Steroid-cationic lipid compounds and their applications

Abstract

The present invention provides a steroid-cationic lipid compound having the structure shown in formula (1) and its uses for producing lipid nanoparticles (LNPs) for delivering therapeutic and / or prophylactic agents. LNPs produced using the steroid-cationic lipid compound of the present invention have relatively good stability and transfection efficiency. These LNPs can be used for the delivery of nucleic acids, such as mRNA, and can efficiently and stably deliver bioactive substances to target cells or organs. At the same time, these LNPs have relatively high stability and can be used in the development of lyophilized mRNA formulations and for the spray inhalation administration of mRNA. Relatively high specific antibody responses are induced in experimental animals, and the compounds have better safety.

Description

[Technical Field]

[0001] This invention relates to the field of biopharmaceuticals, and more specifically to steroid-cationic lipid compounds and their applications in the delivery of their bioactive substances. [Background technology]

[0002] Nucleic acid drugs are primarily used to treat tumors, tissue regeneration, wound healing, pulmonary fibrosis, inflammatory diseases, and microbial infections. They refer to compounds containing nucleotide or deoxynucleotide structures that possess genetic characteristics and pharmacological activity. After nucleic acid drugs are injected into the human body, an efficient and safe drug delivery system is required to deliver them to the lesion site. This drug delivery system must have sufficient time to accurately target the lesion site while avoiding damage to normal cells.

[0003] Currently, drug delivery systems are divided into viral vectors and non-viral vectors. Viral vectors have relatively few applications in nucleic acid drugs due to their immunogenicity, oncogenetic properties, and limited drug carrying capacity. Non-viral vectors, such as polymers and lipids (liposomes or LNPs), can target specific cells by binding nucleic acid drugs to specific ligands and are widely used in current nucleic acid drug research. LNPs are one of the delivery systems with relatively many applications in current nucleic acid drug research, and LNP delivery systems can deliver nucleic acids safely and efficiently. They have advantages such as high nucleic acid encapsulation efficiency, efficient cell transfection, high tissue permeability, and low cytotoxicity and immunogenicity, giving them significant advantages compared to other drug delivery systems. Therefore, LNP delivery systems have great potential for broad development and application.

[0004] In conventional technology, LNP delivery systems are often composed of components such as ionic lipids (cationic lipids), steroids, neutral lipids, polyethylene glycol lipids, and nucleic acid drugs. For example, Patent Document AU2020325221A1 discloses a composition of target cell delivery LNPs comprising (i) ionic lipids, (ii) sterols or lipids of other structures, (iii) noncationic helper lipids or phospholipids, (iv) PEG lipids and (v) drugs encapsulated in LNPs (e.g., nucleic acid molecules), wherein these four components improve the delivery efficiency of target cells in a specific proportion; Patent Document WO2021 / 250263 A1 discloses a composition comprising ionic lipids, phospholipids, sterols, polyethylene glycol lipids and one or more nucleic acids, wherein the composition comprises a specific proportion of less than about 1 mol% of C14-PEG2000 lipids and other lipids; Patent Document CN102712935B discloses lipid particles comprising cationic lipids, neutral lipids, zwitterionic or anionic lipids, polyethylene glycol lipids, sterols and nucleic acids, wherein the above components are assembled into lipid particles having a solid core, the solid core can achieve higher encapsulation efficiency; Patent Document WO 2021 / 055849A1 discloses a lipid having the following structure,

[0005] [ka] ,

[0006] This structure can improve safety, efficacy, and specificity, and Patent Document WO2021 / 026358Al discloses target cell delivery lipid nanoparticles (LNPs) comprising (i) ionic lipids, (ii) sterols or lipids of other structures, (iii) noncationic helper lipids or phospholipids, (iv) payload, and (v) polyethylene glycol lipids, which achieve both safety and efficacy as a drug delivery system. In recent years, it has been discovered that the introduction of cholesterol into ionic lipid compounds can also be used for the delivery of nucleic acid drugs. Patent Document US7514099B2 describes the urethane lipid compound ClinDMA of cholesterol.

[0007] [ka] The present invention discloses that the compound can deliver siRNA by forming a four-component LNP with phospholipids, cholesterol, and polyethylene glycol lipids, or a five-component LNP with phospholipids, DMOBA lipids, cholesterol, and PEG lipids, and Patent Document CN112424214A describes an ionic cationic lipid compound formed from cholesterol and linear olefins (3) [ka] The disclosed compounds deliver nucleic acids by constructing lipid nanoparticles together with cholesterol, DPPC, DOPE, and DMG-PEG200. Both of the above compounds require four or five different lipid excipients to form nucleic acid drug delivery vector formulations, making their construction complex. To adapt to different application scenarios, it is particularly important to develop different delivery systems that can be applied to different administration routes, such as using spray inhalation to treat lung-related diseases or prevent respiratory-associated pathogenic bacterial infections. Currently, stable storage of mRNA-LNPs is a major challenge, and now, lyophilization allows for stable storage of mRNA-LNPs at 2-8°C, and even at room temperature. Thus, the conventional technology requires further development of its components and the structures of each component in order to obtain an LNP delivery system that is safer, more effective, more stable, easier to construct, and can be applied to different administration routes, through optimization of each component in the composition and cationic lipids. [Overview of the Initiative]

[0008] In a first embodiment, the present invention provides a steroid-cationic lipid compound having the structure shown in formula (I),

[0009] [ka] R1 above is selected from -OR4, -NR4R5, -NR4C(=O)R5, -C(=O)OR5, -OC(=O)R5, -OC(=O)OR5, -CN, a nitrogen-containing heterocyclic group or a guanidino group. R4 and R5 above are each independently H, C 1~9 an alkyl group, C 2~9 an alkenyl group, C 2~9 an alkynyl group, C 3~8 a cycloalkyl group, C 3~8 a cycloalkenyl group or C 3~8 a cycloalkynyl group. G1 above is a chemical bond (-), C 1~9 an alkylene group, C 2~9 an alkenylene group, C 3~9 an alkynylene group, C 3~8 a cycloalkylene group or C 3~8 a cycloalkenylene group. The number of consecutive carbon atoms in R1-G1 is less than 10. For example, the number of consecutive carbon atoms in R1-G1 may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. G2 and G3 are each independently a chemical bond (-), C 1~12 an alkylene group, C 2~12 an alkenylene group, C 2~12 an alkynylene group, C 3~12 a cycloalkylene group, C 3~12 a cycloalkenylene group, C 3~12 a cycloalkynylene group or C 6~12 an arylene group. L1 and L2 are each independently a chemical bond (-), -O-, -S-, -O(C=O)O-, -(C=O)NR a -, -NR a (C=O)-, -O(C=O)-, -(C=O)O-, -S-S-, -S(O) x -, -OS(O) x O-, -C(=O)S-, -SC(=O)-, -NR a C(=O)NR b -, -OC(=O)NR a -, -NR a C(=O)O-, -OC(=O)S-, -SC(=O)O-, -P(O)(ORa )O-, -OP(O)(OR a )O- or C 1~12 Selected from one or more combinations of alkylene groups, Here, x is chosen from 0, 1, or 2. Here, R a , R b H and C are independent of each other. 1~12 Alkyl alkyl group, C 2~12 Alkenyl group or C 2~12 Selected by Alkin, The above R2 is selected from naturally occurring or unnaturally occurring steroids. The above R3 is a steroid that exists independently, either naturally or unnaturally, C 6~24 Alkyl alkyl group, C 6~24 Alkenyl group, C 6~24 Alkyne or C 6~24 Selected from alkoxy groups. Furthermore, R1 is selected from -OR4, -NR4R5, pyrazolyl group, imidazolyl group, piperazinyl group, alkylpiperazinyl, piperidinyl group, alkylpiperidinyl, guanidino group, pyrrolyl group, or pyrrolidinyl group, and R4 and R5 are independently H and C, respectively. 1~9 Alkyl alkyl group, C 2~9 Alkenyl group, C 2~9 Alkynyl group, C 3~8 Cycloalkyl groups or C 3~8 Selected from cycloalkenyl groups. The above G1 is a chemical bond (-), C 1~9 Alkylene group, C 2~9 Alkenylene group, C 3~8 Cycloalkylene group or C 3~8 It is a cycloalkenylene group, Preferably, the above G1 is C 1~6 Alkylene group or C 2~6 It is an alkenylene group, The above R1-G1-consecutive carbon atom count is less than 10, and specifically, the above R1-G1-consecutive carbon atom count may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The above G2 and G3 are each independently chemically bonded (-) and -C. 1~12 Alkylene group, -C 2~12 Alkenylene group or -C 2~12 Selected from the alkynylene group, The above L1 and L2 are independently -O(C=O)-, -(C=O)O-, -SS-, -O(C=O)O-, and -NR, respectively. a C(=O)O-, -(C=O)NR a -, -NR a (C=O)-, -C(=O)S-, -SC(=O)-, -OC(=O)NR a - or C 1-12 Selected from one or more combinations of alkylene groups, Here, R a H, C 1~12 Alkyl alkyl group, C 2~12 Alkenyl group or C 2~12 alkynes, Furthermore, the above R1 is selected from -OR4, -NR4R5, imidazolyl group, piperazinyl group, or guanidino group. The above R4 and R5 are H and C, respectively, independently. 1~5 Selected from alkyl groups, The above G1 is an unsubstituted C 1~6 It is an alkylene group, The above R1-G1-consecutive carbon atom count is less than 10, and specifically, the above R1-G1-consecutive carbon atom count may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The above G2 and G3 are each independently of a chemical bond (-), C 1-10 Selected from alkylene groups, The above L1 and L2 are independently -O-, -O(C=O)-, -(C=O)O-, -(C=O)S-, -O(C=O)O-, -NHC(=O)O-, -NHC(=O)-, [ka] They are selected from among them. Furthermore, the above R2 is a sterol, Preferably, the sterol is an animal sterol or its oxidized or reduced form, and / or the sterol is a plant sterol or its oxidized or reduced form, and / or the sterol is a synthetic sterol or its oxidized or reduced form. More preferably, the sterol is selected from cholesterol, oxidized cholesterol, reduced cholesterol, alkyl lithocholate, stigmasterol, stigmastanol, campesterol, ergosterol, or sitosterol. More preferably, the sterol is an oxidized form of cholesterol, a reduced form of cholesterol, an alkyl lithocholate, stigmasterol, stigmastanol, campesterol, ergosterol, or sitosterol. More preferably, the above sterols are selected from avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, dinosterol, epicholesterol, ergosterol, fucosterol, hexahydrolumysterol, hydroxycholesterol, lanosterol, photosterol, algasterol, sitosteranol, sitosterol, stigmathanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, or lithocholic acid. More preferably, the structural formula of the above sterol is as follows:

[0010] [ka] Here, R is C 1~20 It is an alkyl group.

[0011] Furthermore, the above R3 is, [ka] It is selected based on this structure.

[0012] Preferably, the above R3 is [ka] It is selected based on this structure.

[0013] In one embodiment, the compound of formula (I) is [ka] [ka] [ka] They are selected from among them.

[0014] In another embodiment, the present invention further provides lipid nanoparticles comprising a steroid-cationic lipid compound represented by formula (I) described above. In one embodiment, the lipid nanoparticles further comprise polyethylene glycol lipids and at least one helper lipid, the helper lipid being selected from neutral lipids, zwitterionic lipids, or anionic lipids.

[0015] In one embodiment, the polyethylene glycol lipid is selected from 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerylmethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-distearylglycerin (PEG-DSG), PEG-dipalmitoyloleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristoyloxypropyl-3-(PEG-c-DMA). and / or, The above neutral lipids are 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine ( DMPE), 2-dioleoyl group-sn-glycero-3-phospho-(1'-rac-glycero)(DOPG), oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine Selected from lysophosphatidylcholine (DAPC), phosphatidylethanolamine (PE), phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), lysophosphatidylcholine, dilinoleylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), or lysophosphatidylethanolamine.

[0016] In one embodiment, the molar ratio of the steroid-cationic lipid represented by formula (I) to the helper lipid and polyethylene glycol lipid in the lipid nanoparticles is 20-80:20-80:0.5-20, preferably 30-80:30-80:0.5-20, more preferably 40-60:40-60:0.5-5, and more preferably 49.25:49.25:1.5.

[0017] In one embodiment, the lipid nanoparticles have a diameter of 15 nm to 300 nm, preferably a diameter of 60 nm to 102 nm, and more preferably a diameter of 80 nm to 90 nm.

[0018] In one embodiment, the encapsulation efficiency (%) of the lipid nanoparticles is 80% to 100%. In yet another embodiment, the present invention further provides applications in which the aforementioned steroid-cationic lipid compounds and the aforementioned lipid nanoparticles are used to manufacture bioactive substance delivery systems.

[0019] In one embodiment, the bioactive substance is divided into small molecule compounds, nucleic acids, oligopeptides, etc. Preferably, the bioactive substance is nucleic acid, more preferably, the bioactive substance is DNA or RNA, more preferably, the DNA includes non-coding DNA (antisense DNA) or coding DNA, and / or the RNA includes antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, circRNA, and self-replicating mRNA.

[0020] In one embodiment, the above-mentioned bioactive substance is used for the prevention and / or treatment of cancer, inflammation, fibrous diseases, autoimmune diseases, infectious diseases, mental disorders, blood disorders, chromosomal disorders, genetic disorders, connective tissue disorders, digestive disorders, ear, nose, and throat disorders, endocrine disorders, eye diseases, reproductive disorders, heart diseases, kidney diseases, lung diseases, metabolic disorders, oral diseases, musculoskeletal disorders, newborn screening, nutritional deficiencies, parasitic diseases, skin diseases, etc.

[0021] In one embodiment, the bioactive substance delivery system is an mRNA vaccine. In one embodiment, the mRNA vaccine is used to prevent cancer, viral infections, bacterial infections, fungal infections, etc. In one embodiment, the virus includes, but is not limited to, norovirus, Ebola virus, coronavirus (including SARS-CoV-2), cytomegalovirus, dengue virus, Zika virus, coxsackievirus, enterovirus, hepatitis virus, herpes simplex virus, human papillomavirus, influenza virus, Marburg virus, measles virus, poliovirus, rabies virus, rotavirus, and measles virus.

[0022] In yet another embodiment, the present invention further provides lipid nanoparticles comprising the aforementioned lipid nanoparticles and a bioactive substance.

[0023] In one embodiment, the bioactive substance is divided into small molecule compounds, nucleic acids, oligopeptides, etc. Preferably, the bioactive substance is nucleic acid, more preferably, the bioactive substance is DNA or RNA, more preferably, the DNA includes non-coding DNA (antisense DNA) or coding DNA, and / or the RNA includes antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, circRNA, and self-replicating mRNA.

[0024] In one embodiment, the ratio of nitrogen to phosphorus in the lipid nanoparticles is (1-15):1. For example, the nitrogen-to-phosphorus ratio of the lipid nanoparticles may be 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, or 15:1. Preferably, the nitrogen-to-phosphorus ratio of the lipid nanoparticles is (2-10):1, more preferably, the nitrogen-to-phosphorus ratio of the lipid nanoparticles is (4-8):1, and more preferably, the nitrogen-to-phosphorus ratio of the composition is 6:1 or 8:1.

[0025] In yet another embodiment, the present invention further provides applications for the lipid nanoparticles to produce mRNA vaccines.

[0026] In one embodiment, the lipid nanoparticles of the present invention can be manufactured using conventional methods in the art, such as microfluidic control techniques.

[0027] In yet another embodiment, the present invention further provides a drug comprising the aforementioned bioactive substance and lipid nanoparticles.

[0028] Furthermore, the above-mentioned drugs further contain pharmaceutically acceptable excipients.

[0029] Furthermore, the pharmaceutically acceptable excipients described in the present invention include, for example, vectors, adjuvants, and diluents.

[0030] Furthermore, the drug described in the present invention is a gene drug.

[0031] Furthermore, the drug described in the present invention is a liquid formulation or a freeze-dried powder.

[0032] Furthermore, the drugs described in the present invention include oral formulations, intramuscular injection formulations, subcutaneous injection formulations, intravenous injection formulations, spray inhalation formulations, or dry powder inhalation formulations.

[0033] In yet another embodiment, the present invention further provides a method for delivering a bioactive substance, the method comprising administering the drug described in the present invention to a subject requiring it.

[0034] The beneficial effects of this invention are as follows:

[0035] The present invention provides a steroid-cationic lipid compound having the structure shown in formula (I) and its uses, wherein the compound is used in the production of lipid nanoparticles for delivering bioactive substances by mixing a helper lipid and a polyethylene glycol lipid. The three-component LNP produced using the steroid-cationic lipid compound of the present invention has the advantages of a simple manufacturing process, good stability, and high transfection efficiency. By using the three-component LNP for the delivery of nucleic acids such as mRNA, nucleic acid mRNA can be delivered efficiently and stably to target cells or organs, resulting in a relatively high specific antibody response in the animal body and providing better safety.

[0036] The three-component LNP produced using the steroid-cationic lipid compound provided by the present invention exhibits superior stability during the freeze-drying process, making it more suitable for the development and application of mRNA-LNP freeze-dried powder formulations. Simultaneously, the three-component LNP produced using the steroid-cationic lipid compound provided by the present invention maintains structural stability and efficient transfection activity even after spraying, thus offering excellent potential for application in mRNA spray inhalation or freeze-dried powder inhalation. [Brief explanation of the drawing]

[0037] [Figure 1] Figure 1 shows GFP-mRNA-LNP expression in Hep3B cells as detected by fluorescence microscopy. [Figure 2] Figure 2 shows the serum antibody titers determined by ELISA detection after mouse mRNA-LNP immunization. [Figure 3a] Figure 3a shows CD8 T cell immunity mediated by ICS detection after mouse immunomRNA-LNP. [Figure 3b] Figure 3b shows CD4 T cell immunity mediated by ICS detection after mouse immunomRNA-LNP. [Figure 4] Figure 4 shows serum IL-6 expression in mice after high-dose mRNA-LNP administration. [Figure 5] Figure 5 is a comparison chart showing DLS before and after LNP spraying. [Figure 6] Figure 6 shows the GFP fluorescent protein expression in Hep3B cells before and after transfection with GFP-mRNA-LNP detected by fluorescence microscopy. [Figure 7a] Figure 7a shows the living organism and organs after mouse Luc-mRNA-LNP is sprayed and inhaled. [Figure 7b] Figure 7b shows imaging of the living organism and organs after spray inhalation of mouse Luc-mRNA-LNP. [Modes for carrying out the invention]

[0038] The technical concepts in the embodiments of the present invention will be described clearly and completely below, in conjunction with the drawings of the embodiments; however, the embodiments described are merely a part of the embodiments of the present invention, and not all embodiments. All other embodiments that a person skilled in the art could obtain without creative effort based on the embodiments of the present invention fall within the scope of the claims of the present invention.

[0039] definition When used herein, the following words and phrases are intended to have the meanings set forth below, unless otherwise indicated by their context.

[0040] As used herein, the terms “lipid nanoparticle,” “LNP,” or “LNP” refer to nanoscale particles, such as those between 1 nm and 1,000 nm, that contain one or more types of lipid molecules.

[0041] As used herein, the term “gene-drug” typically consists of a vector or delivery system containing an engineered gene construct, the active component of which may be DNA, RNA, genetically modified virus, bacteria, or cells, and achieves the objective of treating and preventing disease by introducing an exogenous gene into target cells or tissues and replacing, compensating for, blocking, or modifying a specific gene.

[0042] As used herein, the term “nucleic acid” means a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, and includes DNA, RNA, and their hybrids.

[0043] As used herein, the terms “lipid compounds” or “lipids” refer to a group of organic compounds, including but not limited to esters of fatty acids, that are typically insoluble in water but soluble in many organic solvents. The organic solvents of the present invention include, but are not limited to, benzene, toluene, pentane, hexane, methanol, ethanol, isopropanol, ether, ethyl acetate, acetone, and carbon tetrachloride.

[0044] As used herein, the term “alkyl group” refers to a saturated linear or branched hydrocarbon group. 1~9 Alkyl alkyl groups include linear or branched alkyl groups containing 1, 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms, for example, C 1~8 , C 2~7 , C 2~8 , C 3~6 or C 4~7 Includes, but is not limited to, alkyl groups, C 6~24The alkyl group includes a linear or branched alkyl group containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbon atoms, for example, C 6~8 , C 6~17 , C 6~22 , C 10~16 , or C 14~17 alkyl group, including but not limited to these, C 1~5 The alkyl group includes a linear or branched alkyl group containing 1, 2, 3, 4 or 5 carbon atoms, for example, C 1~4 , C 2~4 , C 2~3 , C 3~5 , or C 4~5 alkyl group, including but not limited to these, C 1~20 The alkyl group includes a linear or branched alkyl group containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms, for example, C 1~8 , C 1~10 , C 6~22 , C 10~16 , or C 14~17 alkyl group, including but not limited to these.

[0045] As used herein, the term "alkenyl group" refers to an unsaturated linear or branched hydrocarbon group containing one or more unsaturated carbon-carbon double bonds. The unsaturated carbon-carbon double bond may be present at any stable point along the chain. As used herein, C 2~9 The alkenyl group includes a linear or branched alkenyl group containing 2, 3, 4, 5, 6, 7, 8 or 9 carbon atoms, for example, C 1~8 , C 2~7 , C 2~8 , C 3~6 , or C 4~8 linear or branched alkenyl group, including but not limited to these, C 6~24 The alkenyl group includes a linear or branched alkenyl group containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbon atoms, for example, C6~8 、 C 6~17 、 C 6~22 、 C 10~16 or C 14~17 including, but not limited to, an alkenyl group.

[0046] As used herein, the term "alkynyl group" refers to an unsaturated straight-chain or branched-chain hydrocarbon group containing one or more unsaturated carbon-carbon triple bonds. As used herein, C 2~9 alkynyl groups include straight-chain or branched-chain alkynyl groups containing 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms, for example, C 1~8 、 C 2~7 、 C 2~8 、 C 3~6 or C 4~7 including, but not limited to, an alkynyl group.

[0047] As used herein, one or more carbons at other positions of an alkyl group, an alkenyl group, or an alkynyl group, except for the positions of the terminal groups, may be substituted with heteroatoms such as nitrogen, oxygen, sulfur, and their oxides, for example, nitrogen oxides, carbonyl groups, sulfoxides, etc., for example, -(CH2) m -O-(CH2) n -, -(CH2) m -S-(CH2) n -, -(CH2) m -S-S-(CH2) n -, -(CH2) m -CO-(CH2) n -, -(CH2) m -OCO-(CH2) n -, -(CH2) m -OCOO-(CH2) n -(where m and n may be integers from 1 to 9) may be unsubstituted or substituted with one or more heteroatom substituents described herein. In some embodiments, the alkyl group, alkenyl group, or alkynyl group contains no heteroatoms.

[0048] As used herein, the term "alkylene group" refers to a divalent saturated straight-chain or branched-chain hydrocarbon group. 1~9 The alkylene group includes a linear or branched alkylene group containing 1, 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms, for example, C 1~8 , C 2~7 , C 2~8 , C 3~6 or C 4~7 C 1~12 Alkylene groups include linear or branched alkylene groups containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, for example, C 1~8 , C 2~7 , C 2~8 , C 3~6 or C 4~12 C 1~10 The alkylene group includes a linear or branched alkylene group containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, for example, C 1~8 , C 2~7 , C 2~8 , C 3~6 or C 4~10 C 1~6 Alkylene groups include linear or branched alkylene groups containing 1, 2, 3, 4, 5, or 6 carbon atoms, for example, C 1~5 , C 2~4 , C 3~6 or C 4~6 This includes, but is not limited to, alkylene groups.

[0049] As used herein, the term "alkenylene group" refers to an unsaturated straight-chain or branched-chain hydrocarbon group containing one or more unsaturated carbon-carbon double bonds. 2~9 Alkenylene groups include linear or branched alkenylene groups containing 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms, for example, C 2~7 , C 2~8 , C 3~6 or C 4~7C 2~12 Alkenylene groups include linear or branched alkenylene groups containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, for example, C 2~8 , C 2~7 , C 3~6 or C 4~12 C 2~6 Alkenylene groups include linear or branched alkenylene groups containing 2, 3, 4, 5, or 6 carbon atoms, for example, C 2~5 , C 2~4 , C 3~6 or C 4~6 This includes, but is not limited to, alkenylene groups.

[0050] As used herein, the term "alkynylene group" refers to an unsaturated linear or branched hydrocarbon group containing one or more unsaturated carbon-carbon triple bonds. 2~12 Alkynylene groups include linear or branched alkynylene groups containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, for example, C 2~8 , C 2~7 , C 3~6 or C 4~12 C 3~9 The alkylylene group includes linear or branched alkylylene groups containing 3, 4, 5, 6, 7, 8, or 9 carbon atoms, for example, C 3~6 , C 3~8 Some C 4~7 This includes, but is not limited to, an alkynylene group.

[0051] When used herein, an alkylene group, alkenylene group, or alkynylene group may contain one or more cyclic aliphatic groups and / or one or more heteroatoms such as oxygen, nitrogen, or sulfur, and may optionally be substituted with one or more substituents such as alkyl groups, halogen groups, alkoxy groups, hydroxyl groups, amino groups, aryl groups, ethers, esters, or amides. Except for the terminal group positions, one or more carbons at other positions may be substituted with heteroatoms such as nitrogen, oxygen, or sulfur and their oxides such as nitrogen oxides, carbonyl groups, sulfone groups, or sulfoxide groups. In some embodiments, the alkylene group, alkenylene group, or alkynylene group is unsubstituted. In some embodiments, the alkylene group, alkenylene group, or alkynylene group contains no heteroatoms at all.

[0052] As used herein, the term "nitrogen-containing heterocyclic group" means a heterocyclic group that contains a nitrogen atom in its structure and includes, but is not limited to, substituted or unsubstituted azilidinyl groups, azetidinyl groups, β-propiolactam groups, pyrrolyl groups, piperidinyl alkyl groups, pyrazolyl groups, imidazolyl groups, oxazolyl groups, isoxazolyl groups, thiazolyl groups, pyridyl groups, caprolactam groups, pyranyl groups, pyridadinyl groups, pyrimidinyl groups, pyrazinyl groups, piperazinyl groups, piperazinyl alkyl groups, indolyl groups, benzimidazolyl groups, carbazolyl groups, quinolinyl groups, isoquinolinyl groups, pteridinyl groups, acridinyl groups, 7H-prinyl groups, phenadinyl groups, phenothiazinyl groups, or 1H-azafuranil groups.

[0053] As used herein, the term “alkoxy group” means an “alkyl-O-” group, where alkyl is as defined herein. 6~24 The alkoxy group includes linear or branched alkoxy groups containing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms, for example, C 6~8 , C 6~17 , C6~22 , C 10~16 or C 14~17 This includes, but is not limited to, alkoxy groups.

[0054] As used herein, the term "cycloalkyl group" refers to a saturated cyclic hydrocarbon group. 3~8 Cycloalkyl groups include cycloalkyl groups containing 3, 4, 5, 6, 7, or 8 carbon atoms, for example, C 3~7 , C 4~7 or C 3~6 This includes, but is not limited to, cycloalkyl groups.

[0055] As used herein, the term "cycloalkenyl group" refers to a cyclic hydrocarbon group containing at least one carbon-carbon double bond. 3~8 Cycloalkenyl groups include cycloalkenyl groups containing 3, 4, 5, 6, 7, or 8 carbon atoms, for example, C 3~7 , C 4~7 or C 3~6 This includes, but is not limited to, cycloalkenyl groups.

[0056] As used herein, the term "cycloalkynyl group" refers to a cyclic hydrocarbon group containing at least one carbon-carbon triple bond. 3~8 A cycloalkynyl group includes a cycloalkynyl group containing 3, 4, 5, 6, 7, or 8 carbon atoms, for example, C 3~7 , C 4~7 or C 3~6 This includes, but is not limited to, cycloalkynyl groups.

[0057] As used herein, the term "cycloalkylene group" refers to a divalent cycloalkyl group used to link two structures. 3~12 Cycloalkylene groups include cycloalkylene groups containing 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, for example, C 3~8 , C4~7 , C 5~8 or C 6~12 This includes, but is not limited to, cycloalkylene groups, C 3~8 The cycloalkylene group includes a cycloalkyl group containing 3, 4, 5, 6, 7, or 8 carbon atoms, for example, C 3~7 , C 4~7 or C 6~8 This includes, but is not limited to, cycloalkylene groups.

[0058] As used herein, the term "cycloalkenylene group" refers to a divalent cycloalkenyl group used to link two structures. 3~12 The cycloalkenylene group includes a cycloalkenylene group containing 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, for example, C 3~8 , C 4~7 , C 5~8 or C 6~12 This includes, but is not limited to, cycloalkenylene groups, C 3~8 Cycloalkenylene groups include cycloalkenylene groups containing 3, 4, 5, 6, 7, or 8 carbon atoms, for example, C 3~7 , C 4~7 or C 6~8 This includes, but is not limited to, cycloalkenylene groups.

[0059] As used herein, the term "cycloalkylynylene group" refers to a divalent cycloalkylyl group used to link two structures. 3~12 The cycloalkylylene group includes a cycloalkylylene group containing 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, for example, C 3~8 , C 4~7 , C 5~8 or C 6~12 Includes, but is not limited to, cycloalkylene groups, C 3~8 The cycloalkylylene group includes a cycloalkylylene group containing 3, 4, 5, 6, 7, or 8 carbon atoms, for example, C 3~7 , C 4~7 or C6~8 This includes, but is not limited to, cycloalkylene groups.

[0060] As used herein, the term “arylene group” refers to a divalent aryl group used to link two structures, where the aryl group includes monocyclic aromatic groups (e.g., having 4n+2 delocalized electrons) and polycyclic aromatic groups. As used herein, C 6~12 Arylene groups include arylene groups containing 6, 7, 8, 9, 10, 11 or 12 carbon atoms, for example, C 6~8 , C 6~9 , C 6~12 This includes, but is not limited to, allerene groups.

[0061] As used herein, the term “cationic lipid” refers to a lipid molecule that can become positively charged in response to environmental pH values ​​or hydrogen ion activity.

[0062] As used herein, the term "helper lipid" refers to lipids that do not have a positive charge, including neutral lipids, zwitterionic lipids, and anionic lipids that have a negative charge, which have no charge under environmental pH conditions.

[0063] As used herein, the term "polyethylene glycol lipid" refers to a lipid molecule comprising a lipid portion and a polyethylene glycol portion.

[0064] As used herein, the term “delivery system” refers to a formulation or composition that modulates the in vivo distribution of a bioactive ingredient in space, time, and dose.

[0065] Unless otherwise specified, the methods used in this invention are all conventional methods, and unless otherwise specified, the reagents used in this invention are all commercially available products.

[0066] [Examples]

[0067] Example 1 Synthesis of Compound 1 [ka]

[0068] Step 1: Synthesis of 6-bromohexyl 2-hexyldecanoate (1a) 2-Hexyldecanoic acid (2.12 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and 6-bromo-n-hexanol (0.93 g, 5.0 mmol), 4-dimethylaminopyridine (DMAP, 0.21 g, 2.0 mmol), and triethylamine (0.62 g, 6.0 mmol) were added and dissolved under stirring. A solution of EDC.HCl (1.10 g, 6.0 mmol) in dichloromethane was added dropwise, and after the addition was complete, the mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3, and the solution was separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 1a (1.45 g, pale yellow oily substance) with a yield of 70%. MS m / z (ESI): 419.2 [M+1]

[0069] Step 2: Synthesis of 6-((4-hydroxybutyl)amino)hexyl 2-hexyldecanoate (1b)

[0070] Under room temperature conditions, 6-bromohexyl 2-hexyldecanoate 1a (1.28 g, 3 mmol) was dissolved in 20 mL of ethanol, 4-amino-1-butanol (4.00 g, 45 mmol) was added, the temperature was raised to 50°C, and the mixture was stirred for 8 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the mixture was cooled to room temperature, the ethanol was removed at 45°C, the crude product was dissolved in dichloromethane, washed three times with saturated brine, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 1b (1.13 g, pale yellow oily substance). MS m / z (ESI): 428.4 [M+1]

[0071] Step 3: Synthesis of 6-bromohexylcholesteryl carbonate (1c) 6-bromohexanol (0.91 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, 4-dimethylaminopyridine (1.22 g, 10 mmol) was added, and p-nitrophenyl chloroformate (1.11 g, 5.5 mmol) was added in batches. The reaction mixture was stirred at room temperature for 3 hours, cholesterol (2.16 g, 5.6 mmol) was added to the reaction mixture, and the mixture was stirred overnight at room temperature. After TLC indicated that the reaction was complete, the mixture was diluted with 20 mL of dichloromethane, then washed with 30 mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to obtain product 1c (1.83 g, pale yellow oily substance) with a yield of 62%. MS m / z (ESI): 593.3 [M+1]

[0072] Step 4: Synthesis of Compound 1 6-((4-hydroxybutyl)amino)hexyl 2-hexyldecanoate 1b (428 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 6-bromohexylcholesteryl carbonate 1c (711 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, and the residue was washed with dichloromethane. A saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 1 (470 mg, pale yellow oily substance) in a yield of 50%. MS m / z (ESI): 941.4 [M+1] 1H NMR (300MHz, CDCl3): δ 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.20-4.02 (m, 4H), 3.67-3.61 (m, 2H), 3.58-3.50 (m, 2H), 2.47-2.42 (m, 8H), 2.03-1.72 (m, 5H), 1.67-0.85 (m, 82H), 0.70 (s, 3H)

[0073] Example 2 Synthesis of Compound 2 [ka] 6-bromohexylcholesteryl carbonate 1c (1.48 g, 2.5 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 4-amino-1-butanol (89.2 mg, 1.0 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, and the residue was washed with dichloromethane. A saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 2 (679 mg, pale yellow solid) with a yield of 61%. MS m / z (ESI): 1115.2 [M+1] 1H NMR (300MHz, CDCl3): δ 5.40 (t, 2H, J = 5.4 Hz), 4.53-4.40 (m, 2H), 4.15 (t, 4H, J = 5.6 Hz), 3.76 (t, 2H, J = 5.4 Hz), 3.20-3.01 (m, 6H), 2.45-2.31 (m, 4H), 2.02-1.75 (m, 16H), 1.74-1.22 (m, 36H), 1.19-0.87 (m, 44H), 0.70 (s, 6H)

[0074] Example 3 Synthesis of Compound 3 [ka]

[0075] Step 1: Synthesis of 6-((2-(dimethylamino)ethyl)amino)hexyl 2-hexyldecanoate (3a) At room temperature, 6-bromohexyl 2-hexyldecanoate 1a (1.28 g, 3 mmol) was dissolved in 20 mL of ethanol, N,N-dimethylethylenediamine (4.00 g, 45 mmol) was added, the temperature was raised to 50°C, and the mixture was stirred for 8 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the mixture was cooled to room temperature, the ethanol was removed at 45°C, the crude product was dissolved in dichloromethane, washed three times with saturated brine, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 3a (1.20 g, pale yellow oily substance). MS m / z (ESI): 427.4 [M+1]

[0076] Step 2: Synthesis of Compound 3 6-((2-dimethylamino)ethyl)amino)hexyl 2-hexyldecanoate 3a (426 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 6-bromohexylcholesteryl carbonate 1c (711 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, the residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 3 (517 mg, pale yellow oily substance) in a yield of 55%. MS m / z (ESI): 940.0 [M+1] 1H NMR (300MHz, CDCl3): δ 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.20-4.02 (m, 3H), 3.76-3.61 (m, 2H), 3.45-3.38 (m, 3H), 2.94-2.85 (m, 1H), 2.55-2.27 (m, 5H), 2.03-0.85 (m, 70H), 0.70 (s, 3H)

[0077] Example 4 Synthesis of Compound 4 [ka]

[0078] Step 1: Synthesis of 6-((2-hydroxyethyl)amino)hexyl 2-hexyldecanoate (4a) At room temperature, 6-bromohexyl 2-hexyldecanoate 1a (1.28 g, 3 mmol) was dissolved in 20 mL of ethanol, ethanolamine (2.75 g, 45 mmol) was added, the temperature was raised to 50°C, and the mixture was stirred for 8 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the mixture was cooled to room temperature, the ethanol was removed at 45°C, the crude product was dissolved in dichloromethane, washed three times with saturated brine, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 4a (1.01 g, pale yellow oily substance). MS m / z (ESI): 400.4 [M+1]

[0079] Step 2: Synthesis of Compound 4 6-((2-hydroxyethyl)amino)hexyl 2-hexyldecanoate 4a (400 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 6-bromohexylcholesteryl carbonate 1c (711 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, and the residue was washed with dichloromethane. A saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 4 (474 ​​mg, pale yellow oily substance) with a yield of 52%. MS m / z (ESI): 913.1 [M+1] 1H NMR (300MHz, CDCl3): δ 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.20-4.02 (m, 4H), 3.67-3.61 (m, 2H), 3.58-3.50 (m, 4H), 2.47-2.42 (m, 2H), 2.03-1.72 (m, 5H), 1.67-0.85 (m, 82H), 0.70 (s, 3H)

[0080] Example 5 Synthesis of Compound 5 [ka]

[0081] Step 1: Synthesis of 2-hexyldecyl 8-bromooctanoate (5a) 8-bromooctanoic acid (1.12 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and 2-hexyldecanol (1.21 g, 5.0 mmol), DMAP (0.21 g, 2.0 mmol), and triethylamine (0.62 g, 6.0 mmol) were added and dissolved under stirring. A solution of EDC.HCl (1.10 g, 6.0 mmol) in dichloromethane was added dropwise, and after the addition was complete, the mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3, followed by liquid-liquid separation. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 5a (1.52 g, pale yellow oily substance) with a yield of 68%. MS m / z (ESI): 447.2 [M+1]

[0082] Step 2: Synthesis of 8-((4-hydroxybutyl)amino)octanoate 2-hexyldecyl (5b) Under room temperature conditions, 2-hexyldecyl 8-bromooctanoate (1.34 g, 3 mmol) was dissolved in 20 mL of ethanol, ethanolamine (4.00 g, 45 mmol) was added, the temperature was raised to 50°C, and the mixture was stirred for 8 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the mixture was cooled to room temperature, the ethanol was removed at 45°C, the crude product was dissolved in dichloromethane, washed three times with saturated brine, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 5b (1.21 g, pale yellow oily substance). MS m / z (ESI): 456.4 [M+1]

[0083] Step 3: Synthesis of cholesteryl (5c) 8-bromooctanoate 8-bromooctanoic acid (1.12 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and cholesterol (1.93 g, 5.0 mmol), DMAP (0.21 g, 2.0 mmol), and triethylamine (0.62 g, 6.0 mmol) were added and dissolved under stirring. A solution of EDC.HCl (1.10 g, 6.0 mmol) in dichloromethane was added dropwise, and after the addition was complete, the mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3, and the mixture was separated. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 5c (1.77 g, pale yellow oily substance) with a yield of 60%. MS m / z (ESI): 591.4 [M+1]

[0084] Step 4: Synthesis of Compound 5 8-((4-hydroxybutyl)amino)octanoic acid 2-hexyldecyl 5b (455 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 8-bromooctanoic acid cholesteryl 5c (708 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, and the residue was washed with dichloromethane. A saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 5 (464 mg, pale yellow oily substance) with a yield of 48%. MS m / z (ESI): 967.6 [M+1] 1H NMR (300MHz, CDCl3): δ 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.20-4.02 (m, 2H), 3.67-3.61 (m, 2H), 2.64 (t, 6H, J = 5.4 Hz), 2.37-2.25 (m, 6H), 2.10-0.85 (m, 93H), 0.70 (s, 3H)

[0085] Example 6 Synthesis of Compound 6 [ka]

[0086] Step 1: Synthesis of 6-((3-(1H-imidazole-1-yl)propyl)amino)hexyl 2-hexyldecanoate (6a) At room temperature, 6-bromohexyl 2-hexyldecanoate 1a (1.28 g, 3 mmol) was dissolved in 20 mL of ethanol, 1-(3-aminopropyl)imidazole (5.63 g, 45 mmol) was added, the temperature was raised to 50°C, and the mixture was stirred for 8 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the mixture was cooled to room temperature, the ethanol was removed at 45°C, the crude product was dissolved in dichloromethane, washed three times with saturated saline solution, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 6a (1.46 g, pale yellow oily substance). MS m / z (ESI): 464.4 [M+1]

[0087] Step 2: Synthesis of Compound 6 6-((3-(1H-imidazole-1-yl)propyl)amino)hexyl 2-hexyldecanoate 6a (463 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 6-bromohexylcholesteryl carbonate 1c (711 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, the residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 6 (517 mg, pale yellow oily substance) in a yield of 53%. MS m / z (ESI): 976.9 [M+1] 1H NMR (300MHz, CDCl3): δ 7.49 (s, 1H), 7.08 (d, 1H, J = 5.6 Hz), 6.93 (d, 1H, J = 5.6 Hz), 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.20-3.95 (m, 5H), 2.47-2.30 (m, 9H), 2.36-0.85 (m, 87H), 0.70 (s, 3H)

[0088] Example 7 Synthesis of Compound 7 [ka]

[0089] Step 1: Synthesis of 6-bromohexyl undecylcarbamate (7a) 6-bromohexanol (0.91 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and 4-dimethylaminopyridine (1.22 g, 10 mmol) was added. Then, in a batch, p-nitrophenyl chloroformate (1.11 g, 5.5 mmol) was added, and the reaction mixture was stirred at room temperature for 3 hours. Undecylamine (0.96 g, 5.6 mmol) was added to the reaction mixture, and the mixture was stirred overnight at room temperature. After TLC indicated that the reaction was complete, the mixture was diluted with 20 mL of dichloromethane, then washed with 30 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered and concentrated to obtain compound 7a (1.23 g, pale yellow oily substance) in a yield of 65%. MS m / z (ESI): 378.2 [M+1]

[0090] Step 2: Synthesis of 6-((4-hydroxybutyl)amino)hexyl undecylcarbamate (7b) Under room temperature conditions, 6-bromohexyl undecylcarbamate 7a (1.13 g, 3 mmol) was dissolved in 20 mL of ethanol, ethanolamine (4.00 g, 45 mmol) was added, the temperature was raised to 50°C, and the mixture was stirred for 8 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the temperature was lowered to room temperature, the ethanol was removed at 45°C, the crude product was dissolved in dichloromethane, washed three times with saturated brine, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 7b (1.14 g, pale yellow oily substance). MS m / z (ESI): 387.4 [M+1]

[0091] Step 3: Synthesis of Compound 7 6-((4-hydroxybutyl)amino)hexylundecylcarbamate 7b (386 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 6-bromohexylcholesterylcarbonate 1c (711 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, the residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 7 (459 mg, pale yellow oily substance) in a yield of 51%. MS m / z (ESI): 900.4 [M+1] 1H NMR (300MHz, CDCl3): δ 5.42 (t, 1H, J = 5.4 Hz), 4.80-4.71 (m, 1H), 4.51-4.40 (m, 1H), 4.20-4.02 (m, 4H), 3.75-3.61 (m, 2H), 3.20-3.11 (m, 2H), 2.91-2.73 (m, 5H), 2.43-2.36 (m, 2H), 2.10-0.83 (m, 79H), 0.70 (s, 3H)

[0092] Example 8 Synthesis of Compound 8 [ka]

[0093] Step 1: Synthesis of S-undecyl 8-bromooctanthioate (8a) 8-bromooctanoic acid (1.12 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and undecanethiol (0.94 g, 5.0 mmol), DMAP (0.21 g, 2.0 mmol), and triethylamine (0.62 g, 6.0 mmol) were added and dissolved under stirring. A solution of EDC.HCl (1.10 g, 6.0 mmol) in dichloromethane was added dropwise, and after the addition was complete, the mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3, followed by liquid-liquid separation. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 8a (1.30 g, pale yellow oily substance) with a yield of 66%. MS m / z (ESI): 393.2 [M+1]

[0094] Step 2: Synthesis of S-undecyl 8-((4-hydroxybutyl)amino)octanthioate(8b) Under room temperature conditions, S-undecyl 8-bromooctanthioate 8a (1.18 g, 3 mmol) was dissolved in 20 mL of ethanol, ethanolamine (4.00 g, 45 mmol) was added, the temperature was raised to 50°C, and the mixture was stirred for 8 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the mixture was cooled to room temperature, the ethanol was removed at 45°C, the crude product was dissolved in dichloromethane, washed three times with saturated brine, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 8b (1.18 g, pale yellow oily substance). MS m / z (ESI): 402.3 [M+1]

[0095] Step 3: Synthesis of Compound 8 S-Undecyl 8-((4-hydroxybutyl)amino)octanthioate 8b (401 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 6-bromohexylcholesteryl carbonate 1c (711 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, and the residue was washed with dichloromethane. A saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 8 (458 mg, pale yellow oily substance) in a yield of 50%. MS m / z (ESI): 914.9 [M+1] 1H NMR (300MHz, CDCl3): δ 5.42 (t, 1H, J = 5.4 Hz), 4.51-4.40 (m, 1H), 4.20-4.02 (m, 2H), 3.65-3.51 (m, 2H), 2.91-2.83 (m, 2H), 2.60-2.36 (m, 10H), 2.10-0.86 (m, 82H), 0.70 (s, 3H)

[0096] Example 9 Synthesis of Compound 9 [ka]

[0097] Step 1: Synthesis of 6-((4-((tert-butoxycarbonyl)amino)butyl)amino)hexyl 2-hexyldecanoate (9a)

[0098] At room temperature, 6-bromohexyl 2-hexyldecanoate 1a (1.28 g, 3 mmol) was dissolved in 20 mL of ethanol, BOC-1,4-butanediamine hydrochloride (10.1 g, 45 mmol) was added, the temperature was raised to 50°C, and the mixture was stirred for 8 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the mixture was cooled to room temperature, the ethanol was removed at 45°C, the crude product was dissolved in dichloromethane, washed three times with saturated saline solution, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 9a (1.51 g, pale yellow oily substance). MS m / z (ESI): 527.5 [M+1]

[0099] Step 2: Synthesis of Compound 9b 6-((4-((tert-butoxycarbonyl)amino)butyl)amino)hexyl 2-hexyldecanoate 9a (526 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 6-bromohexylcholesteryl carbonate 1c (711 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, the residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 9b (540 mg, pale yellow oily substance) with a yield of 52%. MS m / z (ESI): 1039.9 [M+1]

[0100] Step 3: Synthesis of Compound 9 Compound 9b (1.04 g, 1.0 mmol) was dissolved in dichloromethane, and a dichloromethane solution of trifluoroacetic acid was added under ice bath conditions. The mixture was stirred at room temperature for 16 hours. Trifluoroacetic acid was removed using a rotary evaporator, and the mixture was dissolved in dichloromethane. The mixture was washed twice with saturated sodium bicarbonate solution, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain compound 9 (460 mg, pale yellow oily substance) in a yield of 49%. MS m / z (ESI): 940.1 [M+1] 1H NMR (300MHz, CDCl3): δ 5.40 (t, 1H, J = 5.4 Hz), 4.63-4.40 (m, 1H), 4.19-4.03 (m, 4H), 2.79-2.70 (m, 2H), 2.55-2.28 (m, 10H), 2.02-0.87 (m, 89H), 0.70 (s, 3H)

[0101] Example 10 Synthesis of Compound 10 [ka]

[0102] Step 1: Synthesis of 4-bromobutylcholesteryl carbonate (10a) 4-bromobutanol (0.77 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and 4-dimethylaminopyridine (1.22 g, 10 mmol) was added. Then, in a batch, p-nitrophenyl chloroformate (1.11 g, 5.5 mmol) was added, and the reaction mixture was stirred at room temperature for 3 hours. Cholesterol (2.16 g, 5.6 mmol) was added to the reaction mixture, and the mixture was stirred overnight at room temperature. After TLC indicated that the reaction was complete, the mixture was diluted with 20 mL of dichloromethane, then washed with 30 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered and concentrated to obtain product 10a (1.50 g, pale yellow oily substance) with a yield of 53%. MS m / z (ESI): 565.3 [M+1]

[0103] Step 2: Synthesis of Compound 10 6-((3-(1H-imidazole-1-yl)propyl)amino)hexyl 2-hexyldecanoate 6a (463 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 4-bromobutylcholesteryl carbonate 10a (678 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, the residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 10 (455 mg, pale yellow oily substance) with a yield of 48%. MS m / z (ESI): 948.9 [M+1] 1H NMR (300MHz, CDCl3): δ 7.49 (s, 1H), 7.08 (d, 1H, J = 5.6 Hz), 6.93 (d, 1H, J = 5.6 Hz), 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.20-3.95 (m, 6H), 2.47-2.30 (m, 8H), 2.36-0.85 (m, 83H), 0.70 (s, 3H)

[0104] Example 11 Synthesis of Compound 11 [ka]

[0105] Step 1: Synthesis of 2-bromoethylcholesteryl carbonate (11a) 2-bromoethanol (0.63 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and 4-dimethylaminopyridine (1.22 g, 10 mmol) was added. Then, in a batch, p-nitrophenyl chloroformate (1.11 g, 5.5 mmol) was added, and the reaction mixture was stirred at room temperature for 3 hours. Cholesterol (2.16 g, 5.6 mmol) was added to the reaction mixture, and the mixture was stirred overnight at room temperature. After TLC indicated that the reaction was complete, the mixture was diluted with 20 mL of dichloromethane, then washed with 30 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain product 11a (1.53 g, pale yellow oily substance) with a yield of 57%. MS m / z (ESI): 537.3 [M+1]

[0106] Step 2: Synthesis of Compound 11 6-((3-(1H-imidazole-1-yl)propyl)amino)hexyl 2-hexyldecanoate 6a (463 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 2-bromoethylcholesteryl carbonate 11a (644 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, the residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 11 (506 mg, pale yellow oily substance) in a yield of 55%. MS m / z (ESI): 920.8 [M+1] 1H NMR (300MHz, CDCl3): δ 7.49 (s, 1H), 7.08 (d, 1H, J = 5.6 Hz), 6.93 (d, 1H, J = 5.6 Hz), 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.20-3.95 (m, 6H), 2.74-2.365 (m, 2H), 2.47-2.30 (m, 6H), 2.36-0.85 (m, 79H), 0.70 (s, 3H)

[0107] Example 12 Synthesis of Compound 12 [ka]

[0108] 6-((4-hydroxybutyl)amino)hexyl 2-hexyldecanoate 1b (428 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 4-bromobutylcholesteryl carbonate 10a (679 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, and the residue was washed with dichloromethane. A saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain product 12 (566 mg, pale yellow oily substance) with a yield of 62%. MS m / z (ESI): 913.0 [M+1] 1H NMR (300MHz, CDCl3): δ 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.20-4.02 (m, 4H), 3.67-3.61 (m, 2H), 2.63-2.32 (m, 8H), 2.03-1.72 (m, 5H), 1.67-0.85 (m, 80H), 0.70 (s, 3H)

[0109] Example 13 Synthesis of Compound 13 [ka] 6-((4-hydroxybutyl)amino)hexyl 2-hexyldecanoate 1b (428 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 4-bromobutylcholesteryl carbonate 11a (645 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, and the residue was washed with dichloromethane. A saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain product 13 (451 mg, pale yellow oily substance) with a yield of 51%. MS m / z (ESI): 885.1 [M+1] 1H NMR (300MHz, CDCl3): δ 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.29-4.21 (m, 2H), 4.20-4.02 (m, 2H), 3.67-3.61 (m, 2H), 2.85-2.78 (m, 2H), 2.63-2.32 (m, 6H), 2.03-1.72 (m, 5H), 1.67-0.85 (m, 76H), 0.70 (s, 3H)

[0110] Example 14 Synthesis of Compound 14 [ka]

[0111] Step 1: Synthesis of 2-(hexyloxy)-1-(pentyloxy)ethyl 7-bromoheptanoate (14a) 7-bromoheptanoic acid (1.05 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and 2-(hexyloxy)-1-(pentyloxy)1-ethanol (1.16 g, 5.0 mmol), DMAP (0.21 g, 2.0 mmol), and triethylamine (0.62 g, 6.0 mmol) were added and dissolved under stirring. A solution of EDC.HCl (1.10 g, 6.0 mmol) in dichloromethane was added dropwise, and after the addition was complete, the mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3, followed by liquid-liquid separation. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 14a (1.08 g, pale yellow oily substance) with a yield of 51%. MS m / z (ESI): 423.2 [M+1]

[0112] Step 2: Synthesis of 2-(hexyloxy)-1-(pentyloxy)ethyl 7-((4-hydroxybutyl)amino)heptanoate (14b)

[0113] Under room temperature conditions, 2-(hexyloxy)-1-(pentyloxy)ethyl 7-bromoheptanoate 14a (1.27 g, 3 mmol) was dissolved in 20 mL of ethanol, ethanolamine (4.00 g, 45 mmol) was added, the temperature was raised to 50°C, and the mixture was stirred for 8 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the mixture was cooled to room temperature, the ethanol was removed at 45°C, the crude product was dissolved in dichloromethane, washed three times with saturated brine, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 14b (1.05 g, pale yellow oily substance). MS m / z (ESI): 432.4 [M+1]

[0114] Step 3: Synthesis of Compound 14 2-(hexyloxy)-1-(pentyloxy)ethyl 7-((4-hydroxybutyl)amino)heptanoate 14b (432 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, cholesteryl 8-bromooctanoate 5c (708 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, the residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 14 (490 mg, pale yellow oily substance) with a yield of 52%. MS m / z (ESI): 942.8 [M+1] 1H NMR (300MHz, CDCl3): δ 6.67 (t, 1H, J= 6.3 Hz), 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.20-4.02 (m, 2H), 3.67-3.61 (m, 2H), 3.47-3.31 (m, 4H), 2.64 (t, 6H, J = 5.4 Hz), 2.37-2.25 (m, 6H), 2.10-0.85 (m, 80H), 0.70 (s, 3H)

[0115] Example 15 Synthesis of Compound 15 [ka]

[0116] Step 1: Synthesis of heptadecane-9-yl-3-oxopropanoate (15a) 3-oxopropanoic acid (0.44 g, 5.0 mmol) was dissolved in 20 mL of dichloromethane, and 9-heptadecanol (1.28 g, 5.0 mmol), DMAP (0.21 g, 2.0 mmol), and triethylamine (0.62 g, 6.0 mmol) were added and dissolved under stirring. A solution of EDC.HCl (1.10 g, 6.0 mmol) in dichloromethane was added dropwise, and after the addition was complete, the mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3, followed by liquid-liquid separation. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 15a (0.78 g, pale yellow oily substance) with a yield of 48%. MS m / z (ESI): 327.3 [M+1]

[0117] Step 2: Synthesis of heptadecane-9-yl 3-hydroxypropanoate (15b) Heptadecan-9-yl-3-oxopropanoate 15a (0.78 g, 2.3 mmol) was dissolved in 10 mL of n-butanol, sodium borohydride (266 mg, 7 mmol) was added at 0°C, and the mixture was stirred at room temperature for 12 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the reaction was quenched with saturated ammonium chloride solution, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 15b (0.70 g, pale yellow oily substance). MS m / z (ESI): 329.3 [M+1]

[0118] Step 3: Synthesis of Compound 15c 3-bromopropionic acid (306 mg, 2.0 mmol) was dissolved in 10 mL of dichloromethane, and heptadecan-9-yl 3-hydroxypropanoate 15b (658 mg, 2.0 mmol), DMAP (84 mg, 0.8 mmol), and triethylamine (248 mg, 2.4 mmol) were added and dissolved under stirring. A solution of EDC.HCl (440 mg, 2.4 mmol) in dichloromethane was added dropwise, and after the addition was complete, the mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3, followed by liquid-liquid separation. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 15c (528 mg, pale yellow oily substance) with a yield of 57%. MS m / z (ESI): 463.2 [M+1]

[0119] Step 4: Synthesis of Compound 15d Under room temperature conditions, compound 15c (528 mg, 1.14 mmol) was dissolved in 10 mL of ethanol, ethanolamine (1.5 g, 17 mmol) was added, the temperature was raised to 50°C, and the mixture was stirred for 8 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the mixture was cooled to room temperature, the ethanol was removed at 45°C, the crude product was dissolved in dichloromethane, washed three times with saturated brine, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 15d (480 mg, pale yellow oily substance). MS m / z (ESI): 472.4 [M+1]

[0120] Step 5: Synthesis of Compound 15e 3-oxopropanoic acid (0.44 g, 5.0 mmol) was dissolved in 20 mL of dichloromethane, and cholesterol (1.93 g, 5.0 mmol), DMAP (0.21 g, 2.0 mmol), and triethylamine (0.62 g, 6.0 mmol) were added and dissolved under stirring. A solution of EDC.HCl (1.10 g, 6.0 mmol) in dichloromethane was added dropwise, and after the addition was complete, the mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3, followed by liquid-liquid separation. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 15a (1.03 g, pale yellow oily substance) with a yield of 45%. MS m / z (ESI): 456.4 [M+1]

[0121] Step 6: Synthesis of Compound 15f Compound 15e (1.03 g, 2.2 mmol) was dissolved in 10 mL of n-butanol, sodium borohydride (266 mg, 7 mmol) was added at 0°C, and the mixture was stirred at room temperature for 12 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the reaction was quenched by adding saturated ammonium chloride solution, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 15f (980 mg, pale yellow oily substance). MS m / z (ESI): 459.4 [M+1]

[0122] Step 7: Synthesis of 15g of compound 3-bromopropionic acid (306 mg, 2.0 mmol) was dissolved in 10 mL of dichloromethane, and compound 15f (916 mg, 2.0 mmol), DMAP (84 mg, 0.8 mmol), and triethylamine (248 mg, 2.4 mmol) were added and dissolved under stirring. A solution of EDC.HCl (440 mg, 2.4 mmol) in dichloromethane was added dropwise, and after the addition was complete, the mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3, followed by liquid-liquid separation. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 15 g of compound (629 mg, pale yellow oily substance) with a yield of 53%. MS m / z (ESI): 593.3 [M+1]

[0123] Step 8: Synthesis of Compound 15 Compound 15d (480 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, compound 15 g (600 mg, 1.1 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, the residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 15 (384 mg, pale yellow oily substance) with a yield of 39%. MS m / z (ESI): 984.8 [M+1] 1H NMR (300MHz, CDCl3): δ 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.30 (m, 6H), 3.77-3.71 (m, 4H), 3.47-3.31 (m, 2H), 3.12-3.05 (m, 2H), 2.68-2.60 (m, 8H), 2.37-2.25 (m, 6H), 2.10-0.85 (m, 72H), 0.70 (s, 3H)

[0124] Example 16 Synthesis of Compound 16 [Chemical formula]

[0125] Step 1: Synthesis of Compound 16a Dissolve 2-hexyldecanoic acid (1.28 g, 5.0 mmol) in 20 mL of dichloromethane. Add bis(2-hydroxyethyl) disulfide (1.54 g, 10.0 mmol), DMAP (0.21 g, 2.0 mmol), and triethylamine (0.62 g, 6.0 mmol), and dissolve with stirring. Dropwise add a dichloromethane solution of EDC·HCl (1.10 g, 6.0 mmol). After the addition is complete, stir at room temperature for 16 hours. Add water for quenching, add dilute hydrochloric acid to adjust the pH value to 1 - 3, and perform liquid separation. Wash the organic phase with saturated brine, dry it over anhydrous sodium sulfate, filter, concentrate, and obtain Compound 16a (1.00 g, light yellow oil) by column chromatography, with a yield of 51%. MS m / z (ESI): 393.2 [M+1]

[0126] Step 2: Synthesis of Compound 16b [[ID=第十九]]Dissolve Compound 16a (1.00 g, 2.5 mmol) in 20 mL of dichloromethane. Add triethylamine (0.76 g, 7.5 mmol), lower the temperature to 0 °C in an ice-water bath, add methanesulfonyl chloride (0.72 g, 6.2 mmol), and stir at room temperature for 16 - 20 h. After complete reaction, wash twice with saturated sodium bicarbonate and twice with water. Dry the organic phase over anhydrous sodium sulfate, filter, concentrate, and obtain Compound 16b (1.00 g, light yellow oil), with a yield of 85%. MS m / z (ESI): 471.2 [M+1]

[0127] Step 3: Synthesis of Compound 16c Compound 16b (1.00 g, 2.0 mmol) was dissolved in 20 mL of acetonitrile, potassium carbonate (0.83 g, 6.0 mmol) and 4-aminobutanol (0.18 g, 2.0 mmol) were added, and the mixture was stirred at room temperature for 16 - 20 h. After the reaction was complete, it was filtered through diatomaceous earth, dichloromethane was added to the filtrate, and it was washed twice with saturated sodium bicarbonate and twice with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 16c (658 mg, pale yellow oil), with a yield of 71%. MS m / z (ESI): 464.3 [M+1]

[0128] Step 4: Synthesis of compound 16d Cholesterol (1.93 g, 5.0 mmol) was dissolved in 20 mL of dichloromethane, 4-dimethylaminopyridine (1.22 g, 10 mmol) was added, and then p-nitrophenyl chloroformate (1.11 g, 5.5 mmol) was added batchwise. The reaction mixture was stirred at room temperature for 3 h. Bis(2-hydroxyethyl) disulfide (0.86 g, 5.6 mmol) was added to this reaction mixture, and the mixture was stirred at room temperature overnight. After TLC indicated that the reaction was complete, 20 mL of dichloromethane was added for dilution, and then it was washed with 30 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain product 16d (1.45 g, pale yellow oil), with a yield of 51%. MS m / z (ESI): 567.4 [M+1]

[0129] Step 5: Synthesis of compound 16e Compound 16d (1.42 g, 2.5 mmol) was dissolved in 20 mL of dichloromethane, triethylamine (0.76 g, 7.5 mmol) was added, the temperature was lowered to 0 °C in an ice-water bath, methanesulfonyl chloride (0.72 g, 6.2 mmol) was added, and the mixture was stirred at room temperature for 16 - 20 h. After the reaction was complete, it was washed twice with saturated sodium bicarbonate and twice with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 16e (1.26 g, pale yellow oil), with a yield of 78%. MS m / z (ESI): 645.3 [M+1]

[0130] Step 6: Synthesis of Compound 16 Compound 16e (1.26 g, 2.0 mmol) was dissolved in 20 mL of acetonitrile, and potassium carbonate (0.83 g, 6.0 mmol) and compound 16c (0.93 g, 2.0 mmol) were added. The mixture was stirred at room temperature for 16-20 hours. After complete reaction, the mixture was filtered through diatomaceous earth, dichloromethane was added to the filtrate, and the mixture was washed twice with saturated sodium bicarbonate and twice with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 16 (871 mg, pale yellow oily substance) with a yield of 43%. MS m / z (ESI): 1012.7 [M+1] 1H NMR (300MHz, CDCl3): δ 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.20-4.02 (m, 4H), 3.67-3.61 (m, 2H), 3.58-3.50 (m, 2H), 2.47-2.42 (m, 8H), 2.03-1.72 (m, 5H), 1.67-0.85 (m, 74H), 0.70 (s, 3H)

[0131] Example 17 Synthesis of Compound 17 [ka]

[0132] Step 1: Synthesis of Compound 17a 4-bromobutyric acid (0.84 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and cholesterol (1.94 g, 5.0 mmol), DMAP (0.21 g, 2.0 mmol), and triethylamine (0.62 g, 6.0 mmol) were added and dissolved under stirring. A solution of EDC.HCl (1.10 g, 6.0 mmol) in dichloromethane was added dropwise, and after the addition was complete, the mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3, followed by liquid-liquid separation. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 17a (1.68 g, pale yellow oily substance) with a yield of 63%. MS m / z (ESI): 535.3 [M+1]

[0133] Step 2: Synthesis of Compound 17 Compound 6a (463 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, compound 17a (643 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, the residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 17 (505 mg, pale yellow oily substance) with a yield of 55%. MS m / z (ESI): 918.9 [M+1] 1H NMR (300MHz, CDCl3): δ 7.49 (s, 1H), 7.08 (s, 1H), 6.94 (s, 1H), 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.10-3.92 (m, 4H), 2.48-2.25 (m, 11H), 2.10-0.85 (m, 80H), 0.70 (s, 3H)

[0134] Example 18 Synthesis of Compound 18 [ka]

[0135] Step 1: Synthesis of Compound 18a 2-Hexyldecanoic acid (2.12 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and 6-bromohexylamine hydrochloride (1.09 g, 5.0 mmol), HATU (2.28 g, 6.0 mmol), and DIPEA (1.29 g, 10.0 mmol) were added. The mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3. The mixture was then separated. The organic phase was washed with saturated saline solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 18a (1.40 g, pale yellow oily substance) with a yield of 67%. MS m / z (ESI): 418.3 [M+1]

[0136] Step 2: Synthesis of Compound 18b Under room temperature conditions, compound 18a (1.25 g, 3 mmol) was dissolved in 20 mL of ethanol, 1-(3-aminopropyl)imidazole (5.63 g, 45 mmol) was added, the temperature was raised to 50°C, and the mixture was stirred for 8 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the mixture was cooled to room temperature, the ethanol was removed at 45°C, the crude product was dissolved in dichloromethane, washed three times with saturated brine, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 18b (1.42 g, pale yellow oily substance). MS m / z (ESI): 463.4 [M+1]

[0137] Step 3: Synthesis of Compound 18c 1,4-Dibromobutane (2.16 g, 10.0 mmol) was dissolved in 30 mL of n-butanol, cholesterol (1.94 g, 5.0 mmol) and sodium hydroxide (1.20 g, 30 mmol) were added, and the mixture was heated with stirring under reflux for 5 h. After the reaction was completed, dilute hydrochloric acid was added to adjust the pH to neutral. n-Butanol was removed by a rotary evaporator, diluted with 20 mL of dichloromethane, washed with 30 mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the product 18c (1.25 g, pale yellow oil) was obtained by column chromatography, with a yield of 48%. MS m / z (ESI): 521.3 [M+1]

[0138] Step 4: Synthesis of Compound 18 Compound 18b (463 mg, 1.0 mmol) was dissolved in tetrahydrofuran, acetonitrile, compound 18c (625 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added, and the mixture was stirred at 83 °C for 16 - 20 h. It was cooled to room temperature, filtered, the residue was washed with dichloromethane, saturated sodium hydrogen carbonate solution was added to the obtained filtrate, extracted twice with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain the product 18 (434 mg, pale yellow oil), with a yield of 48%. MS m / z (ESI): 903.9 [M+1] 1H NMR (300MHz, CDCl3): δ 8.01 (s, 1H), 7.49 (s, 1H), 7.08 (s, 1H), 6.94 (s, 1H), 5.40 (t, 1H, J = 5.4 Hz), 4.10 - 3.92 (m, 2H), 3.35 (t, 2H, J = 5.4 Hz), 3.28 - 3.01 (m, 7H), 2.50 - 2.42 (m, 4H), 2.10 - 0.85 (m, 83H), 0.70 (s, 3H)

[0139] Example 19 Synthesis of Compound 19 [ka]

[0140] Step 1: Synthesis of Compound 19a At room temperature, 6-bromohexyl 2-hexyldecanoate 1a (1.28 g, 3 mmol) was dissolved in 20 mL of ethanol, 1-(3-aminopropyl)-4-methylpiperazine (7.07 g, 45 mmol) was added, the temperature was raised to 50°C, and the mixture was stirred for 8 hours while monitoring the progress of the reaction. After the consumption of the starting materials was complete, the mixture was cooled to room temperature, the ethanol was removed at 45°C, the crude product was dissolved in dichloromethane, washed three times with saturated saline solution, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain compound 19a (1.49 g, pale yellow oily substance). MS m / z (ESI): 496.5 [M+1]

[0141] Step 2: Synthesis of Compound 19 Compound 19a (496 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, compound 11a (646 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, the residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 19 (543 mg, pale yellow oily substance) with a yield of 57%. MS m / z (ESI): 952.9 [M+1] 1H NMR (300MHz, CDCl3): δ 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.20-3.95 (m, 6H), 2.74-2.65 (m, 2H), 2.47-2.40 (m, 6H), 2.36-0.85 (m, 90H), 0.70 (s, 3H)

[0142] Example 20 Synthesis of Compound 20 [ka]

[0143] Step 1: Synthesis of compound 20a 4-bromobutanol (0.77 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and 4-dimethylaminopyridine (1.22 g, 10 mmol) was added. Then, in a batch, p-nitrophenyl chloroformate (1.11 g, 5.5 mmol) was added, and the reaction mixture was stirred at room temperature for 3 hours. β-sitosterol (2.32 g, 5.6 mmol) was added to the reaction mixture, and the mixture was stirred overnight at room temperature. After TLC indicated that the reaction was complete, the mixture was diluted with 20 mL of dichloromethane, then washed with 30 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered and concentrated to obtain product 20a (1.63 g, pale yellow oily substance) with a yield of 55%. MS m / z (ESI): 593.3 [M+1]

[0144] Step 2: Synthesis of Compound 20 6-((3-(1H-imidazole-1-yl)propyl)amino)hexyl 2-hexyldecanoate 6a (463 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, compound 20a (713 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, the residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 20 (547 mg, pale yellow oily substance) with a yield of 56%. MS m / z (ESI): 976.9 [M+1] 1H NMR (300MHz, CDCl3): δ 7.49 (s, 1H), 7.08 (d, 1H, J = 5.6 Hz), 6.93 (d, 1H, J = 5.6 Hz), 5.40 (t, 1H, J = 5.4 Hz), 4.53-4.40 (m, 1H), 4.20-3.95 (m, 6H), 2.47-2.30 (m, 8H), 2.36-0.85 (m, 87H), 0.70 (s, 3H)

[0145] Example 21 Synthesis of Compound 21 [ka]

[0146] Step 1: Synthesis of compound 21a 6-bromohexanoic acid (0.975 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and cholesterol (1.93 g, 5.0 mmol), DMAP (0.21 g, 2.0 mmol), and triethylamine (0.62 g, 6.0 mmol) were added and dissolved under stirring. A solution of EDC.HCl (1.10 g, 6.0 mmol) in dichloromethane was added dropwise, and after the addition was complete, the mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3, followed by liquid-liquid separation. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 21a (1.92 g, pale yellow oily substance) with a yield of 68%. MS m / z (ESI): 563.4 [M+1]

[0147] Step 2: Synthesis of Compound 21 8-((4-hydroxybutyl)amino)octanoic acid 2-hexyldecyl 5b (455 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, 6-bromohexanoic acid cholesteryl 21a (676 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, the residue was washed with dichloromethane, saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 21 (422 mg, pale yellow oily substance) in a yield of 45%. MS m / z (ESI): 938.9 [M+1] 1H NMR (400 MHz, CDCl3) δ 5.27 (tt, J = 12.5, 2.0 Hz, 1H), 4.64 (p, J = 14.8 Hz, 1H), 4.31 (ddd, J = 103.0, 24.8, 14.1 Hz, 2H), 3.46 (t, J = 14.8 Hz, 2H), 2.78 - 2.63 (m, 6H), 2.44 - 2.27 (m, 4H), 2.27 - 1.47 (m, 21H), 1.45 - 1.15 (m, 47H), 1.15 - 0.96 (m, 4H), 0.95 - 0.83 (m, 21H).

[0148] Example 22 Synthesis of Compound 24 [ka]

[0149] Compound 6a (463 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, compound 18c (625 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, and the residue was washed with dichloromethane. A saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain product 24 (470 mg, pale yellow oily substance) with a yield of 52%. MS m / z (ESI): 904.9 [M+1] 1H NMR (400 MHz, CDCl3) δ 7.92 (s, 1H), 7.24 - 6.71 (m, 2H), 5.27 (tt, J = 12.5, 2.0 Hz, 1H), 4.17 - 3.98 (m, 4H), 3.57 - 3.31 (m, 3H), 2.79 - 2.63 (m, 4H), 2.47 - 2.38 (m, 4H), 2.37 - 1.77 (m, 7H), 1.75 - 1.00 (m, 58H), 0.96 - 0.81 (m, 21H).

[0150] Example 23 Synthesis of Compound 25 [ka]

[0151] Step 1: Synthesis of Compound 25a 4-bromobutyric acid (0.835 g, 5.0 mmol) was dissolved in 30 mL of dichloromethane, and cholesterol (1.93 g, 5.0 mmol), DMAP (0.21 g, 2.0 mmol), and triethylamine (0.62 g, 6.0 mmol) were added and dissolved under stirring. A solution of EDC.HCl (1.10 g, 6.0 mmol) in dichloromethane was added dropwise, and after the addition was complete, the mixture was stirred at room temperature for 16 hours. Water was added to quench the mixture, and dilute hydrochloric acid was added to adjust the pH to 1-3, followed by liquid-liquid separation. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 25a (1.90 g, pale yellow oily substance) with a yield of 71%. MS m / z (ESI): 535.4 [M+1]

[0152] Step 2: Synthesis of Compound 25 8-((4-hydroxybutyl)amino)octanoic acid 2-hexyldecyl 5b (455 mg, 1.0 mmol) was dissolved in tetrahydrofuran, and acetonitrile, cholesteryl 4-bromobutanoate 21a (643 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), and potassium iodide (332 mg, 2.0 mmol) were added. The mixture was stirred at 83°C for 16-20 hours. After cooling to room temperature, the mixture was filtered, and the residue was washed with dichloromethane. A saturated sodium bicarbonate solution was added to the resulting filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain compound 25 (373 mg, pale yellow oily substance) with a yield of 41%. MS m / z (ESI): 910.9 [M+1] 1H NMR (400 MHz, CDCl3) δ 5.27 (tt, J = 12.5, 2.0 Hz, 1H), 4.68 (p, J = 14.8 Hz, 1H), 4.31 (ddd, J = 103.0, 24.8, 13.0 Hz, 2H), 3.46 (t, J = 14.8 Hz, 2H), 2.79 - 2.62 (m, 4H), 2.43 (td, J = 12.2, 6.0 Hz, 4H), 2.37 - 2.11 (m, 3H), 2.11 - 1.80 (m, 9H), 1.79 - 1.46 (m, 11H), 1.45 - 1.15 (m, 43H), 1.14 - 0.98 (m, 4H), 0.97 - 0.80 (m, 21H).

[0153] Example 24: Encapsulation of mRNA using a 3-component LNP composition and characterization of LNP-mRNA Using GFP mRNA as an example, the construction of a steroid-cationic lipid three-component LNP-mRNA vaccine is demonstrated as follows. The lipid compound of the present invention, helper lipids, and polyethylene glycol lipids were dissolved in ethanol solution in the molar ratios shown in Table 1 to obtain a lipid mixture. mRNA was dissolved in acetate buffer at pH 4.0, and the lipid mixture and mRNA were prepared as mRNA-LNP compositions using the Precision Nanosystems Ignite nanoparticle manufacturing device at a flow rate ratio of 1:3 (see Table 1 for specific formulations). Packaged mRNA-LNPs were dialyzed, concentrated in DPBS by ultrafiltration, and after sterile filtration, samples for subsequent animal experiments were obtained. Encapsulation efficiency, average particle size, PDI, and zeta potential were detected by sampling.

[0154] [Table 1-1] [Table 1-2] Here,

[0155] CN112424214A compound (1) [ka]

[0156] Patent CN112424214A compound (2) [ka]

[0157] Patent CN112424214A compound (3) [ka]

[0158] Patent US7514099B2 compound (CLinDMA): [ka]

[0159] As shown in Table 2, the results indicate that compositions formed from the steroid-cationic lipids of the present invention, helper phosphate esters, polyethylene glycol lipids, and mRNA exhibit relatively good physicochemical properties. For example, the average particle size was between 60 and 100 nm, the PDI was less than 0.2 for all components, they had relatively good polydispersity coefficients, the zeta potential was between ±10 mV, they had relatively low electrostatic properties, and the encapsulation efficiency was 85% or higher for all components. The PDI and mRNA encapsulation efficiency of the three-component lipid nanoparticle formulations provided by the present invention are both superior to those of lipid nanoparticle formulations produced from lipid compounds provided by CN112424214A and US7514099B2. The encapsulation efficiency of LNPs produced from compounds with branched side chains for mRNA is higher than that of LNPs (LNP7 and LNP8) produced from compounds with linear side chains. This demonstrates that the three-component LNPs produced from the steroid-cationic lipid compounds provided by the present invention enable efficient encapsulation of mRNA.

[0160] [Table 2]

[0161] Example 25: Cellular experiments with mRNA-LNPs compositions encoding GFP Cell density is 6.5 × 10 5 Hep3B cells at a concentration of 1 mL / well were inoculated into a 24-well cell culture plate. After 24 hours, 500 ng of GFP mRNA-LNP (prepared according to Example 24) was transfected into each well. The cell culture plates were then cultured in a 37°C, 5% CO2 cell incubator, and an equal volume of physiological saline was transfected into the negative control group. After 24 hours, imaging was performed using a microscope, and the results are shown in Figure 1. The results showed that high expression of GFP-mRNA could be achieved in cells by using the three-component LNP composition formed by the steroid-cationic lipid compound described in the present invention, and that the expression level was significantly higher than that of the control group. Here, LNPs consisting of compounds with a branched lipid chain structure had higher encapsulation and delivery efficiency for mRNA than compounds with a linear lipid chain structure. This is because such a branched lipid structure facilitates transition from the lamellar phase to the inverse hexagonal phase, which is advantageous for mRNA release and improves transfection efficiency.

[0162] Example 26: Animal immunoassay of a novel coronavirus antigen mRNA-LNP composition Using the mRNA for the SARS-CoV-2 protein antigen, SARS-CoV-2 antigen mRNA-LNP was produced according to the lipid formulation shown in Table 1 of Example 24. Female BALB / c mice aged 6-8 weeks were randomly divided into groups of 5 mice and immunized using the hindlimb intramuscular injection route. Immunosampling was performed on day 0 and day 14, with an immunodose of 3 μg (micrograms) of mRNA-LNP. The negative control group was injected with an equal volume of physiological saline. Blood was collected 14 days after immunization, serum was separated, and specific antibody titers against the SARS-CoV-2 virus S protein antigen were detected by ELISA. On day 28, ICS antigen-specific cellular immunity was detected. As shown in Figures 2, 3a, and 3b, the LNP1, LNP5, LNP6, LNP10, and LNP11 experimental groups all showed relatively good humoral and cellular immunity induction (in Figure 2, the left side of each group represents the IgG antibody titer after the first immunization, and the right side represents the IgG antibody titer after the second immunization; in Figures 3a and 3b, from left to right, the titers were TNFα+, TNFγ+, IL2+, IL4+, and IL5+, respectively).

[0163] Implementation 27: Safety evaluation of cholesterol cationic compounds The SARS-CoV-2 antigen mRNA-LNP complex was administered intramuscularly to CD1 mice at a dose of 30 ug, and an equal volume of saline was injected into the negative control group. The safety of the three-component LNPs of compounds 1, 6, and 11 was evaluated by monitoring the systemic inflammatory factor IL-6 in mice. As shown in Figure 4, after high-dose administration, the expression levels of systemic inflammatory factor IL-6 in the three-component LNP experimental groups based on compounds 1, 6, and 11 were significantly lower compared to the four-component LNP of the commercially available compound ALC-0315.

[0164] Example 28: Freeze-drying study of three-component LNPs of cholesterol cationic lipid compounds Using the GFP-mRNA-LNP complex as an example, mRNA-LNP was dissolved in a 20 mM Tris-sodium acetate pH 7.5 buffer solution containing 10% mass fraction sucrose. 300 μL (microliters) of this solution was dispensed into 3 mL vials and lyophilized. The lyophilization procedure was as follows: Pre-freezing stage: -30°C for 2 hours; Desorption drying stage: 25°C for 1 hour; Sublimation drying stage: -40°C for 2 hours, 20°C for 2 hours, controlled to a total vacuum pressure of ≤10 Pa. The mixture was left stable at 2-8°C for 1 month. After redissolution with DNase / RNase-free water, the total mRNA amount, free mRNA concentration, encapsulation efficiency, and mRNA integrity were detected.

[0165] [Table 3-1] [Table 3-2] [Table 3-3]

[0166] The results are shown in Table 3. The three-component LNPs produced from the lipid compounds provided by the present invention showed significantly higher stability after lyophilization (e.g., particle size, PDI, and encapsulation efficiency parameters) compared to the control group. The LNPs produced from the lipid compounds provided by the present invention showed particle size changes of 10 nm or less before and after lyophilization, and the change in encapsulation efficiency was within 6% in all cases. In contrast, the LNPs in the control group showed an increase in particle size of more than 10 nm after lyophilization, and the change in mRNA encapsulation efficiency also exceeded 10% in all cases.

[0167] Example 29: Spray test of three cholesterol cationic lipid compound LNPs Using a GFP-mRNA-LNP complex, mRNA-LNP was sprayed using a nebulizer, the droplets were collected, and particle size, PDI, and potential were detected using a nanogranulometer. Content and encapsulation efficiency were then detected using a Ribogreen detection kit.

[0168] [Table 4]

[0169] As shown in Table 4 and Figure 5, the experimental results for LNP1, LNP4, LNP5, and LNP10 samples showed relatively small changes in key mass properties such as particle size, PDI, and encapsulation efficiency after spraying, maintaining a relatively good particle state after spraying, and the change in encapsulation efficiency was low, within 10%. In contrast, the particle size of the LNP formulations in control groups 3 and 4 after spraying was around 200 nm, and the encapsulation efficiency decreased to around 40%, while the particle structure of the four-component LNP based on ALC-0315 in control group 5 was destroyed after spraying, and the encapsulation efficiency could not be detected. The sprayed samples were transfected into Hep3B cells with 500 ng of mRNA-LNP / 10 5 As shown in Figure 6, GFP expression levels were detected by microscopic imaging of cells / wells 24 hours later. The LNP formulations in control groups 3 and 4 showed relatively weak cell expression in the sprayed samples. The samples from the four groups LNP1, LNP4, LNP5, and LNP10 maintained relatively good cell transfection activity even after spraying, but there was no cell expression of the four LNP components after spraying.

[0170] Example 30: Lung development status after LNP spray inhalation in mice Following the procedure in Example 24, Luc mRNA was encapsulated in LNP4, LNP5, and LNP10 formulations, and 10 ug of Luc-mRNA-LNP complexes were administered by spraying to 7-9 week old C57BL / 6 female mice. Six hours after administration, the mice were anesthetized, a fluorescent substrate was injected intraperitoneally, the mice were sacrificed and dissected, and fluorescence imaging was performed on the heart, liver, spleen, lungs, and kidneys, and the total fluorescence quantum number was counted. The results are shown in Figures 7a and 7b (wherein Figure 7b, from left to right, were LNP4, LNP5, and LNP10, respectively). Very strong fluorescence expression could be detected in the lungs after the mice inhaled Luc-mRNA-LNP. This indicates that LNP4, LNP5, and LNP10 can achieve selective mRNA expression in the lungs of mice after spray inhalation, further demonstrating that the three-component LNP based on cholesterol cationic lipid compounds designed in this way can be used in the application of mRNA spray inhalation therapy.

[0171] Finally, it should be noted that the above embodiments are used solely to illustrate the technical concepts of the present invention and are not intended to limit them. While the present invention has been described in detail with reference to the embodiments described above, those skilled in the art should understand that they may still modify the technical concepts described in the embodiments above, or make equivalent substitutions for some or all of the technical features thereof, and that such modifications or substitutions will not cause the essence of the corresponding technical concepts to deviate from the scope of the technical concepts of the embodiments of the present invention.

Claims

1. A compound represented by formula (1), 【Chemistry 1】 、 Here, R １ is -OR ４ , -NR ４ R ５ , -NR ４ C(=O)R ５ , -C(=O)OR ５ , -OC(=O)R ５ , -OC(=O)OR ５ , -CN, a nitrogen-containing heterocyclic group or a guanidino group, and is selected from R ４ , R ５ H and C are independent of each other. １～９ alkyl group, C ２～９ Alkenyl group, C ２～９ Alkynyl group, C ３～８ Cycloalkyl groups, C ３～８ Cycloalkenyl group or C ３～８ Selected from cycloalkynyl groups, G １ is a chemical bond (-), C １～９ Alkylene group, C ２～９ Alkenylene group, C ３～９ Alkynylene group, C ３～８ Cycloalkylene group or C ３～８ Selected from cycloalkenylene groups, Preferably, R １ -G １ - The number of consecutive carbon atoms is less than 10. G ２ G ３ Each of these is independently a chemical bond (-), C １～１２ Alkylene group, C ２～１２ Alkenylene group, C ２～１２ Alkynylene group, C ３～１２ Cycloalkylene group, C ３～１２ Cycloalkenylene group, C ３～１２ Cycloalkylene group or C ６～１２ Selected from the arylene group, L １ , L ２ These are each independently chemically bonded (-), -O-, -S-, -O(C=O)O-, and -(C=O)NR ａ -, -NR ａ (C=O)-, -O(C=O)-, -(C=O)O-, -S-S-, -S(O) ｘ -, -OS(O) ｘ O-, -C(=O)S-, -SC(=O)-, -NR ａ C(=O)NR ｂ -, -OC(=O)NR ａ -, -NR ａ C(=O)O-, -OC(=O)S-, -SC(=O)O-, -P(O)(OR ａ )O-, -OP(O)(OR ａ ) O- or C １～１２ Selected from one or more combinations of alkylene groups, x is selected from 0, 1, or 2. R ａ , R ｂ H and C are independent of each other. １～１２ alkyl group, C ２～１２ Alkenyl group or C ２～１２ Selected by Alkin, R ２ These are selected from naturally occurring or unnaturally occurring steroids. R ３ C is a steroid that exists naturally or unnaturally. ６～２４ alkyl group, C ６～２４ Alkenyl group, C ６～２４ Alkyne or C ６～２４ A compound selected from alkoxy groups.

2. The aforementioned R １ is, -OR ４ , -NR ４ R ５ , selected from pyrazolyl group, imidazolyl group, piperazinyl group, alkylpiperazinyl, piperidinyl group, alkylpiperidinyl, guanidino group, pyrrolyl group or pyrrolidinyl group, the R ４ , R ５ H and C are independent of each other. １～９ alkyl group, C ２～９ Alkenyl group, C ２～９ Alkynyl group, C ３～８ Cycloalkyl groups or C ３～８ Selected from cycloalkenyl groups, The aforementioned G １ is a chemical bond (-), C １～９ Alkylene group, C ２～９ Alkenylene group, C ３～８ Cycloalkylene group or C ３～８ It is a cycloalkenylene group, Preferably, the G １ C １～６ Alkylene group or -C ２～６ It is an alkenylene group, Preferably, the R １ -G １ - The number of consecutive carbon atoms is less than 10. The aforementioned G ２ G ３ Each of these is independently a chemical bond (-), C １～１２ Alkylene group, C ２～１２ Alkenylene group or -C ２～１２ Selected from the alkynylene group, Said L １ , L ２ is each independently selected from one or more combinations of -O(C=O)-, -(C=O)O-, -S-S-, -O(C=O)O-, -NR ａ C(=O)O-, -(C=O)NR ａ -, -NR ａ (C=O)-, -C(=O)S-, -SC(=O)-, -OC(=O)NR ａ - or C １～１２ and is selected from one or more combinations of alkylene groups, Here, R ａ H, C １～１２ alkyl group, C ２～１２ Alkenyl group or C ２～１２ The compound according to claim 1, characterized in that it is an alkyne.

3. The above R １ is -OR ４ or -NR ４ R ５ and is selected from an imidazolyl group, a piperazinyl group or a guanidino group The aforementioned R ４ , R ５ H and C are independent of each other. １～５ Selected from alkyl groups, The aforementioned G １ is a non-substitutive C １～６ It is an alkylene group, Preferably, the R １ -G １ - The number of consecutive carbon atoms is less than 10. The aforementioned G ２ G ３ Each of these is independently a chemical bond (-), C １－１０ Selected from alkylene groups, The foregoing L １ , L ２ each independently represents -O-, -O(C=O)-, -O(C=O)O-, -(C=O)O-, -(C=O)S-, -NHC(=O)O-, -NHC(=O)-, 【Chemistry 2】 The compound according to claim 1, characterized by being selected from among.

4. The aforementioned R ２ It is a sterol, Preferably, the sterol is an animal sterol or its oxidized or reduced form, and / or the sterol is a plant sterol or its oxidized or reduced form, and / or the sterol is a synthetic sterol or its oxidized or reduced form. More preferably, the sterol is selected from cholesterol, oxidized cholesterol, reduced cholesterol, alkyl lithocholate, stigmasterol, stigmastanol, campesterol, ergosterol, or sitosterol. More preferably, the sterol is an oxidized form of cholesterol, a reduced form of cholesterol, an alkyl lithocholate, stigmasterol, stigmastanol, campesterol, ergosterol, or sitosterol. More preferably, the sterol is selected from avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, dinosterol, epicholesterol, ergosterol, fucosterol, hexahydrolumysterol, hydroxycholesterol, lanosterol, photosterol, algasterol, sitosteranol, sitosterol, stigmathanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, or lithocholic acid. More preferably, the structural formula of the sterol is as follows: 【Transformation 3】 And, Here, R is C １～２０ The compound according to claim 1, characterized by being an alkyl group.

5. The aforementioned R ３ teeth, 【Chemistry 4】 Selected from, Preferably, the R ３ teeth, 【Transformation 5】 A compound according to any one of claims 1 to 4, characterized by being selected from among.

6. The aforementioned compound, 【Chemistry 6-1】 【Chemistry 6-2】 【Transformation 6-3】 A compound according to any one of claims 1 to 5, characterized by being selected from among.

7. Lipid nanoparticles characterized by containing the compound described in any one of claims 1 to 6.

8. The lipid nanoparticle according to claim 7, further comprising a polyethylene glycol lipid and at least one helper lipid, wherein the helper lipid is selected from a neutral lipid, a zwitterionic lipid, or an anionic lipid.

9. The polyethylene glycol lipid is selected from 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerylmethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-distearylglycerin (PEG-DSG), PEG-dipalmitoyloleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA). The helper lipids are 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE). , 2-dioleoyl group-sn-glycero-3-phospho-(1'-rac-glycero) (DOPG), palmitoyloleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC) Lipid nanoparticles according to claim 8, characterized in that they are selected from phosphatidylethanolamine (PE), phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), lysophosphatidylcholine, dilinoleylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), or lysophosphatidylethanolamine.

10. Lipid nanoparticles according to claim 8, characterized in that the molar ratio of the compound according to any one of claims 1 to 6 to the helper lipid and polyethylene glycol lipid is 20 to 80: 20 to 80: 0.5 to 20, and preferably the molar ratio of the compound according to any one of claims 1 to 6 to the helper lipid and polyethylene glycol lipid is 30 to 80: 30 to 80: 0.5 to 20.

11. The lipid nanoparticles according to claim 7, characterized in that the lipid nanoparticles have a diameter of 15 nm to 300 nm.

12. The compounds described in claims 1 to 6 and / or the lipid nanoparticles described in claims 7 to 11 are used in the production of a bioactive substance delivery system.

13. The biologically active substance is DNA or RNA, and the RNA is selected from antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, circRNA, and self-replicating mRNA. Preferably, the bioactive substance delivery system is an mRNA vaccine. Preferably, the mRNA vaccine is a vaccine for the prevention of cancer, viral infection, bacterial infection, or fungal infection. Preferably, the virus is selected from norovirus, Ebola virus, coronavirus, cytomegalovirus, dengue virus, Zika virus, coxsackievirus, enterovirus, hepatitis virus, herpes simplex virus, human papillomavirus, influenza virus, Marburg virus, measles virus, poliovirus, rabies virus, rotavirus and / or measles virus, the use according to claim 12.

14. The lipid nanoparticles according to any one of claims 7 to 11, characterized in that they further contain a bioactive substance.

15. The lipid nanoparticle according to claim 14, wherein the biologically active substance is DNA or RNA, and the RNA is preferably selected from antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, circRNA, and self-replicating mRNA.

16. The lipid nanoparticles according to claim 14 or 15, characterized in that the ratio of nitrogen to phosphorus in the lipid nanoparticles is (1 to 15):

1.

17. A drug comprising lipid nanoparticles according to any one of claims 14 to 16 and a pharmaceutically acceptable excipient.

18. The drug according to claim 17, characterized in that the drug is a liquid formulation or a freeze-dried powder.

19. The drug according to claim 17, characterized in that the drug is an oral preparation, an intramuscular injection preparation, a subcutaneous injection preparation, an intravenous injection preparation, a spray inhalation preparation and / or a dry powder inhalation preparation.

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