some embodiments, the ionizable lipid molecule is a compound represented by formula (1) above or a pharmaceutically acceptable salt thereof.

[0079] In some embodiments, the ionizable lipid molecule further comprises at least one of the following: (1-octylnonyl-8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]octanoate)SM-102, ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)ALC-0315, 1,2-dioleoyl-3-dimethylammonium-propane DODAP, 1,2-dioleoylalkoxy-3-dimethyl Amino-propane DODMA, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane DOBAQ, YSK05, Dlin-DMA, N,N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecen-1-yl-1,3-dioxolane-4-ethylamine Dlin-KC2-DMA, methyl 4-(N,N-dimethylamino)butyrate (dilinyl) ester Dlin-MC3-DMA, etc.

[0080] In some embodiments, the ionizable lipid molecule further includes at least one of the following: SM-102, ALC-0315, DODAP, DODMA, DOBAQ, YSK05, Dlin-DMA, Dlin-KC2-DMA, and Dlin-MC3-DMA.

[0081] In some embodiments, the ionizable lipid molecule further includes at least one of SM-102, ALC-0315, DODAP, and Dlin-DMA.

[0082] In some embodiments, the ionizable lipid molecule further includes at least one of SM-102 and ALC-0315.

[0083] In some embodiments, the polyethylene glycol lipid molecule is selected from at least one of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (PEG-DMG), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterol glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearate, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), and PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA).

[0084] In some embodiments, the polyethylene glycol lipid molecule is selected from at least one of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (PEG-DMG), and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE).

[0085] In some embodiments, the steroidal lipid molecule is selected from at least one of the following: alfalfa sterol, β-sitosterol, brassosterol, ergocalciferol, campesterol, cholesterol, coccidosterol, dehydrocholesterol, sterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, stigmasterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterol, phycosterol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid.

[0086] In some embodiments, the steroidal lipid molecule is selected from at least one of the following: cholesterol, cholesterol, ergocalciferol, dihydrocholesterol, phycosterol, taurine, and deoxycholic acid.

[0087] In some embodiments, the steroidal lipid molecule is selected from at least one of cholesterol, cholesterol, dihydrocholesterol, phycosterol, and deoxycholic acid.

[0088] In some implementations, the steroidal lipid molecule is selected from at least one of cholesterol and dihydrocholesterol.

[0089] In some embodiments, the auxiliary lipid molecule is selected from at least one of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dispalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dispalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE).

[0090] In some embodiments, the auxiliary lipid molecule is selected from at least one of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), and 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE).

[0091] In some embodiments, the auxiliary lipid molecule is selected from at least one of 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC) and 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE).

[0092] In some embodiments, the lipid carrier comprises, by molar percentage, 10% to 70% of ionizable lipid molecules (the compound represented by formula (1) above or a pharmaceutically acceptable salt thereof), 5% to 60% of steroidal lipid molecules, 1% to 60% of auxiliary lipid molecules and 1% to 30% of polyethylene glycol lipid molecules.

[0093] In some embodiments, the molar percentage of the compound represented by formula (1) or its pharmaceutically acceptable salt is 15% to 65%; or the molar percentage is 30% to 60%; or the molar percentage is 45% to 55%; or the molar percentage is 50%.

[0094] In some embodiments, the molar percentage of the steroidal lipid molecules is 10% to 50%; or the molar percentage is 25% to 45%; or the molar percentage is 30% to 40%; or the molar percentage is 38%.

[0095] In some embodiments, the molar percentage of the polyethylene glycol lipid molecules is 1% to 20%; or the molar percentage is 1% to 10%; or the molar percentage is 1% to 5%; or the molar percentage is 2%.

[0096] In some embodiments, the molar percentage of the auxiliary lipid molecule is 5% to 50%; or the molar percentage is 5% to 40%; or the molar percentage is 10% to 30%; or the molar percentage is 10%.

[0097] In some implementations, the molar ratio of ionizable lipid molecules, auxiliary lipid molecules, steroidal lipid molecules, and polyethylene glycol lipid molecules in the lipid carrier is 50:10:38:2, 45:10:42:3, 30:25:30:10, 46:15:40:3, 50:10:38.5:1.5, 50:10:37:3, 50:9:38:3, 39.4:39.4:19.7:1.5, etc.

[0098] [Nucleic Acid Lipid Nanoparticle Composition]

[0099] The present invention provides a nucleic acid lipid nanoparticle composition comprising the above-mentioned lipid carrier and at least one component selected from nucleic acids, small molecule drugs and protein peptides.

[0100] The present invention provides a nucleic acid lipid nanoparticle composition comprising the above-mentioned lipid carrier and nucleic acid.

[0101] In some embodiments, the nucleic acid is selected from at least one of DNA, mRNA, rRNA, siRNA, tRNA, microRNA, antisense nucleic acid, self-replicating RNA, and circular RNA.

[0102] In some implementations, the nucleic acid is mRNA.

[0103] In some embodiments, the nucleic acid is firefly luciferase mRNA, green fluorescent protein (GFP) mRNA, ovalbumin (OVA) mRNA, IL-2 protein mRNA, or H1N1 influenza virus mRNA.

[0104] In some embodiments, the aforementioned small molecule drug is selected from at least one of commonly used small molecule chemotherapeutic drugs in the art (such as doxorubicin, cisplatin, oxaliplatin, amphotericin B, paclitaxel, KRAS inhibitors, etc.).

[0105] In some embodiments, the aforementioned protein peptides are selected from at least one of commonly used peptide and protein drugs in the art, such as insulin, calcitonin, oxytocin, octreotide, leuprorelin, desmopressin, etc.

[0106] In some embodiments, the mass ratio of lipid carrier to nucleic acid in the nucleic acid lipid nanoparticle composition is 5:1 to 50:1; or the mass ratio is 10:1 to 30:1.

[0107] In some embodiments, the nucleic acid is mRNA, and the mass ratio of lipid carrier to nucleic acid in the nucleic acid lipid nanoparticle composition is 20:1 to 30:1; or the mass ratio is 20:1, 25:1 or 30:1.

[0108] In some embodiments, the particle size of the nucleic acid lipid nanoparticle composition is 30–500 nm; or the particle size is 30–200 nm.

[0109] In some implementations, the particle size can be approximately 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, etc.

[0110] In some embodiments, the encapsulation efficiency of nucleic acids in the nucleic acid lipid nanoparticle composition is greater than 50%. Exemplarily, the encapsulation efficiency can be about 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.

[0111] [Preparation method of nucleic acid-loaded lipid nanoparticles]

[0112] This invention provides a method for preparing lipid nanoparticles loaded with nucleic acids, comprising the following steps:

[0113] (A1) Ionizable lipid molecules (the compound represented by formula (1) or its pharmaceutically acceptable salt), polyethylene glycol lipid molecules, auxiliary lipid molecules and steroidal lipid molecules are mixed in the proportions described above and dissolved in a solvent to obtain an organic phase lipid solution;

[0114] (A2) Dissolve the nucleic acid in a buffer solution of appropriate pH to obtain an aqueous nucleic acid solution;

[0115] (A3) The organic phase lipid solution and the aqueous phase nucleic acid solution are uniformly mixed using a microfluidic device according to the mass ratio and a certain volume ratio described above to prepare a lipid nanoparticle solution loaded with nucleic acid.

[0116] In some embodiments, the solvent used to dissolve the lipid molecules in step (A1) is methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide, or N,N-dimethylformamide; or the solvent is ethanol, tetrahydrofuran, or acetone; or the solvent is ethanol.

[0117] In some embodiments, the buffer solution in step (A2) is an acetic acid / sodium acetate solution or a citric acid / sodium citrate solution; or the buffer solution is a citric acid / sodium citrate solution.

[0118] In some embodiments, the pH of the buffer solution in step (A2) is 3 to 9; or the pH of the buffer solution is 4 to 6; or the pH of the buffer solution is 5.

[0119] In some embodiments, the concentration of the buffer solution in step (A2) is 1 mM to 1 M; or the concentration of the buffer solution is 20 mM to 500 mM; or the concentration of the buffer solution is 100 mM.

[0120] In some embodiments, the volume ratio of the organic phase lipid solution to the aqueous phase nucleic acid solution in step (A3) is 1:1 to 1:10; or the volume ratio is 1:1 to 1:5; or the volume ratio is 1:3.

[0121] In some implementations, the microfluidic device in step (A3) can be a microfluidic device conventionally used in the art, such as INano. TM L / L+, Myanna or BT, Precision NanoSystems.

[0122] [Liposome Composition]

[0123] The present invention also provides a liposome composition comprising the above-described lipid carrier and at least one component selected from nucleic acids, small molecule drugs and protein peptides.

[0124] In some implementations, the nucleic acid is selected from at least one of DNA, mRNA, rRNA, siRNA, tRNA, microRNA, antisense nucleic acid, self-replicating RNA, and circular RNA.

[0125] In some embodiments, the aforementioned small molecule drug is selected from at least one of commonly used small molecule chemotherapeutic drugs in the art (such as doxorubicin, cisplatin, oxaliplatin, amphotericin B, paclitaxel, KRAS inhibitors, etc.).

[0126] In some embodiments, the aforementioned protein peptides are selected from at least one of commonly used peptide and protein drugs in the art, such as insulin, calcitonin, oxytocin, octreotide, leuprorelin, desmopressin, etc.

[0127] In some embodiments, in the above-described liposome composition, the mass ratio of the lipid carrier to the nucleic acid is 5:1 to 50:1; or, the mass ratio is 10:1 to 30:1; or, the mass ratio is 20:1, 25:1, or 30:1.

[0128] In some embodiments, in the above-described liposome composition, the mass ratio of the lipid carrier to the small molecule drug or protein peptide is 2:1 to 100:1; or, the mass ratio is 5:1 to 50:1; or, the mass ratio is 10:1 to 30:1; or, the mass ratio is 20.5:1.

[0129] [Preparation method of liposome composition]

[0130] This invention provides a method for preparing a liposome composition, comprising the following steps:

[0131] (A1) Ionizable lipid molecules (the compound represented by formula (1) or its pharmaceutically acceptable salt), polyethylene glycol lipid molecules, auxiliary lipid molecules and steroidal lipid molecules are mixed in the proportions described above and dissolved in a solvent to obtain an organic phase lipid solution;

[0132] (A2) Add the above organic phase lipid solution to a round-bottom flask, and then slowly evaporate the solvent using a rotary evaporator to form a uniform lipid film;

[0133] (A3) Disperse small molecule drugs or protein peptides in a solvent, add the resulting solution to a lipid film, and gently agitate until a uniform liposome suspension is formed.

[0134] (A4) The liposome suspension is poured into an ultrasonic processor for processing, and the size of the liposomes is further controlled by using a sieve with a pore size.

[0135] In some embodiments, the solvent used to dissolve the lipid molecules in step (A1) is chloroform, dichloromethane, methanol, ethanol, tetrahydrofuran, acetone, dimethyl sulfoxide, N,N-dimethylformamide, or a mixture of the above solvents; or the solvent is a mixture of chloroform and methanol.

[0136] The solvent in step (A3) is selected from distilled water, PBS buffer, citrate buffer, acetate buffer or phosphate buffer.

[0137] The ultrasonic processing frequency in step (A4) is 5 to 100 Hz, or the ultrasonic processing frequency is 60 Hz.

[0138] [Nucleic acid lipid nanoparticle formulations, liposome formulations, uses and treatment methods]

[0139] The present invention provides a nucleic acid lipid nanoparticle formulation comprising the above-mentioned nucleic acid lipid nanoparticle composition and pharmaceutically acceptable excipients.

[0140] The present invention provides a liposome formulation comprising the above-described liposome composition and pharmaceutically acceptable excipients.

[0141] The present invention also provides the use of the compound represented by formula (1) above or a pharmaceutically acceptable salt thereof, the lipid carrier above, the nucleic acid lipid nanoparticle composition above or the liposome composition above in the preparation of nucleic acid drugs, vaccines, polypeptide drugs and small molecule drugs.

[0142] The present invention also provides the use of the compound represented by formula (1) above or a pharmaceutically acceptable salt thereof, the lipid carrier, the nucleic acid lipid nanoparticle composition or the liposome composition above in the preparation of a medicament for treating or preventing diseases (e.g., inflammatory diseases, viral infections and cancer) in a subject.

[0143] The present invention also provides the compound represented by formula (1) above or a pharmaceutically acceptable salt thereof, the above lipid carrier, the above nucleic acid lipid nanoparticle composition or the above liposome composition for in vivo delivery of nucleic acid drugs, vaccines, polypeptide drugs and small molecule drugs.

[0144] The present invention also provides compounds represented by formula (1) above or pharmaceutically acceptable salts thereof, the above lipid carriers, the above nucleic acid lipid nanoparticle compositions or the above liposome compositions for treating or preventing diseases (e.g., inflammatory diseases, viral infections and cancer) in subjects.

[0145] The present invention also provides a method for in vivo delivery of nucleic acid drugs, vaccines, peptide drugs and small molecule drugs, the method comprising administering the above-mentioned nucleic acid lipid nanoparticle composition, the above-mentioned liposome composition, the above-mentioned nucleic acid lipid nanoparticle formulation or the above-mentioned liposome formulation to a subject in need.

[0146] The present invention also provides a method for treating or preventing diseases (e.g., inflammatory diseases, viral infections, and cancer) or conditions in a subject, the method comprising administering the above-described nucleic acid lipid nanoparticle composition, the above-described liposome composition, the above-described nucleic acid lipid nanoparticle formulation, or the above-described liposome formulation to a subject in need.

[0147] The term "inflammatory disease" includes autoimmune diseases, allergic diseases, and inflammatory conditions, such as those selected from arthritis, ankylosing spondylitis, inflammatory bowel disease, ulcerative colitis, gastritis, pancreatitis, Crohn's disease, celiac disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, rheumatic fever, gout, organ or transplant rejection, acute or chronic graft-versus-host disease, chronic allogeneic graft rejection, Bechtel's disease, uveitis, psoriasis, dermatitis, atopic dermatitis, dermatomyositis, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, Sjögren's syndrome, and blistering diseases (e.g., pemphigus vulgaris), antibody-mediated vasculitis syndromes including ANCA-associated vasculitis, purpura, and immune complex vasculitis (stage I or II cancer or infection). Allergic diseases may include contact dermatitis, celiac disease, asthma, hypersensitivity to house dust mites, pollen and related allergens, and beryllium poisoning.

[0148] The term "viral infection" includes, but is not limited to, retroviral infection, hepatitis virus infection, COVID-19 infection, Zika virus infection, dengue virus infection, etc.

[0149] The term "cancer" includes, but is not limited to, primary lung cancer (including non-small cell lung cancer and small cell lung cancer), metastatic lung cancer (lung metastasis of liver cancer, lung metastasis of breast cancer, lung metastasis of colon cancer, lung metastasis of melanoma, etc.), and cancers in other parts of the body (such as melanoma).

[0150] In some embodiments, the above-described nucleic acid lipid nanoparticle composition, liposome composition, nucleic acid lipid nanoparticle formulation, or liposome formulation is administered via one of the following routes: oral, intranasal, intravenous, intraperitoneal, intramuscular, intra-articular, intralesional, intratracheal, subcutaneous, or intradermal. In some embodiments, the above-described nucleic acid lipid nanoparticle composition, liposome composition, nucleic acid lipid nanoparticle formulation, or liposome formulation is administered, for example, via enteral or parenteral administration. In some embodiments, the nucleic acid lipid nanoparticle composition or nucleic acid lipid nanoparticle formulation is administered to the subject at a dose of about 0.001 mg / kg to about 10 mg / kg.

[0151] The present invention has the following beneficial effects:

[0152] 1. A lipid carrier containing the compound of formula (1) or a pharmaceutically acceptable salt thereof binds tightly to nucleic acid or small molecule drugs, thereby achieving high loading rate and stable protection of nucleic acid or small molecule drugs.

[0153] 2. The LNPs formed have good biocompatibility and are more stable; they can improve the efficiency of nucleic acid delivery in vivo, thus achieving efficient expression of nucleic acids.

[0154] 3. These lipid nanoparticles are suitable for nucleic acid delivery with different molecular weights and sequences, and have universality.

[0155] 4. The technology of this invention is simple to synthesize, the raw materials are inexpensive, and it is suitable for large-scale production. Attached Figure Description

[0156] Figure 1 This shows Basic LNP@mRNA Luc And the I-1LNP@mRNA of the present invention Luc I-2LNP@mRNA Luc I-3LNP@mRNA Luc I-4LNP@mRNA Luc Particle size distribution.

[0157] Figure 2 This demonstrates the use of Basic LNP@mRNA OVA And the I-1LNP@mRNA of the present invention OVA I-2LNP@mRNA OVA I-3LNP@mRNA OVA I-4LNP@mRNA OVA The proportion of OVA-H-2Kb positive cells after in vitro transfection of DC2.4 cells, where **p<0.01; ***p<0.001; ****p<0.0001.

[0158] Figure 3 This demonstrates the use of Basic LNP@mRNA Luc And the I-1LNP@mRNA of the present invention Luc I-2LNP@mRNA Luc I-3LNP@mRNA Luc I-4LNP@mRNA Luc The expression level of luciferase after intramuscular injection in the leg of mice was determined, with **p<0.01; ***p<0.001; ****p<0.0001.

[0159] Figure 4 This demonstrates the use of Basic LNP@mRNA Luc And the I-1LNP@mRNA of the present invention Luc I-2LNP@mRNA Luc I-3LNP@mRNA Luc I-4LNP@mRNA Luc The expression level of luciferase after intramuscular injection in the leg of large animals, where ***p<0.001; ****p<0.0001.

[0160] Figure 5 This demonstrates the use of Basic LNP@mRNA OVA And the I-1LNP@mRNA of the present invention OVA I-2LNP@mRNA OVA I-3LNP@mRNA OVA I-4LNP@mRNA OVA Tumor growth curves in mice after injection treatment.

[0161] Figure 6 This demonstrates the use of Basic LNP@mRNA OVA And the I-1LNP@mRNA of the present invention OVA I-2LNP@mRNA OVA I-3LNP@mRNA OVA I-4LNP@mRNA OVA Survival rate curves of mice after injection treatment.

[0162] Figure 7 This demonstrates the use of Basic LNP@mRNA OVA And the I-1LNP@mRNA of the present invention OVA I-2LNP@mRNA OVA I-3LNP@mRNA OVA I-4LNP@mRNA OVA The expression of IFN-γ in tumor tissues of mice after injection treatment was shown, with ***p<0.001; ****p<0.0001.

[0163] Figure 8 This demonstrates the use of Basic LNP@mRNA OVA And the I-1LNP@mRNA of the present invention OVA I-2LNP@mRNA OVA I-3LNP@mRNA OVA I-4LNP@mRNA OVA The expression of TNF-α in tumor tissues of mice after injection treatment was shown, with ***p<0.001; ****p<0.0001.

[0164] Figure 9 The particle size distribution of liposomes constructed using compound I-1 as an ionizable lipid component is shown. Detailed Implementation

[0165] I. Definition

[0166] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the related terms and laboratory procedures used herein are those widely used and standard practices in their respective fields. To better understand this disclosure, definitions and explanations of related terms are provided below.

[0167] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "containing" shall be understood to include the stated elements or components without excluding other elements or other components.

[0168] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0169] The term "pharmaceutically acceptable salt" refers to the salt of the compounds in this application, prepared by reacting a compound with a relatively non-toxic acid or base, as discovered in this application, with a specific substituent. When the compounds in this application contain relatively acidic functional groups, a base addition salt can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. When the compounds in this application contain relatively basic functional groups, an acid addition salt can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Certain specific compounds in this application contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

[0170] The pharmaceutically acceptable salts of this application can be synthesized from parent compounds containing an acid radical or a base using conventional chemical methods. Generally, the salts are prepared by reacting these compounds, in their free acid or base form, with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof.

[0171] The compounds disclosed herein may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers are included, such as enantiomers and diastereomers. The compounds containing asymmetric carbon atoms of this disclosure can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents. Racemic, diastereomer, and enantiomers are all included within the scope of this disclosure.

[0172] In this disclosure, This refers to the location where the substituents are bonded.

[0173] The term “optional” or “optionally” means that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0174] When the lower and upper limits of a numerical range are disclosed, any value or subrange falling within that range is specifically disclosed. In particular, each numerical range of parameters disclosed herein (e.g., in the form of "about a to b", or equivalently "above a and below b", or equivalently "about a to b") should be understood to encompass every value and subrange therein. For example, "C..." 1-4 "This should be understood as encompassing any subrange and every point value, such as C." 2-4 C 3-4 C 1-2 C 1-3 C 1-4 And so on, as well as C1, C2, C3, C4, etc. For example, "5-10 yuan" should be understood as covering any sub-range and each point value, such as 5-6 yuan, 5-7 yuan, 5-8 yuan, 5-9 yuan, 6-7 yuan, 6-8 yuan, etc., as well as 5, 6, 7, 8, 9, 10 yuan, etc.

[0175] When any variable (e.g., R) n When a substituent appears more than once in the composition or structure of a compound, its definition is independent in each case. Therefore, for example, if a group is substituted by 1-5 Rs, the group can optionally be substituted by up to 5 Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0176] The terms "substituted" or "substituted" refer to the substitution of one or more hydrogen atoms on a particular atom or group by a substituent, provided that the valence state of the particular atom or group is normal and the substituted compound is stable. When the substituent is an oxo group (i.e., =O), it means that two hydrogen atoms are substituted. Unless otherwise specified, the type and number of substituents can be arbitrary on a chemically feasible basis. Substituents can be selected from one, two or more of the following: deuterium, halogen groups, cyano, nitro, -C(=O)R, -C(=O)OR', -OC(=O)R", imide, amide, hydroxyl, substituted or unsubstituted amino, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted haloalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted aryloxy, substituted or unsubstituted heteroaryl, etc., but not limited to these.

[0177] The term "independently" means that at least two groups (or ring systems) in a structure with the same or similar value ranges can have the same or different meanings under specific circumstances. For example, if substituent X and substituent Y are independently hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl, then when substituent X is hydrogen, substituent Y can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl; similarly, when substituent Y is hydrogen, substituent X can be hydrogen, halogen, hydroxyl, cyano, alkyl, or aryl.

[0178] The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0179] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain or branched saturated hydrocarbon groups, which have the indicated number of carbon atoms. For example, the term "C..." 1-10 "Alkyl" refers to alkyl groups having 1 to 10 carbon atoms, including C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, and C1 alkyl. 10 Alkyl groups, examples of which include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, n-hexyl, 2-hexyl, 3-hexyl, etc. Alkyl groups may be optionally substituted or unsubstituted.

[0180] The term "alkylene" refers to a straight-chain or branched divalent saturated aliphatic hydrocarbon group to which the two groups (or segments) are attached can be connected to the same carbon atom or different carbon atoms. For example, the term "C" as used in this article... 1-10 "Alkylene" refers to an alkylene group having 1-10 carbon atoms (such as methylene, 1,1-ethylene, 1,2-ethylene, 1,2-propylene, 1,3-butylene, etc.). Alkylenes can be optionally substituted or unsubstituted.

[0181] The term "alkenyl" refers to a monovalent, straight-chain or branched alkane group consisting only of carbon and hydrogen atoms, containing at least one double bond, and connected to other segments via a single bond. This includes (but is not limited to) vinyl, propenyl, allyl, isopropenyl, butenyl, and isobutenyl groups. For example, "C..." 2-30 "Alkenyl" refers to a monovalent straight-chain or branched hydrocarbon group containing 2 to 30 carbon atoms and having at least one carbon-carbon double bond. Alkenyl groups can be optionally substituted or unsubstituted.

[0182] The term "heterocyclic group" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic, e.g., fused, bridged, or spirocyclic) non-aromatic group whose ring atoms consist of a carbon atom and at least one heteroatom selected from N, O, and S, wherein the S atom is optionally substituted to form S(=O), S(=O)2, or S(=O)(=NR). x ), R x Independently selected from H or C 1-4 Alkyl groups. If the valence requirement is met, the heterocyclic group can be connected to the rest of the molecule via any one ring atom. For example, the term "5-8 membered heterocyclic group" refers to a heterocyclic group having 5 to 8 ring atoms. Common heterocyclic groups include (but are not limited to) ethylene oxide, aziridine propane, aziridine butane, oxadiazine, tetrahydrofuranyl, dioxadienyl, pyrrolyl, pyrrolidone, imidazoyl, pyrazolyl, tetrahydropyranyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dithiaalkyl, or trithiaalkyl. The heterocyclic groups in this invention are optionally substituted with one or more substituents described in this invention.

[0183] The term "sub-heterocyclic group" refers to a divalent heterocyclic group. For example, the term "5- to 10-membered sub-heterocyclic group" as used herein refers to a divalent heterocyclic group having 5 to 10 ring atoms, such as... The heterocyclic group can be optionally substituted or unsubstituted.

[0184] The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated π-electron system. For example, the term "C..." 6-10 "Aryl" refers to an aryl group having 6 to 10 carbon atoms. Common aryl groups include (but are not limited to) phenyl, naphthyl, anthraceneyl, phenanthryl, acenaphthene, azulel, fluorenyl, indene, pyrene, etc. The aryl group in this invention may optionally be substituted by one or more substituents described in this invention.

[0185] The term "aryl" refers to a divalent aryl group, for example, the term "C" as used herein. 6-10 "Arylene" refers to a divalent aryl group having 6-10 carbon atoms, for example... etc. The aryl group can be optionally substituted or unsubstituted.

[0186] The term "heteroaryl" refers to an aromatic group that is a monocyclic or fused polycyclic ring with a conjugated π-electron system, whose ring atoms consist of a carbon atom and at least one heteroatom selected from N, O, and S. If the valence requirements are met, a heteroaryl can be attached to the rest of the molecule through any one of its ring atoms. For example, the term "5-10-membered heteroaryl" refers to a heteroaryl having 5 to 10 ring atoms. Common heteroaryl groups include (but are not limited to) thiophene, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl and its benzo[a] derivatives, pyrrolopyridinyl, pyrrolopyrazinyl, pyrazolopyridinyl, imidazopyridinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, purine, etc. The heteroaryl group in this invention is optionally replaced by one or more substituents described in this invention (such as halogens, C...). 1-6 Alkyl groups, etc., are substituted.

[0187] The term "hybrid aryl" refers to a divalent heteroaryl group. For example, the term "5- to 8-membered heteroaryl" as used herein refers to a divalent heteroaryl group having 5 to 8 ring atoms, such as... Etc. The heteroaryl group can be optionally substituted or unsubstituted.

[0188] The term "pharmaceuticalally acceptable excipient" refers to an excipient administered co-administered with the above-described nucleic acid lipid nanoparticle composition, which, to the extent of reasonable medical judgment, is suitable for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio. Examples include, but are not limited to: carriers, diluents, binders, absorbents, colorants, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, thermosensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0189] The tocopherols and tocotrienols mentioned in this article are all natural tocopherols and tocotrienols, including α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol, or δ-tocotrienol.

[0190] Unless otherwise specified, the divalent groups (e.g., L) mentioned herein 1 L 2 and L 3 G 1 G 2 and G 3 L 4 L 5 and L 6 G 4 G5 and G 6 The specific groups listed in the definition do not specify the connection direction. For example, -OC(=O)- includes both -OC(=O)- with O connected to the left group and -C(=O)O- with O connected to the right group.

[0191] II. Specific Implementation Examples

[0192] The present invention will be specifically described below through embodiments. These embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are within the scope of protection of the present invention.

[0193] All reagents and instruments used in the examples are commercially available conventional products. Unless otherwise specified, all conditions were performed under conventional conditions or conditions recommended by the manufacturer. The term "room temperature" as used in this invention refers to 20℃ ± 5℃. When used to modify a numerical value or range, the term "about" as used in this invention refers to the value or range and a range of errors acceptable to those skilled in the art, such as ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, etc.

[0194] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0195] The abbreviations used in this article have the following meanings:

[0196] abbreviation meaning abbreviation meaning DMF N,N'-Dimethylformamide DMAP 4-Dimethylaminopyridine EDC 1-Ethyl-(3-dimethylaminopropyl)carbodiimide DCM dichloromethane <![CDATA[NEt3]]> Triethylamine EA Ethyl acetate NHS N-hydroxysuccinimide <![CDATA[CuSO4·5H2O]]> Copper sulfate pentahydrate MeOH methanol

[0197] Example 1: Synthesis method of compound I-1

[0198]

[0199] Compound 1 (1.0 eq) and compound 2 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH:NEt3 = 100:10:1) to give compound 3 (yield: 48%). Compound 3 (1.0 eq) and compound 4 (1.0 eq) were dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH:CH3COOH = 100:10:1) to give compound 5 (yield: 71%). Compound 5 (1.0 eq) and compound 6 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was purified by column chromatography (DCM:MeOH = 8:1) to obtain compound I-1 (yield: 60%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 3.33–3.25 (m, 3H), 2.77–2.62 (m, 5H), 2.57–2.50 (t, 2H), 2.44–2.35 (m, 4H), 2.27–2.17 (m, 8H), 2.17 (s, 3H), 1.74–1.66 (m, 4H), 1.66–1.61 (m, 5H), 1.61–1.45 (m, 6H), 1.45–1.08 (m, 36H), 1.07–0.97 (m, 2H), 0.94–0.79 (m, 15H). HRMS: m / z calculated value: 1746.4621 (M+H) + ), Measured value: 1746.4634.

[0200] Example 2: Synthesis method of compound I-2

[0201]

[0202] Compounds 8 (1.0 eq) and 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water, the organic layer was collected, and the solvent was evaporated under reduced pressure to give compound 10 (yield: 90%). Compounds 11 (1.0 eq) and 12 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH:NEt3 = 100:10:1) to give compound 13 (yield: 60%). Compounds 13 (1.0 eq) and 10 (2.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water, the organic layer was collected, 20% TFA (1.0 eq) was added dropwise, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water, the organic layer was collected, and the solvent was evaporated under reduced pressure to give compound 14 (yield: 90%). Compounds 15 (1.0 eq) and 16 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound 17 (yield: 48%). Compounds 14 (1.0 eq) and 17 (2.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound I-2 (yield: 47%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 4.31–4.15 (m, 3H), 4.10–3.99 (m, 8H), 2.98–2.45 (m, 20H), 2.22 (s, 6H), 2.19 (s, 6H), 2.17 (s, 6H), 1.96–1.84 (ddd, J = 13.6, 7.5, 5.2Hz, 2H), 1.75–0.96 (m, 114H), 0.93–0.77 (m, 36H). HRMS: m / z calculated value: 1895.5197 (M+H) + ), Measured value: 1895.5193.

[0203] Example 3: Synthesis method of compound I-3

[0204]

[0205] Compound 19 (4.0 eq) and compound 20 (3.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH:CH3COOH = 10:1) to give compound 21 (yield: 53%). Compound 21 (1.0 eq) and compound 22 (2.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound 23 (yield: 47%). Compounds 23 (1.0 eq) and 24 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound 25 (yield: 51%). Compounds 25 (1.0 eq), 6 (1.0 eq), and 26 (1.0 eq) were dissolved in DCM, and NEt3 (1.0 eq) was added dropwise. After the reaction was complete, the mixture was purified by column chromatography (DCM:EA = 5:1) to give compound I-3 (yield: 46%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 4.42–4.34 (t, 2H), 4.31–4.24 (t, 6H), 4.09–4.03 (t, 4H), 3.98–3.79 (m, 2H), 3.02–2.92 (m, 8H), 2.87–2.51 (m, 30H), 2.22–2.19 (s, 3H), 2.19–2.17 (s, 3H), 2.16–2.13 (s, 3H), 1.72–0.97 (m, 10⁷H), 0.92–0.78 (m, 24H). HRMS: m / z calculated value: 1874.3417 (M+H) + ), Measured value: 1874.3409.

[0206] Example 4: Synthesis method of compound I-4

[0207]

[0208] Compounds 28 (1.0 eq) and 16 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 30:1) to give compound 29 (yield: 59%). Compounds 30 (1.0 eq) and 31 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 25:1) to give compound 32 (yield: 59%). Compounds 32 (2.0 eq) and 33 (1.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 15:1) to give compound 34 (yield: 48%). Compound 34 (2.0 eq) and compound 29 (1.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was completed, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound I-4 (yield: 48%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 4.15–3.78 (m, 10H), 2.88–2.60 (m, 12H), 2.45–2.39 (t, 4H), 2.36–2.28 (m, 12H), 2.23 (s, 6H), 2.20 (s, 6H), 2.17 (s, 6H), 2.01–0.96 (m, 167H), 0.92–0.78 (m, 37H). HRMS: m / z calculated value: 2242.8749 (M+H) + ), Measured value: 2242.5933.

[0209] Example 5: Synthesis method of compound I-5

[0210]

[0211] Compound 6 (1.0 eq) and compound 36 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 37 (yield: 64%). Compound 37 (1.0 eq) and compound 38 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 25:1) to give compound 39 (yield: 49%). Compound 1 (1.0 eq) and compound 38 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 25:1) to give compound 40 (yield: 76%). Compounds 41 (2.0 eq), 40 (2.0 eq), and 39 (1.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was completed, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound I-5 (yield: 49%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 4.33–4.24 (q, 6H), 3.59–3.51 (m, 4H), 3.32–3.26 (m, 2H), 3.04–2.88 (m, 8H), 2.79–2.56 (m, 12H), 2.49–2.26 (m, 6H), 2.23–2.20 (s, 6H), 2.20–2.17 (s, 6H), 2.16–2.13 (s, 6H), 1.98–1.80 (m, 6H), 1.74–0.97 (m, 130H), 0.94–0.77 (m, 36H). HRMS: m / z calculated value: 2033.7333 (M+H) + ), Measured value: 2033.7408.

[0212] Example 6: Synthesis of Compound I-6

[0213]

[0214] Compound 16 (1.0 eq) and compound 43 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound 44 (yield: 64%). Compound 16 (1.0 eq) and compound 45 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound 46 (yield: 64%). Compounds 46 (1.0 eq), 47 (1.0 eq), and 44 (1.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 48 (yield: 39%). Compounds 48 (1.0 eq) and 45 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:EA = 5:1) to give compound I-6 (yield: 62%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 6.84–6.75 (d, 2H), 5.42–5.30 (m, 4H), 5 .16–5.02(m,6H),4.20–3.86(m,6H),2.85–2.60(m,14H),2.56–2.44(t,2H ),2.34–2.27(m,6H),2.25–2.22(d,6H),2.20–2.18(d,6H),2.16–1.83(m, 30H),1.81–1.16(m,68H),0.95–0.86(t,3H).HRMS:m / z calculated value:1537.1426(M+H + ), Measured value: 1537.1464.

[0215] Example 7: Synthesis of Compound I-7

[0216]

[0217] Compounds 50 (1.0 eq) and 51 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 52 (yield: 43%). Compounds 52 (1.0 eq) and 53 (1.0 eq) were dissolved in DMF, and CuSO4·5H2O (1.0 eq) and ascorbic acid (3.0 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water, and the organic layer was collected. The solvent was evaporated under reduced pressure to give compound 54 (yield: 66%). Compounds 54 (1.0 eq) and 55 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 56 (yield: 47%). Compound 56 (1.0 eq) and compound 12 (L-lysine, 1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH:NEt3 = 100:10:1) to give compound 57 (yield: 57%). Compound 57 (1.0 eq) and compound 58 (1.0 eq) were dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound I-7 (yield: 48%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 7.48 (s, 1H), 6.72 (s, 1H), 5.38–5.27 (m, 4H), 4.36–4.04 (m, 9H), 3.15–2.61 (m, 16H), 2.38–1.83 (m, 25H), 1.82–0.95 (m, 85H), 0.94–0.77 (m, 21H). HRMS: m / z calculated value: 1554.2492 (M+H) + ), Measured value: 1554.2389.

[0218] Example 8: Synthesis of Compound I-8

[0219]

[0220] Compound 15 (2.0 eq) and compound 60 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 61 (yield: 39%). Compound 61 (2.0 eq) and compound 12 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 62 (yield: 54%). Compound 62 (2.0 eq) and compound 58 (2.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound I-8 (yield: 54%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 5.40–5.22 (m, 4H), 4.32–4.16 (m, 5H), 3.22–2.49 (m, 22H), 2.29–2.12 (m, 22H), 2.06–1.86 (m, 10H), 1.83–1.10 (m, 97H), 1.09–0.96 (m, 4H), 0.93–0.77 (m, 30H). HRMS: m / z calculated value: 1844.4989 (M+H) + ), Measured value: 1844.4931.

[0221] Example 9: Synthesis of Compound I-9

[0222]

[0223] Compounds 64 (3.0 eq) and 65 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 66 (yield: 60%). Compounds 66 (1.0 eq) and 67 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 68 (yield: 70%). Compounds 68 (1.0 eq) and 15 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound I-9 (yield: 70%). 1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 6.78–6.68 (s, 4H), 5.39–5.28 (m, 6H), 5.15–5.05 (m,3H),4.33–4.24(s,8H),4.20–4.06(m,10H),2.96–2.62(m,6H),2.22–2.20(s,3H) ,2.19–2.18(s,3H),2.16–2.14(s,3H),2.14–1.85(m,25H),1.80–1.73(m,5H),1.67–1.59(m,18H),1.41–1.23(m,64H),0.92–0.86(m,9H). HRMS: m / z calculated value: 1628.2423(M+H) + ), Measured value: 1628.2567.

[0224] Example 10: Synthesis of Compound I-10

[0225]

[0226] Compound 70 (1.0 eq) and compound 64 (3.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 71 (yield: 67%). Compound 71 (1.0 eq) and compound 48 (3.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound I-10 (yield: 60%). 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 6.83–6.74 (q, 1H), 5.42–5.24 (m, 8H), 4.15–3.86 (m,12H),3.56–3.49(s,8H),3.30–3.21(s,4H),2.86–2.60(m,6H),2.55–2.45(t,J=6 0Hz, 2H), 2.36–2.27 (m, 6H), 2.25–2.21 (s, 3H), 2.21–2.18 (d, J = 0.8Hz, 3H), 2.10–1.86 (m, 17H), 1.79–0.95 (m, 133H), 0.94–0.79 (m, 24H). HRMS: m / z calculated value: 2035.7126 (M+H) + ), Measured value: 2035.7264.

[0227] Example 11: Synthesis of Compound I-11

[0228]

[0229] Compound 6 (1.0 eq) and compound 4 (1.0 eq) were dissolved in DCM, and EDC (4.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 73 (yield: 58%). Compound 73 (1.0 eq) and compound 2 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (4.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 74 (yield: 64%). Compounds 74 (1.0 eq) and 75 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (4.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound I-11 (yield: 57%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 3.32–3.23 (m, 6H), 2.79–2.62 (m, 10H), 2.57–2.50 (t, 4H), 2.44–2.36 (m, 8H), 2.27–2.11 (m, 24H), 1.95–1.87 (m, 2H), 1.72–0.97 (m, 90H), 0.94–0.77 (m, 27H). HRMS: m / z calculated value: 1620.3213 (M+H) + ), Measured value: 1620.3298.

[0230] Example 12: Synthesis of Compound I-12

[0231]

[0232] Compounds 22 (1.0 eq) and 20 (1.0 eq) were dissolved in DCM, and EDC (4.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 77 (yield: 57%). Compounds 77 (3.0 eq) and 19 (1.0 eq) were dissolved in DCM, and EDC (4.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 78 (yield: 42%). Compounds 78 (1.0 eq), 26 (1.0 eq), and 6 (1.0 eq) were dissolved in DCM, and NEt3 (1.0 eq) was added dropwise. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound I-12 (yield: 64%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 4.41–4.34 (t, 2H), 4.31–4.25 (t, 6H), 4.10–4.03 (t, 6H), 3.02–2.94 (m, 8H), 2.88–2.52 (m, 30H), 2.22–2.19 (s, 3H), 2.19–2.17 (s, 3H), 2.16–2.13 (s, 3H), 1.73–0.96 (m, 87H), 0.93–0.77 (m, 21H). HRMS: m / z calculated value: 1706.1539 (M+H) + ), Measured value: 1706.1601.

[0233] Example 13: Synthesis of Compound I-13

[0234]

[0235] Compound 80 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the solvent was evaporated under reduced pressure to give compound 81 (yield: 90%). Compound 81 (1.0 eq) and compound 12 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the solvent was evaporated under reduced pressure to give compound 82 (yield: 70%). Compound 17 (2.0 eq) and compound 83 (1.0 eq) were dissolved in acetonitrile, potassium carbonate (1.0 eq) was added, and the mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 84 (yield: 56%). Compound 84 (1.0 eq) and compound 82 (1.0 eq) were dissolved in DCM, and EDC (4.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound I-13 (yield: 58%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 4.33–4.24 (t, 2H), 4.23–4.15 (m, 1H), 4.09–4.03 (m, 8H), 2.97–2.90 (m, 2H), 2.88–2.79 (m, 4H), 2.76–2.56 (m, 10H), 2.54–2.48 (t, 4H), 2.23 (s, 6H), 2.20 (s, 6H), 2.17 (s, 6H), 1.94–1.87 (m, 2H), 1.73–0.96 (m, 118H), 0.92–0.77 (m, 30H). HRMS: m / z calculated value: 1867.4884 (M+H) + ), Measured value: 1867.4884.

[0236] Example 14: Synthesis of Compound I-14

[0237]

[0238] Compound 22 (2.0 eq) and compound 31 (1.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound 86 (yield: 64%). Compound 86 (2.0 eq) and compound 33 (1.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound 87 (yield: 53%). Compound 87 (2.0 eq) and compound 17 (1.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound I-14 (yield: 53%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 4.15–4.03 (m, 6H), 3.98–3.91 (t, 4H), 2.87–2.79 (m, 4H), 2.77–2.62 (m, 8H), 2.44–2.38 (m, 4H), 2.35–2.27 (m, 12H), 2.23 (s, 6H), 2.20 (s, 6H), 2.17 (s, 6H), 1.95–1.87 (m, 2H), 1.81–0.96 (m, 136H), 0.92–0.79 (m, 30H). HRMS: m / z calculated value: 2074.6871 (M+H) + ), Measured value: 2074.7503.

[0239] Example 15: Synthesis of Compound I-15

[0240]

[0241] Compound 89 (1.0 eq) and compound 16 (3.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound 90 (yield: 46%). Compound 91 (1.0 eq), compound 90 (2.0 eq), and compound 17 (2.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:EA = 5:1) to give compound I-15 (yield: 49%). 1¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 4.15–4.04 (m, 8H), 2.90–2.62 (m, 12H), 2.60–2.57 (s, 8H), 2.56–2.44 (m, 16H), 2.36–2.27 (p, 2H), 2.23–2.20 (s, 6H), 2.20–2.18 (s, 6H), 2.17–2.14 (s, 6H), 1.95–1.86 (m, 2H), 1.71–0.97 (m, 158H), 0.93–0.78 (m, 36H). HRMS: m / z calculated value: 2242.9477 (M+H) + ), Measured value: 2242.9507.

[0242] Example 16: Synthesis of Compound I-16

[0243]

[0244] Compound 16 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 6 h. After the reaction was complete, the mixture was washed with water, and the organic layer was collected. Then, compound 93 (1.0 eq) was added to give compound 94 (yield: 66%). Compound 47 (1.0 eq), compound 94 (2.0 eq), and compound 17 (2.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:EA = 5:1) to give compound 95 (yield: 50%). Compound 47 (1.0 eq), compound 32 (2.0 eq), and compound 17 (2.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:EA = 5:1) to give compound 96 (yield: 42%). Compounds 50 (1.0 eq) and 97 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH:NEt3 = 100:10:1) to give compound 98 (yield: 43%). Compounds 98 (1.0 eq), 95 (1.0 eq), 9 (3.0 eq), and 96 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:EA = 5:1) to give compound I-16 (yield: 39%). 1H NMR(400MHz, CDCl3,293K)δ(ppm):4.31–4.24(t,6H),4.14–3.79(m,24H),3.20– 3.11(q,4H),3.02–2.96(t,6H),2.89–2.80(m,6H),2.76–2.62(m,12H),2.54–2. 47(t,6H),2.38–2.27(m,14H),2.23–2.20(s,9H),2.20–2.17(s,9H),2.05–1.85 (m,7H),1.78–0.97(m,222H),0.95–0.78(m,48H).HRMS:m / z calculated value:3647.8792(M+H + ), Measured value: 3647.8898.

[0245] Example 17: Synthesis of Compound I-17

[0246]

[0247] Compound 6 (1.0 eq) and compound 100 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 101 (yield: 40%). Compound 102 (1.0 eq) and compound 100 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 103 (yield: 64%). Compounds 101 (1.0 eq), 50 (1.0 eq), and 103 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound 104 (yield: 41%). Compounds 104 (1.0 eq) and 105 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 106 (yield: 49%). Compound 106 (2.0 eq) and compound 107 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was purified by column chromatography (DCM:EA = 5:1) to obtain compound I-17 (yield: 45%). 1¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 4.33–4.19 (m, 16H), 4.03–3.72 (m, 8H), 3.04–2.86 (m, 32H), 2.83–2.57 (m, 24H), 2.39–2.34 (s, 6H), 2.22–2.20 (s, 6H), 2.20–2.17 (s, 6H), 1.94–1.87 (M, 2H), 1.72–0.96 (m, 10²H), 0.92–0.78 (m, 36H). HRMS: m / z calculated value: 2682.5755 (M+H) + ), Measured value: 2682.5629.

[0248] Example 18: Synthesis of Compound I-18

[0249]

[0250] Compounds 24 (1.0 eq) and 109 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 110 (yield: 54%). Compounds 110 (1.0 eq) and 16 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:EA = 5:1) to give compound 111 (yield: 54%). Compounds 45 (1.0 eq) and 112 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:EA = 5:1) to give compound 113 (yield: 78%). Compounds 114 (1.0 eq) and 83 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:EA = 5:1) to give compound 115 (yield: 78%). Compounds 115 (1.0 eq) and 111 (1.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 15:1) to give compound 116 (yield: 72%). Compounds 116 (1.0 eq) and 113 (1.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound 117 (yield: 72%). Compounds 117 (3.0 eq) and 9 (3.0 eq) were dissolved in DCM and stirred at room temperature for 6 h. After the reaction was complete, the mixture was washed with water, and the organic layer was collected. Compound 118 (1.0 eq) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH:NEt3 = 100:10:1) to give compound 119 (yield: 40%). Compounds 119 (1.0 eq) and 120 (2.0 eq) were dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. After the reaction was complete, the mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound I-18 (yield: 43%). 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 6.86–6.74 (t, 3H), 6.15–6.07 (t, 3H), 5.23–5.03 (m, 12H),4.33–4.22(t,6H),4.15–3.77(m,48H),3.72–3.62(m,24H),3.22–3.08(m,12H),2. 97–2.89(t, 6H), 2.84–2.45(m, 54H), 2.34–2.27(t, 6H), 2.24–2.21(s, 9H), 2.20–2.18(s, 9H), 2.18–1.84(m, 45H), 1.81–1.55(m, 90H), 1.51–1.20(m, 222H), 0.92–0.85(m, 26H). HRMS: m / z calculated value: 5556.3061(M+H) + ), Measured value: 5556.3437.

[0251] Example 19: Synthesis of Compound I-19

[0252]

[0253] Compound 60 (1.0 eq) and compound 122 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water, and the organic phase was collected to give compound 123 (yield: 76%). Compound 123 (1.0 eq), compound 26 (1.0 eq), and compound 124 (1.0 eq) were dissolved in DCM, and NEt3 was added dropwise. The mixture was stirred at room temperature for 12 h to give compound 125 (yield: 63%). Compound 125 (1.0 eq) and compound 126 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 127 (yield: 54%). Compound 127 (1.0 eq) was dissolved in DCM, and 10% TFA was added at 0°C. The mixture was stirred at room temperature for 3 h. After the reaction was complete, the mixture was washed with water and the organic phase was collected to give compound 128 (yield: 78%). Compound 128 (2.0 eq) and compound 100 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound I-19 (yield: 67%). 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 6.79–6.73 (t, 2H), 5.49–5.26 (m, 18H), 5.15–5.00 (m, 6H), 4.41–4.23 (m, 8H), 3.03–2.86 (m, 16H), 2.79–2.56 ( m,24H),2.17–2.14(s,6H),2.14–1.78(m,43H),1.75–1.68(q,4H),1.66– 1.58(m,32H),1.52–1.43(m,4H),1.39–1.18(m,18H),0.92–0.86(t,6H). HRMS: m / z calculated value: 2060.3237 (M+H) + ), Measured value: 2060.3264.

[0254] Example 20: Synthesis of Compound I-20

[0255]

[0256] Compound 130 (2.0 eq) and compound 50 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 131 (yield: 60%). Compound 131 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the solvent was evaporated under reduced pressure to give compound 132 (yield: 91%). Compound 132 (1.0 eq) and compound 60 (1.0 eq) were dissolved in DCM, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the solvent was evaporated under reduced pressure to give compound 133 (yield: 67%). Compound 133 (1.0 eq) and compound 9 (2.0 eq) were dissolved in DCM, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the solvent was evaporated under reduced pressure to give compound 134 (yield: 76%). Compound 134 (1.0 eq) and compound 135 (2.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was completed, the solvent was evaporated under reduced pressure to give compound I-20 (yield: 91%). 1¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 5.21–5.13 (m, 3H), 4.98–4.91 (m, 1H), 4.53–4.42 (m, 2H), 4.27–4.13 (m, 2H), 3.76–3.55 (m, 180H), 3.36 (s, 3H), 2.88–2.58 (m, 12H), 2.25–2.23 (s, 6H), 2.21–2.18 (s, 6H), 2.15–2.13 (s, 6H), 2.11–0.98 (m, 55H), 0.86–0.80 (m, 12H). HRMS: m / z calculated value: 3513.7162 (M+H) + ), Measured value: 3513.7848. 1 H NMR(500MHz,Chloroform-d)δ5.44–5.32(m,8H),4.33–4.18(dt,J=17.9,6.2Hz,1 8H),3.18–3.10(q,J=5.9Hz,6H),3.04–2.92(m,18H),2.88–2.52(m,30H),2.37–2. 27(t,J=8.5Hz,8H),2.23–2.20(s,12H),2.20–2.16(s,12H),2.08–1.99(m,8H),1 .97–1.87(ddd,J=13.7,7.6,5.2Hz,4H),1.81–0.97(m,170H),0.93–0.77(m,54H).

[0257] Example 21: Synthesis of Compound I-21

[0258]

[0259] Compounds 137 (1.0 eq) and 44 (2.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 138 (yield: 43%). Compounds 138 (1.0 eq) and 17 (2.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound I-21 (yield: 38%). 1¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 5.21–5.13 (m, 3H), 4.98–4.91 (m, 1H), 4.53–4.42 (m, 2H), 4.27–4.13 (m, 2H), 3.76–3.55 (m, 180H), 3.36 (s, 3H), 2.88–2.58 (m, 12H), 2.25–2.23 (s, 6H), 2.21–2.18 (s, 6H), 2.15–2.13 (s, 6H), 2.11–0.98 (m, 55H), 0.86–0.80 (m, 12H). HRMS: m / z calculated value: 2222.7912 (M+H) + ), Measured value: 2222.7892. 1 H NMR(500MHz,Chloroform-d)δ5.45–5.29(m,8H),5.15–5.03(m,6H),4.11–4.02(t,J=6.2Hz,4H), 3.98–3.90(t,J=6.2Hz,4H),2.88–2.80(td,J=8.8,1.0Hz,4H),2.77–2.61(m,16H),2.53–2.45(m, 4H),2.39–2.27(m,20H),2.23–2.20(s,6H),2.19–2.17(s,6H),2.17–1.84(m,40H),1.81–1.72(d dd,J=13.7,7.5,5.3Hz,2H),1.68–1.56(m,36H),1.52–1.18(m,74H),0.93–0.85(t,J=6.3Hz,6H).

[0260] Example 22: Synthesis of compound I-22

[0261]

[0262] Compounds 140 (1.0 eq), 46 (1.0 eq), and 44 (2.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 141 (yield: 43%). Compounds 141 (3.0 eq) and 142 (1.0 eq) were dissolved in DCM, and EDC (4.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound I-22 (yield: 45%). 1H NMR (400MHz, CDCl3, 293K) δ (ppm): 6.82–6.75 (m, 3H), 5.43–5.31 (m, 12H), 5.19–5. 01(m,9H),4.16–4.00(m,12H),3.98–3.90(t,6H),3.69–3.60(s,6H),2.85–2.57(m ,24H), 2.37–2.27(m,24H), 2.24–2.21(s,9H), 2.21–2.18(d,9H), 2.16–1.84(m,52H), 1.80–1.72(m,3H), 1.67–1.55(m,60H), 1.51–1.18(m,106H), 0.91–0.86(m,9H). HRMS: m / z calculated value: 3354.5579(M+H) + ), Measured value: 3355.0977.

[0263] Example 23: Synthesis of compound I-23

[0264]

[0265] Compound 56 (3.0 eq) and compound 144 (1.0 eq) were dissolved in DCM, and EDC (4.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound I-23 (yield: 45%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 7.50–7.36 (s, 6H), 6.74–6.59 (q, 6H), 4.35–4.10 (m, 48H), 3.40–3.31 (s, 12H), 3.07–2.93 (m, 36H), 2.89–2.60 (m, 72H), 2.38–2.14 (m, 60H), 2.02–1.83 (m, 18H), 1.81–0.96 (m, 210H), 0.93–0.75 (m, 90H). HRMS: m / z calculated value: 2940.0953 (M+H). + ), Measured value: 2940.0865.

[0266] Example 24: Synthesis of compound I-24

[0267]

[0268] Compounds 128 (2.0 eq) and 146 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the solution was purified by column chromatography (DCM:MeOH = 10:1) to give compound 147 (yield: 50%). Compounds 147 (1.0 eq) and 148 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the solution was purified by column chromatography (DCM:MeOH = 10:1) to give compound 149 (yield: 54%). Compounds 149 (1.0 eq) and 9 (1.0 eq) were dissolved in DCM, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the solvent was evaporated under reduced pressure to give compound 150 (yield: 71%). Compound 150 (2.0 eq) and compound 151 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the solvent was evaporated under reduced pressure to give compound 152 (yield: 56%). Compound 152 (1.0 eq), compound 9 (1.0 eq), and compound 153 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the solvent was evaporated under reduced pressure to give compound I-24 (yield: 61%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 6.88–6.83 (d, 4H), 5.47–5.30 (m, 36H), 5.23–4.90 (m, 16H), 4.43–4.03 (m, 28H), 3.20–3.09 (m, 14H), 3.02–2.92 (m, 16H), 2.78–2.44 (m, 48H), 2.37–2.27 (m, 12H), 2.25–2.22 (s, 12H), 2.21–2.16 (d, 12H), 2.16–1.18 (m, 279H), 0.96–0.84 (m, 18H). HRMS: m / z calculated value: 5160.6786 (M+H) + ), Measured value: 5160.9791.

[0269] Example 25: Synthesis of Compound I-25

[0270]

[0271] Compound 95 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the solvent was evaporated under reduced pressure to give compound 155 (yield: 82%). Compound 155 (1.0 eq) and compound 156 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the solvent was evaporated under reduced pressure to give compound I-25 (yield: 62%).1 H NMR (400MHz, CDCl3, 293K) δ (ppm): 4.15–4.01 (m, 12H), 3.34–3.27 (m, 4H), 3.19–3.11 (q, 4H), 2.88–2.61 (m, 12H), 2.53–2.47 ( t,4H),2.35–2.27(t,8H),2.24–2.20(s,6H),2.20–2.17(s,6H),2.07–1.85(m,6H),1.80–0.96(m,138H),0.93–0.78(m,30H). HRMS:m / z calculated value:2222.8083(M+H + ), Measured value: 2222.8976.

[0272] Example 26: Synthesis of Compound I-26

[0273]

[0274] Compound 73 (1.0 eq) and compound 2 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 8:1) to give compound 158 (yield: 43%). Compound 158 (1.0 eq) and compound 75 (1.0 eq) were dissolved in DCM, and NHS (1.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to give compound 159 (yield: 53%). Compound 159 (2.0 eq) and compound 9 (2.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water to obtain the organic phase. Compound 160 (1.0 eq) was added and dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 10:1) to obtain compound I-26 (yield: 45%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 4.24–4.15 (t, 4H), 3.81–3.62 (m, 8H), 3.34–3.23 (m, 12H), 2.82–2.63 (m, 16H), 2.57–2.36 (m, 12H), 2.29–2.09 (m, 26H), 1.97–1.86 (m, 2H), 1.77–0.96 (m, 119H), 0.94–0.75 (m, 30H). HRMS: m / z calculated value: 2264.8301 (M+H) + ), Measured value: 2264.8483.

[0275] Example 27: Synthesis of Compound I-27

[0276]

[0277] Compounds 95 (1.0 eq) and 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and the organic phase was collected. Compound 97 (1.0 eq) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 8:1) to give compound 162 (yield: 45%). Compounds 162 (3.0 eq) and 163 (1.0 eq) were dissolved in DCM, and EDC (2.0 eq) and DMAP (0.1 eq) were added. After the reaction was complete, the mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound I-27 (yield: 48%). 1 ¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 4.19–3.95 (m, 30H), 3.19–3.09 (q, 6H), 2.89–2.62 (m, 18H), 2.53–2.42 (t, 6H), 2.35–2.28 (t, 12H), 2.24–2.20 (s, 9H), 2.20–2.17 (s, 9H), 2.05–1.85 (m, 9H), 1.78–0.97 (m, 213H), 0.93–0.77 (m, 45H). HRMS: m / z calculated value: 3563.7853 (M+H) + ), Measured value: 3564.0947.

[0278] Example 28: Synthesis of Compound I-28

[0279]

[0280] Compound 17 (2.0 eq) and compound 91 (1.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound 165 (yield: 56%). Compound 165 (2.0 eq) and compound 166 (1.0 eq) were dissolved in acetonitrile, and potassium carbonate (1.0 eq) was added. The mixture was stirred at 60 °C for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to give compound I-28 (yield: 64%). 1¹H NMR (400MHz, CDCl₃, 293K) δ (ppm): 4.11–4.04 (t, 4H), 3.48–3.39 (t, 8H), 2.88–2.80 (m, 4H), 2.77–2.62 (m, 8H), 2.61–2.44 (m, 24H), 2.23–2.19 (s, 6H), 2.19–2.17 (s, 6H), 1.96–1.86 (m, 2H), 1.72–0.96 (m, 10⁷H), 0.93–0.77 (m, 30H). HRMS: m / z calculated value: 1822.5509 (M+H) + ), Measured value: 1822.5906.

[0281] Example 29: Synthesis of Compound I-29

[0282]

[0283] Compound 95 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. After the reaction was complete, the mixture was washed with water and the organic phase was collected. Compound 168 (1.0 eq) was added and stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 20:1) to obtain compound I-29 (yield: 47%). 1 H NMR(400MHz, CDCl3,293K)δ(ppm):4.13–4.03(m,18H),3.19–3.09(m,12H),2.88–2.61(m,18H),2.55–2.47(m,12H),2 .35–2.28(t,12H),2.23–2.19(s,9H),2.20–2.18(s,9H),2.06–1.86(m,9H),1.80–0.96(m,214H),0.94–0.78(m,46H). HRMS: m / z calculated value: 3432.8111(M+H + ), Measured value: 3432.7990.

[0284] Example 30: Synthesis method of compound I-30

[0285]

[0286] Compound 160 (1.0 eq) and compound 9 (1.0 eq) were dissolved in DCM and stirred at room temperature for 12 h. Then, compound 2 (1.0 eq) was added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH:NEt3 = 100:10:1) to give compound 170 (yield: 41%). Compound 73 (1.0 eq) and compound 170 (1.0 eq) were dissolved in DCM. NHS (2.0 eq), EDC (2.0 eq), and DMAP (0.1 eq) were added, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the mixture was purified by column chromatography (DCM:MeOH = 25:1) to give compound 171 (yield: 51%). Compounds 171 (1.0 eq) and 172 (1.0 eq) were dissolved in DCM, and NHS (2.0 eq), EDC (2.0 eq) and DMAP (0.1 eq) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was purified by column chromatography (DCM:MeOH = 25:1) to give compound I-30 (yield: 61%). 1 H NMR(400MHz, CDCl3,293K)δ(ppm):4.24–4.17(t,4H),3.77–3.72(t,4H),3.70–3 .66(s,4H),3.34–3.21(m,12H),2.80–2.62(m,16H),2.57–2.49(m,4H),2.44–2. 28(m,10H),2.26–2.20(d,10H),2.20–2.17(s,6H),2.17–2.12(s,6H),1.95–1.8 6(m,2H),1.75–0.96(m,138H),0.93–0.76(m,36H).HRMS:m / z calculated value:2388.9553(M+H + ), Measured value: 2388.9474.

[0287] Example 31: Preparation and characterization of basic LNPs carrying mRNA and LNPs of the present invention

[0288] Ionizable lipid molecules (ALC-0315), DSPC, cholesterol, and polyethylene glycol lipid molecules (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. Firefly luciferase mRNA was dissolved in a 100mM sodium citrate buffer solution at pH 5.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier was mixed with mRNA at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol was then removed by ultrafiltration to obtain basic lipid nanoparticles encapsulating mRNA (represented as Basic LNP@mRNA). Luc ).

[0289] The compounds synthesized in Examples 1-30 (I-1 to I-30), DSPC, cholesterol, and polyethylene glycol lipid molecules (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA, firefly luciferase mRNA, was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier was mixed with the mRNA at a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration to obtain the lipid nanoparticles of the present invention encapsulating mRNA (named according to the compounds synthesized in Examples 1-30. For example, if compound I-1 is used, the lipid nanoparticles can be represented as I-1LNP@mRNA). Luc ).

[0290] The particle size distribution of the obtained basic lipid nanoparticles and the lipid nanoparticles of this invention was characterized using dynamic light scattering (DLS). Specifically, Basic LNP@mRNA... Luc I-1LNP@mRNA Luc I-2LNP@mRNA Luc I-3LNP@mRNA Luc and I-4LNP@mRNA Luc The particle size distribution is as follows Figure 1 As shown.

[0291] DLS results (Table 1) show that Basic LNP@mRNA Luc Compared with the LNP@mRNA of the present invention prepared based on I-1 to I-30 Luc In comparison, there was no significant difference in hydrated particle size, and both met the usable standards.

[0292] LNP@mRNA Luc Encapsulation efficiency determination

[0293] The slightly white solution obtained above was dialyzed against an appropriate volume of PBS solution for 4 hours. The filtrate was collected, and the mRNA content in the filtrate was determined using Nanodrop. The encapsulation efficiency was calculated using the following formula:

[0294] Encapsulation efficiency = mRNA 总量 -mRNA 滤液 / mRNA 总量 .

[0295] The results (Table 1) show that the LNP@mRNA prepared according to the present invention... Luc Both exhibit good mRNA encapsulation efficiency.

[0296] Table 1

[0297]

[0298]

[0299] Example 32: LNP@mRNA of the present invention Luc In vitro transfection efficiency experiment

[0300] The compounds synthesized in Examples 1-30 (I-1 to I-30), DSPC, cholesterol, and polyethylene glycol lipid molecules (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA, firefly luciferase mRNA, was dissolved in a 100 mM sodium citrate buffer solution at pH 5.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier was mixed with the mRNA at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol was then removed by ultrafiltration. The I-1LNP@mRNA of this invention, encapsulating the mRNA, was obtained. Luc To I-30LNP@mRNA Luc Using an ionizable lipid molecule (ALC-0315) instead of the compounds synthesized in Examples 1-30, Basic LNP@mRNA encapsulating mRNA was obtained in the same manner. Luc .

[0301] DC2.4 cells were fed at a rate of 1×10⁻⁶. 6 Cells were seeded at high density in 12-well plates and incubated in DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C in a 5% CO2 atmosphere. The medium was replaced with fresh medium after 24 hours of incubation. PBS and Basic LNP@mRNA were then added to the cells. Luc And the LNP@mRNA of the present invention Luc (The mRNA dose per well was 2 μg). After incubating the cells for 24 hours, the cells were lysed, and the firefly luciferase detection kit (Luciferase Reporter Gene Assay Kit, Yeasen) was added. After thorough mixing, the RLU (Relative Light Unit) was measured using the Luminescence mode of a multi-functional microplate reader to reflect the LNP@mRNA levels in each group. Luc The in vitro transfection efficiency.

[0302] The results (Table 2) show that Basic LNP@mRNA Luc In vitro transfection efficiency is low, while the LNP@mRNA of this invention... Luc In vitro transfection efficiency was significantly higher than that of Basic LNP@mRNA. Luc There has been a significant improvement, and the transfection effect is excellent.

[0303] Table 2

[0304]

[0305] Example 33: LNP@mRNA of the present invention OVA Antigen presentation efficiency experiment

[0306] The compounds synthesized in the examples (I-1, I-2, I-3, I-4), DSPC, cholesterol, and polyethylene glycol lipid molecules (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was chicken ovalbumin mRNA (OVA mRNA), dissolved in a 100mM sodium citrate buffer solution at pH 6.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier was mixed with the mRNA at a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration to obtain I-1LNP@mRNA encapsulating the mRNA. OVA I-2LNP@mRNA OVA I-3LNP@mRNA OVA and I-4LNP@mRNA OVA Using ALC-0315 instead of the compound synthesized in the examples, Basic LNP@mRNA encapsulating mRNA was obtained in the same manner. OVA .

[0307] DC2.4 cells were loaded at 5 × 10⁻⁶ 5 Cells were seeded at high density in 24-well plates and incubated in DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C in a 5% CO2 atmosphere. After 24 hours of incubation, the medium was replaced with fresh medium. PBS and Basic LNP@mRNA were then added to the cells. OVA I-1LNP@mRNA OVA I-2LNP@mRNA OVA I-3LNP@mRNA OVA and I-4LNP@mRNA OVA (The mRNA dose per well was 1 μg). After incubating the cells for 24 hours, flow cytometry analysis was performed to detect the proportion of OVA-H-2Kb positive cells and to compare the antigen presentation efficiency of each group.

[0308] The results show ( Figure 2 ), using Basic LNP@mRNA OVA After in vitro transfection, the proportion of OVA-H-2Kb positive cells was low, indicating that DC2.4 cells were transfected via Basic LNP@mRNA. OVAAfter transfection with OVA mRNA, the antigen presentation efficiency was very limited. However, using I-1LNP@mRNA... OVA I-2LNP@mRNA OVA I-3LNP@mRNA OVA and I-4LNP@mRNA OVA After transfection with OVA mRNA in vitro, the proportion of OVA-H-2Kb positive cells increased significantly, demonstrating that the LNP@mRNA of the present invention can significantly improve antigen presentation efficiency.

[0309] Example 34: LNP@mRNA of the present invention Luc In vivo expression experiment in mice

[0310] The compounds synthesized in the examples (I-1, I-2, I-3, I-4), DSPC, cholesterol, and polyethylene glycol lipid molecules (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA, firefly luciferase mRNA, was dissolved in a 100mM sodium citrate buffer solution at pH 6.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier was mixed with the mRNA at a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration to obtain I-1LNP@mRNA encapsulating the mRNA. Luc I-2LNP@mRNA Luc I-3LNP@mRNA Luc and I-4LNP@mRNA Luc Using ALC-0315 instead of the compound synthesized in the examples, Basic LNP@mRNA encapsulating mRNA was obtained in the same manner. Luc .

[0311] Subsequently, the five LNP@mRNAs prepared Luc C57BL / 6J mice (4-6 weeks old, male, weighing approximately 18-20g) were administered an intramuscular injection in the thigh at a dose of 5 μg mRNA per mouse. Six hours later, the substrate (fluorescein sodium salt, D-Luciferin, 150 mg / kg, Yeasen) was injected intraperitoneally into the mice, followed by in vivo fluorescence imaging using a small animal fluorescence imaging system. Bioluminescence imaging was performed using Spectrum (PerkinElmer).

[0312] The results show ( Figure 3 The LNP@mRNA of this invention Luc With Basic LNP@mRNA Luc Compared to the previous version, the expression level of luciferase was significantly increased, and the LNP@mRNA of this invention showed a significant improvement. LucBoth methods can achieve successful delivery and efficient expression of nucleic acids in vivo.

[0313] Example 35: LNP@mRNA of the present invention Luc Large animal in vivo expression experiments

[0314] The compounds synthesized in the examples (I-1, I-2, I-3, I-4), DSPC, cholesterol, and polyethylene glycol lipid molecules (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA, firefly luciferase mRNA, was dissolved in a 100mM sodium citrate buffer solution at pH 6.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier was mixed with the mRNA at a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration to obtain I-1LNP@mRNA encapsulating the mRNA. Luc I-2LNP@mRNA Luc I-3LNP@mRNA Luc and I-4LNP@mRNA Luc Using ALC-0315 instead of the compound synthesized in the examples, Basic LNP@mRNA encapsulating mRNA was obtained in the same manner. Luc .

[0315] Subsequently, the five LNP@mRNAs prepared Luc Rabbits (6 weeks old, male, approximately 650g) were administered an intramuscular injection in the thigh at a dose of 25 μg mRNA per rabbit. Six hours later, the substrate (fluorescein sodium salt, D-Luciferin, 150 mg / kg, Yeasen) was injected intraperitoneally, followed by in vivo fluorescence imaging using a small animal fluorescence imaging system. Spectrum (PerkinElmer) was used to perform local bioluminescence imaging.

[0316] The results show ( Figure 4 The LNP@mRNA of this invention Luc With Basic LNP@mRNA Luc Compared to the previous version, the expression level of luciferase was significantly increased, and the LNP@mRNA of this invention showed a significant improvement. Luc Both methods can achieve successful delivery and efficient expression of nucleic acids in vivo.

[0317] Example 36: LNP@mRNA of the present invention OVA Tumor growth inhibition rate experiment

[0318] The compounds synthesized in the examples (I-1 to I-30), DSPC, cholesterol, and polyethylene glycol lipid molecules (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was chicken ovalbumin mRNA (OVA mRNA), dissolved in a 100mM sodium citrate buffer solution at pH 6.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier was mixed with the mRNA at a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration to obtain the LNP@mRNA of the present invention encapsulating the mRNA. OVA Using ALC-0315 instead of the compound synthesized in the examples, Basic LNP@mRNA encapsulating mRNA was obtained in the same manner. OVA .

[0319] C57BL / 6J mice (4-6 weeks old, male, weighing approximately 18-20g) were used with B16-OVA cells (1×10⁻⁶). 6 Tumor models were established via subcutaneous injection. The day of tumor bearing was recorded as day 0. On day 9, PBS and Basic LNP@mRNA were injected intramuscularly into the thigh. OVA And the LNP@mRNA of the present invention OVA The mRNA injection dose was 5 μg. The tumor volume of mice was measured on day 0 and day 21. The tumor growth inhibition rate was calculated according to the formula TGI (Tumor Growth Inhibition) = (1 - Tumor volume of treatment group / Tumor volume of PBS) * 100%.

[0320] The results (Table 3) show that Basic LNP@mRNA OVA The tumor growth inhibition rate was approximately 46.9%, while the LNP@mRNA of this invention... OVA The tumor growth inhibition rate reached approximately 60-80%, compared to Basic LNP@mRNA. OVA The significant improvement demonstrates the effectiveness of the LNP@mRNA of this invention. OVA It can significantly enhance the in vivo expression efficiency of mRNA.

[0321] Table 3

[0322]

[0323] Example 37: Injection of the LNP@mRNA of the present invention OVA Tumor growth curve after

[0324] The compounds synthesized in the examples (I-1, I-2, I-3, or I-4), DSPC, cholesterol, and polyethylene glycol lipid molecules (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was chicken ovalbumin mRNA (OVA mRNA), dissolved in a 100mM sodium citrate buffer solution at pH 6.0. The organic phase solution and aqueous phase solution were mixed at a volume ratio of 1:3, and the lipid carrier and mRNA were mixed at a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration to obtain the LNP@mRNA of the present invention encapsulating the mRNA. OVA Using ALC-0315 instead of the compounds in the examples, Basic LNP@mRNA encapsulating mRNA was obtained in the same manner. OVA .

[0325] C57BL / 6J mice (4-6 weeks old, male, weighing approximately 18-20g) were used with B16-OVA cells (1×10⁻⁶). 6 Tumor models were established via subcutaneous injection. The day of tumor bearing was recorded as day 0. On day 9, PBS and Basic LNP@mRNA were injected intramuscularly into the thigh. OVA And the LNP@mRNA of the present invention OVA The mRNA injection dose was 5 μg, and the tumor volume changes in mice were recorded over 30 days.

[0326] The results show ( Figure 5 ), accepting the LNP@mRNA of the present invention OVA The tumors in the treated mice grew slowly, and the treatment effect was significantly better than that of mice receiving Basic LNP@mRNA. OVA The LNP@mRNA of the present invention was demonstrated in treated mice. OVA It can significantly enhance the in vivo expression efficiency of mRNA and effectively inhibit tumor growth.

[0327] Example 38: LNP@mRNA of the present invention OVA Survival experiment in mouse tumor model

[0328] The compounds synthesized in the examples (I-1, I-2, I-3, I-4), DSPC, cholesterol, and polyethylene glycol lipid molecules (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was chicken ovalbumin mRNA (OVA mRNA), dissolved in a 100mM sodium citrate buffer solution at pH 6.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier was mixed with the mRNA at a mass ratio of 25:1 to obtain a slightly white solution. Ethanol was then removed by ultrafiltration. The I-1LNP@mRNA of the present invention, encapsulating the mRNA, was obtained.OVA I-2LNP@mRNA OVA I-3LNP@mRNA OVA and I-4LNP@mRNA OVA Using ALC-0315 instead of the compounds synthesized in the above examples, Basic LNP@mRNA encapsulating mRNA was obtained using the same method. OVA .

[0329] C57BL / 6J mice (4-6 weeks old, male, weighing approximately 18-20g) were used with B16-OVA cells (1×10⁻⁶). 6 Tumor models were established via subcutaneous injection. The day of tumor bearing was recorded as day 0. PBS and BasicLNP@mRNA were administered intramuscularly to the thigh twice, on days 9 and 15, respectively. OVA And the LNP@mRNA of the present invention OVA Each mRNA injection dose was 5 μg, and the survival status of mice was recorded for 60 days.

[0330] The results show ( Figure 6 The median survival time of mice in the PBS group was 20 days, and they received Basic LNP@mRNA. OVA The median survival time of the treated tumor model mice was 35 days, after receiving the LNP@mRNA of this invention. OVA The survival of the treated tumor model mice was significantly prolonged, with a survival rate of 40%-100% at the end of the 60-day observation period.

[0331] Example 39: LNP@mRNA of the present invention OVA Antitumor immune response experiment

[0332] The compounds synthesized in the examples (I-1, I-2, I-3, I-4), DSPC, cholesterol, and polyethylene glycol lipid molecules (ALC-0159) were dissolved in ethanol at a molar ratio of 50:10:38:2. The mRNA was chicken ovalbumin mRNA (OVA mRNA), dissolved in a 100mM sodium citrate buffer solution at pH 6.0. The volume ratio of the organic phase solution to the aqueous phase solution was 1:3, and the lipid carrier was mixed with the mRNA at a mass ratio of 25:1 to obtain a slightly white solution. The ethanol was then removed by ultrafiltration to obtain the LNP@mRNA of the present invention encapsulating the mRNA. OVA Using ALC-0315 instead of the compounds synthesized in the above examples, Basic LNP@mRNA encapsulating mRNA was obtained using the same method. OVA .

[0333] C57BL / 6J mice (4-6 weeks old, male, weighing approximately 18-20g) were used with B16-OVA cells (1×10⁻⁶). 6 Tumor models were established via subcutaneous injection. The day of tumor bearing was recorded as day 0. On day 9, PBS and Basic LNP@mRNA were injected intramuscularly into the thigh. OVA And the LNP@mRNA of the present invention OVA The mRNA injection dose was 5 μg. After 21 days, the mice were sacrificed, the tumor tissue was completely removed, ground, and subjected to ELISA to detect the expression levels of IFN-γ and TNF-α in the tumor tissue, and the anti-tumor immune response capabilities of the two LNP@mRNAs were compared.

[0334] The results showed that the LNP@mRNA of the present invention was accepted OVA IFN-γ in tumor tissue of treated mice Figure 7 ) and TNF-α ( Figure 8 The expression level of ) was higher, and the treatment effect was significantly better than that of receiving Basic LNP@mRNA. OVA The LNP@mRNA of the present invention was demonstrated in treated mice. OVA It can significantly enhance the in vivo expression efficiency of mRNA.

[0335] Example 40: The compounds of the present invention can be used to construct liposomes.

[0336] 100 mg of compound I-1, 80 mg of DSPC, 20 mg of cholesterol, and 5 mg of PEG-DSPE were dissolved in an equal volume of chloroform (5 mL) and methanol (5 mL). The solvent was evaporated at 45 °C using a rotary evaporator until a homogeneous lipid film was formed. 10 mL of distilled water was added to resuspend the film, and the mixture was gently agitated until a homogeneous emulsion was formed. The emulsion was then subjected to an ultrasonic treatment at 60 Hz for 1 minute to form liposomes. The resulting liposome solution was frozen at -20 °C for 24 hours and then thawed at room temperature. This process was repeated three times to improve the stability of the liposomes. The liposomes were then extruded through a 100 nm polycarbonate membrane using an extrusion device, ensuring a particle size of approximately 100 nm. The particle size distribution of the obtained I-1 lipsomes is shown below. Figure 9 As shown.

[0337] Example 41: The liposomes of the present invention can be used to encapsulate small molecule drugs (taking paclitaxel as an example).

[0338] 100 mg of the compound from the examples (I-1, I-2, I-3, or I-4), 80 mg of DSPC, 20 mg of cholesterol, and 5 mg of PEG-DSPE (molar ratio approximately 39.4:39.4:19.7:1.5) were dissolved in a mixed solvent of 5 mL chloroform and 5 mL methanol. The solvent was evaporated at 45 °C using a rotary evaporator until a homogeneous lipid film was formed. 10 mg of paclitaxel was then dispersed in 10 mL of distilled water. The paclitaxel solution was added to the lipid film and gently agitated until a homogeneous emulsion was formed. The film was then sonicated at 60 Hz for 1 minute to allow the lipid molecules to form primary liposomes encapsulating paclitaxel. The resulting liposome solution was frozen at -20 °C for 24 hours and then thawed at room temperature. This freeze-thaw process was repeated three times to improve the stability of the liposomes and the encapsulation efficiency of paclitaxel. The liposomes were extruded through a 200 nm polycarbonate membrane using an extrusion apparatus, ensuring an average diameter of approximately 200 nm. Unencapsulated paclitaxel was separated by centrifugation at 10,000 rpm for 30 minutes. The concentration of paclitaxel in the supernatant was measured, and the encapsulation efficiency was calculated, as shown in Table 4. The liposomes prepared according to this invention all exhibited good encapsulation efficiency for paclitaxel.

[0339] Table 4

[0340] Liposome name Paclitaxel encapsulation efficiency I-1 Lipsome 79.3％ I-2Lipsome 82.4％ I-3 Lipsome 64.5％ I-4 Lipsome 81.6％

[0341] The foregoing description of specific exemplary embodiments of this disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit this disclosure to the precise form disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of this disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments and various different options of this disclosure.

Claims

1. A compound of formula (1) or a pharmaceutically acceptable salt thereof, in, X is selected from N, CH, or Selected from L 1 L 2 and L 3 Independently selected from single bonds, C 1-10 Alkylene, C 6-10 aryl or -C 1-10 Alkylene-NR d -C 1-10 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens; R d Selected from C 1-10 Alkyl, -C 1-10 Alkylene-NHC(=O)-C 1-30 Alkyl, -C 1-10 Alkylene-NHC(=O)-C 2-30 alkenyl or -C 1-10 Alkylene-OC(=O)-C 1-6 Alkylene-SC 1-6 Alkylene-C(=O)OC 1-30 alkyl; L 4 L 5 and L 6 Independently selected from single bonds, C 1-10 Alkylene, -C 1-6 Alkylene-SC 1-6 alkylene-, -C 1-6 Alkylene-SSC 1-6 alkylene- or -C 1-10 Alkylene-NR e -C 1-10 Alkylene -, wherein the alkylene is unsubstituted or converted by one or more OH, NH2, halogen, -NHC(=O)OC 1-30 Alkyl group, -NHC(=O)OC 2-30 alkenyl, -NHC(=O)-C 1-30 Alkyl group, -NHC(=O)-C 2-30 alkenyl, -C 1-10 Alkylene-C(=O)NH-C 1-10 Alkylene-NR e -C 1-10 Alkylene-OC(=O)-C 2-30 alkenyl, -C(=O)OC 1-30 Alkyl group, -C(=O)OC 2-30 alkenyl, -OC(=O)-C 1-30 Alkyl or -OC(=O)-C 2-30 Alkenyl substitution; R e Selected from -C 1-10 Alkylene-OC(=O)-C(=O)-R f -C 1-10 Alkylene-C(=O)OC 1-30 Alkyl, -C 1-10 Alkylene-OC(=O)-C 1-30 Alkyl, -C 1-10 Alkylene-C(=O)OC 2-30 alkenyl, -C 1-10 Alkylene-OC(=O)-C 2-30 alkenyl, -C 1-10 Alkylene-OC(=O)-L b -C(=O)OC 1-30 Alkyl, -C 1-10 Alkylene-OC(=O)-C 1-20 Alkylene-C(=O)-R f -C 1-10 Alkylene-OC(=O)-C 1-10 alkylene-(5-8-membered heteroaryl)-C 1-10 Alkyl, -C 1-10 Alkylene-OC(=O)NH-C 1-30 Alkyl, -C 1-10 Alkylene-NHC(=O)-C 1-30 Alkyl or -C 1-10 Alkylene-OC 1-30 alkyl; R f Selected from L b Selected from or -C 1-10 Alkylene-SSC 1-10 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens; n is selected from 1, 2, 3, 4, 5, 6, 7 or 8; G 1 G 2 and G 3 Independently selected from single bonds, -NR 3 -, -O-, -S-, -C(=O)-, -OC(=O)-, -OC(=O)O-, -C(=O)C(=O)O-, -NR 3 C(=O)-、-NR 3 C(=O)O-, 5 to 10 membered heterocyclic groups, -OC(=O)NR 3 -L a -C(=O)O-、-OC(=O)NR 3 -L a -NR 3 -C(=O)O-、-OC(=O)-L a -OC(=O)-NR 3 -、-NR 3 C(=O)-L a -C(=O)-NR 3 -、-NR 3 C(=O)OL a -C(=O)-NR 3 -or-C(=O)OL a -OC(=O)-NR 3 -; G 4 G 5 and G 6 Independently selected from single bonds, -NR 3 -, -O-, -C(=O)-, -OC(=O)-, -OC(=O)O-, -C(=O)C(=O)O-, -NR 3 C(=O)-、-NR 3 C(=O)O-、-C(=O)-L a -C(=O)-、-OC(=O)-L a -C(=O)-、-C(=O)OL a -C(=O)NR 3 -、-C(=O)OL a -OC(=O)NR 3 -、-C(=O)-L a -C(=O)NR 3 -, 5-8 quinone heteroaryl or -C(=O)NR 3 -L a -OC(=O)C(=O)-; Each R 3 Independently selected from H and C 1-30 Alkyl, -C 1-10 Alkylene-OC(=O)-C 1-30 Alkyl, -C 1-10 Alkylene-OC(=O)-C 2-30 alkenyl, -C 1-10 Alkylene-C(=O)OC 1-30 Alkyl, -C 1-10 Alkylene-C(=O)OC 2-30 alkenyl or -C 1-10 Alkylene-OC(=O)-C 1-8 Alkylene-SC 1-8 Alkylene-C(=O)OC 1-30 alkyl; Each L a Independently selected from single bonds, C 1-20 Alkylene, -[OCH2CH2] n -、-C 1-10 Alkylene-SSC 1-10 alkylene-, -C 1-10 Alkylene-SC 1-10 alkylene-, -C 1-10 Alkylene-C(=O)-C 1-10 alkylene- or -C 1-10 Alkylene-NR e -C 1-10 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens; R 1 and R 2 Selected independently C 1-30 Alkyl or C 2-30 Alkenyl group, wherein the alkyl or alkenyl group is unsubstituted or substituted by one or more OH, NH2 or halogens.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein, L 1 L 2 and L 3 Independently selected from single bonds, C 1-8 Alkylene, C 6-10 aryl or -C 1-8 Alkylene-NR d -C 1-8 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens; R d Selected from C 1-8 Alkyl, -C 1-8 Alkylene-NHC(=O)-C 8-26 Alkyl, -C 1-8 Alkylene-NHC(=O)-C 8-26 alkenyl or -C 1-8 Alkylene-OC(=O)-C 1-4 Alkylene-SC 1-4 Alkylene-C(=O)OC 8-26 alkyl; Or, L 1 L 2 and L 3 Independently selected from single bonds, C 1-6 alkylene, phenylene or -C 1-6 Alkylene-NR d -C 1-6 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens; R d Selected from C 1-6 Alkyl, -C 1-6 Alkylene-NHC(=O)-C 10-20 Alkyl or -C 1-6 Alkylene-OC(=O)-C 1-4 Alkylene-SC 1-4 Alkylene-C(=O)OC 10-20 alkyl; Or, L 1 L 2 and L 3 Independently selected from single bonds, -CH2-, -CH2CH2-, 3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein, L 4 L 5 and L 6 Independently selected from single bonds, C 1-8 Alkylene, -C 1-4 Alkylene-SC 1-4 alkylene-, -C 1-4 Alkylene-SSC 1-4 alkylene- or -C 1-8 Alkylene-NR e -C 1-8 Alkylene -, wherein the alkylene is unsubstituted or converted by one or more OH, NH2, halogen, -NHC(=O)OC 8-26 Alkyl group, -NHC(=O)OC 8-26 alkenyl, -NHC(=O)-C 8-26 Alkyl group, -NHC(=O)-C 8-26 alkenyl, -C 1-8 Alkylene-C(=O)NH-C 1-8 Alkylene-NR e -C 1-8 Alkylene-OC(=O)-C 8-26 alkenyl, -C(=O)OC 8-26 Alkyl group, -C(=O)OC 8-26 alkenyl, -OC(=O)-C 8-26 Alkyl or -OC(=O)-C 8-26 Alkenyl substitution; R e Selected from -C 1-8 Alkylene-OC(=O)-C(=O)-R f -C 1-8 Alkylene-C(=O)OC 8-26 Alkyl, -C 1-8 Alkylene-OC(=O)-C 8-26 Alkyl, -C 1-8 Alkylene-C(=O)OC 8-26 alkenyl, -C 1-8 Alkylene-OC(=O)-C 8-26 alkenyl, -C 1-8 Alkylene-OC(=O)-L b -C(=O)OC 8-26 Alkyl, -C 1-8 Alkylene-OC(=O)-C 1-16 Alkylene-C(=O)-R f -C 1-8 Alkylene-OC(=O)-C 1-8 alkylene-(5-6-membered heteroaryl)-C 4-10 Alkyl, -C 1-8 Alkylene-OC(=O)NH-C 8-26 Alkyl, -C 1-8 Alkylene-NHC(=O)-C 8-26 Alkyl or -C 1-8 Alkylene-OC 8-26 alkyl; R f Selected from L b Selected from or -C 1-8 Alkylene-SSC 1-8 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens; n is selected from 1, 2, 3, 4, 5, or 6; Or, L 4 L 5 and L 6 Independently selected from single bonds, -CH2-, -CH2-CH2-, 4. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein, G 1 G 2 and G 3 Independently selected from single bonds, -NR 3 -, -O-, -S-, -C(=O)-, -OC(=O)-, -NR 3 C(=O)-、-NR 3 C(=O)O-, 5- to 8-membered heterocyclic groups, -OC(=O)NR 3 -L a -C(=O)O-、-OC(=O)NR 3 -L a -NR 3 -C(=O)O-、-OC(=O)-L a -OC(=O)-NR 3 -、-NR 3 C(=O)-L a -C(=O)-NR 3 -、-NR 3 C(=O)OL a -C(=O)-NR 3 -or-C(=O)OL a -OC(=O)-NR 3 -; Or, G 1 G 2 and G 3 Independently selected from single bonds, -NH-, -O-, -S-, -C(=O)-, -OC(=O)-, -NHC(=O)-, -OC(=O)NH-, 5. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein, G 4 G 5 and G 6 Independently selected from single bonds, -NR 3 -, -O-, -C(=O)-, -OC(=O)-, -C(=O)C(=O)O-, -NR 3 C(=O)-、-NR 3 C(=O)O-、-C(=O)-L a -C(=O)-、-OC(=O)-L a -C(=O), -C(=O)-L a -C(=O)NR 3 -, 5-6 quinone heteroaryl, -C(=O)-NR 3 -C 1-8 Alkylene-NR e -C 1-8 Alkylene -OC(=O)C(=O)-; Or, G 4 G 5 and G 6 Independently selected from -O-, -C(=O)-, -OC(=O)-, -NHC(=O)-, -NHC(=O)O-, 6. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein, Each R 3 Independently selected from H and C 8-26 Alkyl, -C 1-8 Alkylene-OC(=O)-C 8-26 Alkyl, -C 1-8 Alkylene-OC(=O)-C 8-26 alkenyl, -C 1-8 Alkylene-C(=O)OC 8-26 Alkyl, -C 1-8 Alkylene-C(=O)OC 8-26 alkenyl or -C 1-8 Alkylene-OC(=O)-C 1-6 Alkylene-SC 1-6 Alkylene-C(=O)OC 8-26 alkyl; Or, each R 3 Independently selected from H and C 10-22 Alkyl, -C 1-6 Alkylene-OC(=O)-C 10-22 Alkyl, -C 1-6 Alkylene-OC(=O)-C 10-22 alkenyl, -C 1-6 Alkylene-C(=O)OC 10-22 Alkyl, -C 1-6 Alkylene-C(=O)OC 10-22 alkenyl or -C 1-6 Alkylene-OC(=O)-C 1-4 Alkylene-SC 1-4 Alkylene-C(=O)OC 10-22 alkyl.

7. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein, Each L a Independently selected from single bonds, C 1-16 Alkylene, -[OCH2CH2] n -、-C 1-8 Alkylene-SSC 1-8 alkylene-, -C 1-8 Alkylene-SC 1-8 alkylene-, -C 1-8 Alkylene-C(=O)-C 1-8 alkylene- or -C 1-8 Alkylene-NR e -C 1-8 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens; Or, each L a Independently selected from single bonds, C 1-12 Alkylene, -[OCH2CH2] n -、-C 1-6 Alkylene-SSC 1-6 alkylene-, -C 1-6 Alkylene-SC 1-6 alkylene-, -C 1-6 Alkylene-C(=O)-C 1-6 alkylene- or -C 1-6 Alkylene-NR e -C 1-6 Alkylene - where the alkylene is unsubstituted or substituted with one or more OH, NH2 or halogens; Or, each L a Independently selected from single bonds, -CH2-, -CH2CH2-, 8. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein, R 1 and R 2 Selected independently C 1-24 Alkyl or C 8-24 Alkenyl group, wherein the alkyl or alkenyl group is unsubstituted or substituted by one or more OH, NH2 or halogen; Or, R 1 and R 2 Selected independently -CH3, 9. A compound or a pharmaceutically acceptable salt thereof, said compound being selected from the following:

10. A lipid carrier comprising an ionizable lipid molecule, a polyethylene glycol lipid molecule, a steroidal lipid molecule, and an accessory lipid molecule, wherein the ionizable lipid molecule comprises a compound according to any one of claims 1-9 or a pharmaceutically acceptable salt thereof.

11. The lipid carrier according to claim 10, wherein, The polyethylene glycol lipid molecule is selected from at least one of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (PEG-DMG), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterol glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearate, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), and PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA); The steroidal lipid molecules are selected from at least one of the following: alfalfa sterol, β-sitosterol, brassosterol, ergocalciferol, campesterol, cholesterol, coccidol, dehydrocholesterol, sterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, serotonol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol, lanosterol, photosterol, phycosterol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, and lithocholic acid; The auxiliary lipid molecule is selected from at least one of the following: 1,2-distearyl-sn-glycerol-3-phosphate choline DSPC, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline DPPC, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine DOPE, 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine DPPE, 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine DMPE, 2-dioleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol)DOPG, oleoylphosphatidylcholine POPC, and 1-palmitoyl-2-oleoylphosphatidylethanolamine POPE.

12. The lipid carrier according to claim 10 or 11, wherein, The lipid carrier comprises, by molar percentage, 10%-70% ionizable lipid molecules, 5%-60% steroidal lipid molecules, 1%-60% accessory lipid molecules and 1%-30% polyethylene glycol lipid molecules.

13. A lipid nanoparticle composition comprising a lipid carrier according to any one of claims 10-12 and at least one component selected from nucleic acids, small molecule drugs and protein peptides.

14. A liposome composition comprising a lipid carrier according to any one of claims 10-12 and at least one component selected from nucleic acids, small molecule drugs and protein peptides.

15. The lipid nanoparticle composition according to claim 13 or the liposome composition according to claim 14, wherein, The nucleic acid is selected from at least one of DNA, mRNA, rRNA, siRNA, tRNA, microRNA, antisense nucleic acid, self-replicating RNA, and circular RNA.

16. The lipid nanoparticle composition according to claim 13 or the liposome composition according to claim 14, wherein, The mass ratio of the lipid carrier to nucleic acid is 5:1 to 50:1; or, the mass ratio of the lipid carrier to nucleic acid is 10:1 to 30:1; or, the mass ratio of the lipid carrier to nucleic acid is 20:1, 25:1 or 30:

1.

17. The lipid nanoparticle composition according to claim 13 or the liposome composition according to claim 14, wherein, The mass ratio of the lipid carrier to the small molecule drug or protein peptide is 2:1 to 100:1; or, the mass ratio is 5:1 to 50:1; or, the mass ratio is 10:1 to 30:1; or, the mass ratio is 20.5:

1.

18. Use of the compound of any one of claims 1-9 or a pharmaceutically acceptable salt thereof, the lipid carrier of any one of claims 10-12, the lipid nanoparticle composition of claim 13, or the liposome composition of claim 14 in the preparation of nucleic acid drugs or vaccines.

19. Use of the compound of any one of claims 1-9 or a pharmaceutically acceptable salt thereof, the lipid carrier of any one of claims 10-12, the lipid nanoparticle composition of claim 13, or the liposome composition of claim 14 in the preparation of a medicament for treating or preventing inflammatory diseases, viral infections, and cancer in a subject.

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