Steroid-cationic lipid compound and use thereof

Abstract

Provided in the present invention are a steroid-cationic lipid compound having a structure as shown in formula (I) and the use thereof. The compound can be used in the preparation of a lipid nanoparticle (LNP) for delivering a therapeutic agent and / or a preventive agent. The LNP prepared using the steroid-cationic lipid compound of the present invention has relatively good stability and transfection efficiency. The use of the LNP for delivering a nucleic acid, e.g., an mRNA, can realize efficient and stable delivery of biologically active substances to target cells or organs, and can induce a high level of specific antibody responses and cellular immune responses in experimental animals. Moreover, the compound has better safety.

Description

Steroid-cationic lipid compound and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a steroid-cationic lipid compound and application thereof in delivery of bioactive substances. BACKGROUND

[0002] Nucleic acid drugs mainly refer to compounds containing nucleotide or deoxynucleotide structures with genetic properties and pharmacological activities, and can be used for treatment of tumors, tissue regeneration, wound healing, pulmonary fibrosis, inflammatory diseases, microbial infections, etc. After being injected into the human body, the nucleic acid drugs need to be delivered to the lesion site by a high-efficiency and safe drug delivery system. The drug delivery system needs to stay for a sufficient time to accurately target the lesion site, while avoiding damage to normal cells.

[0003] At present, drug delivery systems can be divided into viral vectors and non-viral vectors. Due to immunogenicity, tumorigenicity, and limited drug loading capacity, viral vectors are less used in nucleic acid drugs. Non-viral vectors, such as polymers (polymer), lipids (liposome or LNP), can combine nucleic acid drugs with specific ligands to target specific cells, and are more commonly used in current nucleic acid drugs. LNP is one of the most commonly used delivery systems for nucleic acid drugs, and LNP delivery system can safely and effectively deliver nucleic acids. LNP delivery system has the advantages of high nucleic acid encapsulation efficiency, effective transfection of cells, strong tissue penetration, low cytotoxicity and immunogenicity, and has great advantages compared with other drug delivery systems. Therefore, LNP delivery system has broad development and application prospects.

[0004] In the prior art, LNP delivery systems are often made of ionizable lipids (cationic lipids), steroids, neutral lipids, PEG lipids, nucleic acid drugs, etc. For example: patent document AU2020325221A1 discloses a composition of LNP for target cell delivery, including (i) ionizable lipids; (ii) sterols or other structural lipids; (iii) non-cationic helper lipids or phospholipids; (iv) PEG lipids and (v) agents encapsulated in LNP (such as nucleic acid molecules), the four components are in a specific ratio to enhance the delivery efficiency of target cells; patent document WO2021 / 250263A1 discloses a composition including ionizable lipids, phospholipids, sterols, PEG lipids and one or more nucleic acids, and discloses specific percentages of less than about 1 mol% C14-PEG2000 lipids and other lipids; patent CN102712935B discloses a lipid particle, which comprises: cationic lipids; neutral lipids, zwitterionic lipids or anionic lipids; PEG lipids; sterols and nucleic acids, and the above components are assembled into a lipid particle with a solid core, which has a higher coating efficiency; patent document WO2021 / 055849A1 discloses a lipid with the following structure: This structure can improve its safety, effectiveness and specificity; patent document WO2021 / 026358Al discloses a target cell delivery lipid nanoparticle (LNP): including (i) ionizable lipids (ii) sterols or other structural lipids; (iii) non-cationic helper lipids or phospholipids; (iv) effective load; (v) PEG lipids, as a drug delivery system, taking into account safety and effectiveness. In recent years, it has been found that the introduction of cholesterol into ionizable lipid compounds can also be used to deliver nucleic acid drugs. Patent document US7514099B2 discloses a cholesteric amino lipid compound CLinDMA This compound can be used to deliver siRNA with phospholipids, cholesterol, PEG lipids to form four-component LNP or with phospholipids, DMOBA lipids, cholesterol and PEG lipids to form five-component LNP; patent document CN112424214A discloses an ionizable cationic lipid compound formed by cholesterol and linear olefins (3) This compound is used to construct lipid nanoparticles with cholesterol, DPPC, DOPE and DMG-PEG200 to deliver nucleic acids. Patent CN117069785A also discloses a cholesteric cationic compound 5 The four-component LNP thereof with auxiliary phospholipids, cholesterol and PEG lipids can be used for the delivery of mRNA. The above cholesterol-based cationic lipid compounds all require 4 or even 5 different lipid excipients to form nucleic acid drug delivery carrier formulations, which are complex to construct. The prior art has optimized each component in the composition, as well as the cationic lipids, and further development of the components and structures of each component is needed to further obtain a safer, more effective, stable, and simple-to-construct LNP delivery system that can be suitable for different administration routes at the same time.

[0005] SUMMARY

[0006] In a first aspect, the present application provides a steroid-cationic lipid compound represented by formula (I)

[0007] or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, wherein,

[0008] R1, R2 are each independently selected from H, OR', wherein R' is selected from H, C1-C9 alkyl, cycloalkyl, C2-C9 alkenyl, C2-C9 alkynyl, C 3-8 cycloalkyl, C 3-8 cycloalkenyl or C 3-8 cycloalkynyl;

[0009] L1, L2 are each independently selected from a combination of one or more of 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)(OR a )O-, -OP(O)(OR a )O- or C 1-12 alkylene;

[0010] x is selected from 0, 1 or 2;

[0011] R a , R b are each independently selected from H, C 1-12 alkyl, C 2-12 alkenyl or C2-12 alkynyl;

[0012] R3 is selected from a naturally occurring or non-naturally occurring steroid;

[0013] R4 is selected from C 6-24 alkyl, C 6-24 alkenyl, C 6-24 alkyne or C 6-24 alkoxy;

[0014] m, n, o, p, q are each independently selected from any integer between 0-12;

[0015] with the proviso that the compound:

[0016] Further, R1, R2 are each independently selected from H, OH, C1-C9 alkoxy;

[0017] L1, L2 are each independently selected from any one of a 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-; preferably, L1, L2 are each independently selected from any one of -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)-; more preferably, L1, L2 are each independently selected from any one of -O-, -S-, -O(C=O)O-, -O(C=O)-, -(C=O)O-, -S-S-;

[0018] x is selected from 0, 1 or 2;

[0019] R a , R b are each independently selected from H, C 1-12 alkyl, C2-12 alkenyl or C 2-12 alkynyl;

[0020] R3 is selected from a naturally occurring or non-naturally occurring steroidal;

[0021] R4 is selected from an optionally substituted C 6-24 alkyl, C 6-24 alkenyl; preferably, R4 is selected from an optionally substituted C 6-24 alkyl;

[0022] m, n, o, p, q are each independently selected from any integer between 0 and 12.

[0023] Further, said R3 is a sterol;

[0024] Preferably, said sterol is an animal sterol, or an oxidized or reduced form thereof; and / or, said sterol is a plant sterol, or an oxidized or reduced form thereof; and / or, said sterol is a synthetic sterol, or an oxidized or reduced form thereof;

[0025] More preferably, said sterol is selected from cholesterols, oxidized forms of cholesterols, reduced forms of cholesterols, alkyl lithocholates, stigmasterols, stigmastanol, campesterol, ergosterol or sitosterol;

[0026] More preferably, said sterol is an oxidized form of cholesterols, a reduced form of cholesterols, alkyl lithocholates, stigmasterols, stigmastanol, campesterol, ergosterol or sitosterol;

[0027] More preferably, said sterol is selected from avenasterol, beta-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, desmosterol, desmosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, fucosterol, epicholesterol, ergosterol, fucosterol, hexahydrophotosterol, hydroxycholesterol; lanosterol, photosterols, phytosterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid or lithocholic acid;

[0028] More preferably, said sterol has the following structure:

[0029] wherein R" is a C 1-20 alkyl group.

[0030] Further, said R4 is selected from the following structure:

[0031] Preferably, said R4 is selected from the following structure:

[0032] In some embodiments, the present application provides a lipid compound represented by formula (II-1), (II-2), (II-3), (II-4):

[0033] or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof;

[0034] wherein, m, n, o, p, q are each independently selected from any integer between 0-12;

[0035] R1, R2 are each independently H, OR5, wherein R5 is selected from H, C1-C6 alkyl, cycloalkyl, alkenyl, alkynyl;

[0036] L1, L2 are each independently selected from a combination of one or more of a bond (-), -O-, -S-, -O(C=O)O-, -O(C=O)-, -(C=O)O-;

[0037] R4 is selected from C 6-24 alkyl, C 6-24 alkenyl, C 6-24 alkynyl, or C 6-24 alkoxy.

[0038] In some embodiments, L1 is selected from any one of -O-, -O(C=O)O-, -O(C=O)-, -(C=O)O-; preferably, L1 is selected from -(C=O)O-.

[0039] In some embodiments, L2 is selected from any one of -O-, -O(C=O)O-, -O(C=O)-, -(C=O)O-, -S-S-; preferably, L2 is selected from -O(C=O)-.

[0040] In some embodiments, R4 is selected from preferably, R4 is selected from more preferably, R4 is selected from most preferably, R4 is selected from

[0041] In some embodiments, m is selected from 2 or 3 or 4; preferably, m is selected from 3.

[0042] In some embodiments, p and q are each independently selected from 1, 2, 3, 4, 5, 6, 7, or 8; preferably, p is selected from 4, 5, 6, 7, or 8.

[0043] In some embodiments, in the compound of Formula II-1 described herein, L1is selected from -(C=0)0-; L2is selected from -0(C=0)-; R4is selected from m is selected from 3; p is selected from 2; q is selected from any integer between 4-8.

[0044] In some embodiments, the present application provides a compound selected from the following, or a pharmaceutically acceptable salt thereof.

[0045] In another aspect, the present application also provides a lipid nanoparticle comprising the aforementioned steroid-cationic lipid compound of Formula (I).

[0046] In one embodiment, the lipid nanoparticle further comprises a polyethylene glycol lipid and at least one helper lipid; the helper lipid is selected from a neutral lipid, a zwitterionic lipid, or an anionic lipid.

[0047] In one embodiment, the polyethylene glycol lipid is selected from 2-[(polyethylene glycol)-2000]-N,N-tetracosanoylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycero- methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)] (PEG-DSPE), PEG-disteraryl glycerol (PEG-DSG), PEG- dipalmitoyl, PEG-dioleoyl, PEG-distearoyl, PEG-diaclylglycerol amide (PEG-DAG), PEG- dipalmitoyl phosphatidyl ethanolamine (PEG-DPPE), or PEG-1,2-dimyristoyloxypropyl-3- (PEG-c-DMA);

[0048] and / or,

[0049] The neutral lipid is selected from 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), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg 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-eicosenoyl-sn-glycero-3-phosphocholine (DEPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), or lysophosphatidylethanolamine.

[0050] In one embodiment, the molar ratio of the steroid-cationic lipid of Formula (I) to helper lipid in the lipid nanoparticle is 1:2-5:1, and the molar ratio of the polyethylene glycol lipid to total lipid is 0.5%-20%; preferably, the molar ratio of the steroid-cationic lipid of Formula (I) to helper lipid in the lipid nanoparticle is 1:1-4:1, and the molar ratio of the polyethylene glycol lipid to total lipid is 0.5%-5%; more preferably, the molar ratio of the steroid-cationic lipid of Formula (I) to helper lipid is 1:1, 2:1, or 3:1, and the molar ratio of the polyethylene glycol lipid to total lipid is 0.5%-5%; more preferably, the molar ratio of the steroid-cationic lipid of Formula (I) to helper lipid is 1:1 or 2:1, and the molar ratio of the polyethylene glycol lipid to total lipid is 1.5%-2.5%.

[0051] In one embodiment, the lipid nanoparticle has a diameter of 15 nm-300 nm; preferably, the lipid nanoparticle has a diameter of 60 nm-120 nm; more preferably, the lipid nanoparticle has a diameter of 60 nm-100 nm.

[0052] In one embodiment, the encapsulation efficiency (%) of the lipid nanoparticle is 80-100%.

[0053] In still another aspect, the present application also provides a use of the aforementioned steroid-cationic lipid compound and the aforementioned lipid nanoparticle in the preparation of a bioactive substance delivery system.

[0054] In one embodiment, the bioactive substance can be a small molecule compound, a nucleic acid, an oligopeptide, etc.; preferably, the bioactive substance is a 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, IncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, circRNA, and self-replicating mRNA.

[0055] In one embodiment, the bioactive substance is used for preventing and / or treating cancer, inflammation, fibrotic disease, autoimmune disease, infection, psychiatric disorder, hematological disease, chromosomal disease, genetic disease, connective tissue disease, digestive disease, ear-nose-throat disease, endocrine disease, eye disease, reproductive disease, heart disease, kidney disease, lung disease, metabolic disorder, oral disease, musculoskeletal disease, neonatal screening, nutritional disease, parasitic disease, skin disease, etc.

[0056] In one embodiment, the bioactive substance delivery system is an mRNA vaccine.

[0057] In one embodiment, the mRNA vaccine can be used for preventing cancer, viral infection, bacterial infection, fungal infection, etc. In one embodiment, the virus includes, but is not limited to, norovirus, Ebola virus, coronavirus (including novel coronavirus 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, etc.

[0058] In still another aspect, the present application also provides a lipid nanoparticle comprising the aforementioned lipid nanoparticle and a bioactive substance.

[0059] In one embodiment, the bioactive substance can be a small molecule compound, a nucleic acid, an oligopeptide, etc.; preferably, the bioactive substance is a nucleic acid; more preferably, the bioactive substance is DNA or RNA; more preferably, the DNA comprises non-coding DNA (antisense DNA) or coding DNA, and / or the RNA comprises antisense RNA, saRNA, mRNA, IncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, circRNA and self-replicating mRNA.

[0060] In one embodiment, the nitrogen to phosphorus ratio of the lipid nanoparticle is (1-15): 1. For example, the nitrogen to phosphorus ratio of the lipid nanoparticle can 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 nanoparticle is (2-10):1; more preferably, the nitrogen to phosphorus ratio of the lipid nanoparticle is (4-10):1; more preferably, the nitrogen to phosphorus ratio of the composition is 6:1 or 8:1.

[0061] In another aspect, the present application also provides use of the lipid nanoparticle for preparing an mRNA vaccine.

[0062] In one embodiment, the lipid nanoparticle of the present application can be prepared by using conventional methods in the art, such as microfluidic technology.

[0063] In another aspect, the present application also provides a medicament comprising the aforementioned bioactive substance and the lipid nanoparticle.

[0064] Further, the medicament further comprises a pharmaceutically acceptable excipient.

[0065] Further, the pharmaceutically acceptable excipient of the present application comprises, for example, a carrier, an adjuvant, a diluent, etc.

[0066] Further, the medicament of the present application is a gene drug.

[0067] Further, the medicament of the present application is a liquid preparation or a lyophilized powder.

[0068] Further, the medicament of the present application is an oral preparation, a muscle injection preparation, a subcutaneous injection preparation, an intravenous injection preparation, an aerosol inhalation preparation or a dry powder inhalation preparation, a microneedle patch.

[0069] In still another aspect, the present application also provides a method for delivering a bioactive substance, which comprises administering the medicament of the present application to an individual in need thereof.

[0070] Advantages of the present application:

[0071] The present application provides a steroid-cationic lipid compound having a structure shown in formula (I) and its use, which is mixed with a helper lipid and a polyethylene glycol lipid to prepare a lipid nanoparticle for delivering a bioactive substance. The three-component LNP prepared by using the steroid-cationic lipid compound of the present application has the advantages of simple preparation process, good stability and high transfection efficiency. The three-component LNP is used for delivering nucleic acid, such as mRNA, which can efficiently and stably deliver the nucleic acid mRNA to target cells or organs and induce a higher specific antibody response in animals. At the same time, the three-component LNP prepared by using the compound provided by the present application to deliver mRNA vaccine can induce a higher CD8+T cell immune response, thereby having greater advantages for vaccines, such as tumor vaccines, which require a high level of CD8+T cells. Moreover, the three-component LNP prepared by using the cholesteric lipid compound provided by the present application is injected intramuscularly, and the mRNA is mainly expressed at the injection site rather than in the liver, spleen and other organs, which reduces the systemic non-target exposure of the drug and improves the safety of the drug. BRIEF DESCRIPTION OF DRAWINGS

[0072] Fig. 1 shows the expression of Luc-mRNA-LNP in Hep3B cells detected by an enzyme marker;

[0073] Fig. 2 shows the animal live imaging and organ ex vivo imaging of mice after intramuscular injection of mRNA-LNP;

[0074] Fig. 3 shows the ELISA detection of serum antigen-specific antibody titers of mice after immunization with mRNA-LNP;

[0075] Fig. 4a shows the ICS detection of TNFα+CD8+T cell immunity of mice after immunization with mRNA-LNP;

[0076] Fig. 4b shows the ICS detection of IFNγ+CD8+T cell immunity of mice after immunization with mRNA-LNP;

[0077] Fig. 4c shows the ICS detection of IL2+CD8+T cell immunity of mice after immunization with mRNA-LNP; DETAILED DESCRIPTION

[0078] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application.

[0079] Definitions

[0080] As used in this specification, unless the context indicates otherwise, the following words and phrases are generally intended to have the meanings set forth below.

[0081] As used herein, the term "lipid nanoparticle", "Lipid Nanoparticle" or "LNP" refers to a particle having a nanometer scale, e.g., 1 nm to 1,000 nm, which comprises one or more types of lipid molecules.

[0082] As used herein, the term "gene medicine" generally consists of a carrier or delivery system containing an engineered gene construct, the active ingredient of which can be DNA, RNA, genetically engineered viruses, bacteria or cells, by introducing exogenous genes into target cells or tissues, replacing, compensating, blocking, correcting specific genes, to achieve the purpose of treating and preventing diseases.

[0083] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, and includes DNA, RNA and their hybrids.

[0084] As used herein, the term "lipid compound" or "lipid" refers to a group of organic compounds, which includes but is not limited to esters of fatty acids, and is generally characterized by being poorly soluble in water but soluble in many organic solvents, including but not limited to: benzene, toluene, pentane, hexane, methanol, ethanol, isopropanol, diethyl ether, ethyl acetate, acetone, carbon tetrachloride.

[0085] As used herein, the term "alkyl" refers to a saturated straight-chain or branched-chain hydrocarbon group. As used herein, C 1-9 Alkyl includes straight-chain or branched-chain alkyl groups containing 1, 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms, including but not limited to C 1- 8, C 2-7 , C 2-8 , C 3-6 or C 4-7 alkyl; C 6-24Alkyl includes straight-chain or branched-chain alkyl groups 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 including but not limited to C 6- 8, C 6-17 , C 6-22 , C 10-16 or C 14-17 alkyl; C 1-5 Alkyl includes straight-chain or branched-chain alkyl groups containing 1, 2, 3, 4, or 5 carbon atoms, for example including but not limited to C 1-4 , C 2-4 , C 2-3 , C 3-5 or C 4-5 alkyl; C 1-20 Alkyl includes straight-chain or branched-chain alkyl groups 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 including but not limited to C 1-8 , C 1-10 , C 6-22 , C 10-16 or C 14-17 alkyl.

[0086] As used herein, the term "alkenyl" refers to an unsaturated straight-chain or branched-chain hydrocarbon group containing one or more unsaturated carbon-carbon double bonds. The unsaturated carbon-carbon double bond can be present at any stable point along the chain. As used herein, C 2-9 Alkenyl includes straight-chain or branched-chain alkenyl groups containing 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms, for example including but not limited to C 1-8 , C 2- 7, C 2-8 , C 3-6 or C 4-8 straight-chain or branched-chain alkenyl; C 6-24 Alkenyl includes straight-chain or branched-chain alkenyl 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 including but not limited to C 6-8 , C 6-17 , C 6-22 , C 10-16 or C 14-17 alkenyl.

[0087] As used herein, the term "alkynyl" 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 includes straight-chain or branched-chain alkynyl groups containing 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms, for example including but not limited to C1-8 , C 2-7 , C 2-8 , C 3-6 or C 4-7 alkynyl.

[0088] As used herein, one or more carbons of other positions of the removed end group position of alkyl, alkenyl, alkynyl can be replaced with a heteroatom such as nitrogen, oxygen, sulfur and oxides thereof such as nitric oxide, carbonyl, (sul)fur groups, 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 - (wherein m, n can be an integer from 1 to 9) can be unsubstituted or substituted with one or more heteroatom substituents as described herein. In certain embodiments, no heteroatoms are included in the alkyl, alkenyl, or alkynyl group.

[0089] As used herein, the term "alkylene" refers to a saturated, divalent linear or branched hydrocarbon group. As used herein, C 1-9 alkylene includes straight chain or branched chain alkylene groups containing 1, 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms, for example, including but not limited to C 1-8 , C 2-7 , C 2-8 , C 3-6 or C 4-7 alkylene; C 1-12 alkylene includes straight chain or branched chain alkylene groups containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, for example, including but not limited to C 1-8 , C 2-7 , C 2-8 , C 3-6 or C 4-12 alkylene; C 1-10 alkylene includes straight chain or branched chain alkylene groups containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, for example, including but not limited to C 1-8 , C 2-7 , C 2-8 , C 3-6 or C 4-10 alkylene; C1-6 Alkylene includes straight-chain or branched-chain alkylene groups containing 1, 2, 3, 4, 5, or 6 carbon atoms, for example including but not limited to C 1-5 , C 2-4 , C 3-6 , or C 4-6 alkylene groups.

[0090] As used herein, alkylene groups can contain one or more cyclic aliphatic groups and / or one or more heteroatoms such as oxygen, nitrogen, or sulfur, and can be optionally substituted with one or more substituents such as alkyl, halo, alkoxy, hydroxyl, amino, aryl, ether, ester, or amide. One or more carbons in positions other than the terminal group positions can be replaced with a heteroatom such as nitrogen, oxygen, sulfur, and oxides thereof such as nitrogen oxides, carbonyl, sulfone, or sulfoxide. In certain embodiments, the alkylene group is unsubstituted. In certain embodiments, the alkylene group does not include any heteroatoms.

[0091] As used herein, the term "alkoxy" refers to an "alkyl-O-" group, wherein alkyl is as defined herein. As used herein, C 6-24 Alkoxy includes straight-chain or branched-chain 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 including but not limited to C 6-8 , C 6-17 , C 6-22 , C 10-16 , or C 14-17 alkoxy groups.

[0092] As used herein, the term "cycloalkyl" refers to a saturated cyclic hydrocarbon group. As used herein, C 3-8 Cycloalkyl includes cycloalkyl groups containing 3, 4, 5, 6, 7, or 8 carbon atoms, for example including but not limited to C 3-7 , C 4-7 , or C 3-6 cycloalkyl groups.

[0093] As used herein, the term "cycloalkenyl" refers to a cyclic hydrocarbon group containing at least one carbon-carbon double bond. As used herein, C 3-8 Cycloalkenyl includes cycloalkenyl groups containing 3, 4, 5, 6, 7, or 8 carbon atoms, for example including but not limited to C 3-7 , C 4- , or C 3-6 cycloalkenyl groups.

[0094] As used herein, the term "cycloalkynyl" refers to a cyclic hydrocarbon group containing at least one carbon-carbon triple bond. As used herein, C 3-8Cycloalkynyl groups include cycloalkynyl groups containing 3, 4, 5, 6, 7, or 8 carbon atoms, for example including but not limited to C 3-7 , C 4-7 , or C 3-6 alkynyl groups.

[0095] As used herein, the term "cationic lipid" refers to a lipid molecule that is positively charged in response to environmental pH or hydrogen ion activity.

[0096] As used herein, the term "helper lipid" refers to a lipid that is not positively charged under environmental pH conditions, which includes neutral lipids that are not charged, zwitterionic lipids, and negatively charged anionic lipids.

[0097] As used herein, the term "polyethylene glycol lipid" refers to a lipid molecule that comprises a lipid moiety and a polyethylene glycol moiety.

[0098] As used herein, the term "delivery system" refers to a formulation or composition that modulates the spatial, temporal, and dosage distribution of a biologically active ingredient within a biological organism.

[0099] It should be noted that the methods used in the present application, if not specified, are conventional methods, and the reagents used in the present application, if not specified, are commercially available products.

[0100] Example 1 Synthesis of compound 4

[0101] Step 1: Synthesis of compound la

[0102] Dissolve 2-hexyldecanoic acid (12.8 g, 50 mmol) in 30 mL of dichloromethane, add 6-bromo-n-hexanol (9.0 g, 50 mmol), DMAP (2.1 g, 20 mmol), triethylamine (6.2 g, 60 mmol), and stir to dissolve. Add a solution of EDC.HCL (11.0 g, 60 mmol) in dichloromethane dropwise, and stir at room temperature for 16 hours after the addition is complete. Quench with water, add dilute hydrochloric acid, adjust the pH to 1-3, and separate the layers. Wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to give compound la (14.5 g, pale yellow oil) in 70% yield.

[0103] MS m / z (ESI): 419.3 [M+1] + .

[0104] Step 2: Synthesis of compound lb

[0105] Compound 1a (12.6 g, 30 mmol) was added to a mixture of 25 mL of tetrahydrofuran and 50 mL of acetonitrile at room temperature, and then 1-(3- aminopropyl)imidazole (15.0 g, 120 mmol), potassium carbonate (8.5 g, 60 mmol), and potassium iodide (0.5 g, 3 mmol) were sequentially added. The mixture was stirred at 40-50°C for 16 h, and the reaction progress was monitored. After compound 1a was completely consumed, the mixture was cooled to room temperature, and the solvent was removed at 45°C. The crude product was dissolved in dichloromethane, washed with saturated brine twice, dried over anhydrous sodium sulfate, concentrated, and then separated and purified by silica gel column chromatography (DCM / MeOH 60:1-40:1) to obtain compound 1b (7.2 g, yellowish oil) at a yield of 52%.

[0106] MS m / z (ESI): 464.4 [M+1] + .

[0107] Step 3: Synthesis of compound 1c

[0108] Cholesterol (11.6 g, 30 mmol) was added to 60 mL of dichloromethane at room temperature, and then acrylic acid (4.3 g, 60 mmol) and dicyclohexyl carbodiimide (12.4 g, 60 mmol) were sequentially added. Triethylamine (6.1 g, 60 mmol) was added dropwise to the system, and the mixture was stirred for 16 h. After cholesterol was completely consumed, the mixture was cooled to room temperature, and the dichloromethane was evaporated. Compound 1c (9.2 g, white solid) was obtained by crystallization using a mixture of methyl tert-butyl ether and acetonitrile at a yield of 67%.

[0109] MS m / z (ESI): 455.4 [M+1] + .

[0110] Step 4: Synthesis of compound 4

[0111] Compound 1b (7.0 g, 15 mmol) and compound 1c (8.2 g, 18 mmol) were added to a mixture of 100 mL of tetrahydrofuran and 100 mL of acetonitrile at room temperature, and then the mixture was stirred at 40-50°C for 16 h. The solvent was removed at 45°C, and the crude product was dissolved in dichloromethane, washed with saturated brine twice, dried over anhydrous sodium sulfate, concentrated, and then separated by silica gel column chromatography (DCM / MeOH 100:1-50:1) to obtain compound 5 (6.8 g, yellowish oil) at a yield of 50%.

[0112] MS m / z (ESI): 905.6 [M+1]+;

[0113] 1H NMR (300 MHz, CDC13): δ 7.51 (s, 1H), 7.08 (s, 1H), 6.95 (s, 1H), 5.38-5.39 (m, 1H), 4.62-4.66 (m, 1H), 4.09 (t, 2H, J = 6.6 Hz), 4.01 (t, 2H, J = 6.9 Hz), 2.75-2.77 (m, 2H), 2.32-2.40 (m, 9H), 1.90-2.01 (m, 7H), 0.88-1.62 (m, 71H), 0.70 (s, 3H).

[0114] Example 2 Synthesis of compound 7

[0115] Step 1: Synthesis of compound 2a

[0116] Dissolve 2-hexyldecanoic acid (12.8 g, 50 mmol) in 30 mL of dichloromethane, add 8-bromo-1-octanol (10.5 g, 50 mmol), DMAP (2.4 g, 20 mmol), and stir to dissolve. Add a solution of EDC-HCL (11.5 g, 60 mmol) in dichloromethane dropwise, and stir at room temperature for 16 hours after the addition is complete. Quench with water, add dilute hydrochloric acid, adjust the pH to 1-3, and separate the layers. Wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate to give compound 2a (14.1 g, pale yellow oil) in 63% yield.

[0117] MS m / z (ESI): 447.3 [M+1] + .

[0118] Step 2: Synthesis of compound 2b

[0119] Add compound 2a (13.4 g, 30 mmol) to a mixture of 25 mL of tetrahydrofuran and 50 mL of acetonitrile at room temperature, then add 1-(3-aminopropyl)imidazole (15 g, 120 mmol), potassium carbonate (8.3 g, 60 mmol), and potassium iodide (0.5 g, 3 mmol) in sequence, warm to 40-50°C, stir for 16 hours, monitor the reaction progress, and cool to room temperature after compound 2a is completely consumed. Remove the solvent at 45°C, dissolve the crude product in dichloromethane, wash twice with saturated brine, dry the organic phase over anhydrous sodium sulfate, concentrate to give the crude product, and separate compound 2b (7.4 g, pale yellow oil) in 50% yield by silica gel column chromatography (DCM / MeOH 60:1-40:1).

[0120] MS m / z (ESI): 492.5 [M+1] + .

[0121] Step 3: Synthesis of compound 7

[0122] Compound 2b (7.4 g, 15 mmol), compound 1c (8.0 g, 18 mmol) were added into 15 mL tetrahydrofuran, 30 mL acetonitrile mixed solution at room temperature, heated to 70-80 °C, stirred for 16 h, 45 °C to remove the solvent, dissolved in dichloromethane, washed with saturated brine twice, the organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product, then separated by silica gel column chromatography (DCM / MeOH 100:1-50:1), compound 9 (6.3 g, light yellow oil), yield 45%.

[0123] MS m / z (ESI): 932.8 [M+1] + ;

[0124] 1 H NMR (300 MHz, CDC13): δ 7.51 (s, 1H), 7.08 (s, 1H), 6.95 (s, 1H), 5.33-5.35 (m, 1H), 4.58-4.69 (m, 1H), 4.08 (t, 2H, J = 6.6 Hz), 4.01 (t, 2H, J = 6.9 Hz), 2.77 (br, 2H), 2.32-2.42 (m, 9H), 1.86-1.93 (m, 8H), 0.87-1.64 (m, 74H), 0.70 (s, 3H).

[0125] Example 3 Synthesis of compound 8

[0126] Step 1: Synthesis of compound 3a

[0127] 2-hexyl decanoic acid (12.8 g, 50 mmol) was dissolved in 30 mL dichloromethane, 4-bromo-1-butanol (7.7 g, 50 mmol), DMAP (2.4 g, 20 mmol) were added and stirred to dissolve. A solution of EDC-HCL (11.5 g, 60 mmol) in dichloromethane was added dropwise, and after the addition was completed, it was stirred at room temperature for 16 hours. Quench with water, add dilute hydrochloric acid, adjust the pH to 1-3, separate the organic phase. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give compound 3a (13.0 g, light yellow oil), yield 66%.

[0128] MS m / z (ESI): 391.2 [M+1] + .

[0129] Step 2: Synthesis of compound 3b

[0130] Compound 3a (11.7 g, 30 mmol) was added to a mixture of 25 mL of tetrahydrofuran and 50 mL of acetonitrile at room temperature, followed by the addition of 1-(3- aminopropyl)imidazole (15 g, 120 mmol), potassium carbonate (8.3 g, 60 mmol), and potassium iodide (0.5 g, 3 mmol). The mixture was stirred at 40-50 °C for 16 h. The reaction was monitored by TLC. After the complete consumption of compound 3a, the mixture was allowed to cool to room temperature. The solvent was removed at 45 °C. The crude product was dissolved in dichloromethane and washed with saturated brine twice. The organic phase was dried over anhydrous sodium sulfate. The crude product was concentrated and purified by column chromatography on silica gel (DCM / MeOH 60:1-40:1) to give compound 3b (7.0 g, light yellow oil) in 53% yield.

[0131] MS m / z (ESI): 436.4 [M+1] + .

[0132] Step 3: Synthesis of compound 8

[0133] Compound 3b (7.0 g, 15 mmol) and compound 1c (8.0 g, 18 mmol) were added to a mixture of 15 mL of tetrahydrofuran and 30 mL of acetonitrile at room temperature. The mixture was stirred at 70-80 °C for 16 h. The solvent was removed at 45 °C. The crude product was dissolved in dichloromethane and washed with saturated brine twice. The organic phase was dried over anhydrous sodium sulfate. The crude product was concentrated and purified by column chromatography on silica gel (DCM / MeOH 100:1-50:1) to give compound 10 (6.6 g, light yellow oil) in 50% yield.

[0134] MS m / z (ESI): 876.5 [M+1] + ;

[0135] 1 H NMR (300 MHz, CDC13): δ 7.52 (s, 1H), 7.08 (s, 1H), 6.95 (s, 1H), 5.33-5.35 (m, 1H), 4.58-4.69 (m, 1H), 4.09 (t, 2H, J = 6.6 Hz), 4.00 (t, 2H, J = 6.9 Hz), 2.76 (br, 2H), 2.32-2.44 (m, 9H), 1.86-1.92 (m, 7H), 0.87-1.64 (m, 67H), 0.70 (s, 3H).

[0136] Example 4: Synthesis of compound 9

[0137] Step 1 : Synthesis of compound 4a

[0138] Dissolve 1,3-dibromopropane (2.16 g, 10.0 mmol) in 30 mL of n-butanol, add cholesterols (1.94 g, 5.0 mmol) and sodium hydroxide (1.20 g, 30 mmol), stir at reflux for 5 h. After completion of the reaction, adjust the pH to neutral by adding dilute hydrochloric acid. Remove n-butanol by rotary evaporation, dilute with 20 mL of dichloromethane, then wash with 30 mL of saturated brine, dry the organic phase over anhydrous sodium sulfate, filter and concentrate, and purify by column chromatography to obtain the product 4a (1.25 g, pale yellow oil), yield 48%.

[0139] MS m / z (ESI): 521.3 [M+1] + .

[0140] Step 2: Synthesis of compound 9

[0141] Dissolve compound 4a (463 mg, 1.0 mmol) in tetrahydrofuran, add acetonitrile, compound 1b (625 mg, 1.2 mmol), potassium carbonate (550 mg, 4.0 mmol), potassium iodide (332 mg, 2.0 mmol), stir at 83 °C for 16-20 h. Cool to room temperature, filter, wash the filter residue with dichloromethane, add saturated sodium bicarbonate solution to the resulting filtrate, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and purify by column chromatography to obtain the product 11 (434 mg, pale yellow oil), yield 48%.

[0142] MS m / z (ESI): 903.9 [M+1] + ;

[0143] 1 H NMR (300 MHz, CDC13): δ 8.02 (s, 1H), 7.45 (s, 1H), 7.12 (s, 1H), 6.96 (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, 81H), 0.70 (s, 3H).

[0144] Example 5 Synthesis of compound 11

[0145] Step 1 : Synthesis of compound 5a

[0146] To a solution of 2-(oxirane-2-yl)ethan-1-ol (2.64 g, 30 mmol) in 80 mL of dichloromethane was added 4-dimethylaminopyridine (7.3 g, 60 mmol) followed by portion wise addition of p-nitrophenyl chloroformate (6.6 g, 33 mmol) and the reaction stirred at room temperature for 3 h. To this reaction was added cholesterol (13.5 g, 35 mmol) and the mixture stirred at room temperature overnight. After completion of the reaction as indicated by TLC, the reaction was diluted with 50 mL of dichloromethane and washed with 60 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give the product 5a (9.3 g, pale yellow oil) in 62% yield.

[0147] MS m / z (ESI): 501.4 [M+1] + .

[0148] Step 2: Synthesis of compound 5b

[0149] To compound 5a (7.5 g, 15 mmol) was added 1-(3-aminopropyl)imidazole (7.5 g, 60 mmol) at room temperature and the reaction was heated to 70-80 °C and stirred for 16 h. After completion of the reaction as indicated by monitoring the consumption of compound 5a, the reaction was cooled to room temperature and the crude product was dissolved in dichloromethane and washed with saturated brine twice. The organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product which was purified by column chromatography on silica gel (DCM / MeOH 40:1-20:1) to give compound 5b (3.8 g, pale yellow oil) in 41% yield.

[0150] MS m / z (ESI): 626.5 [M+1] + .

[0151] Step 3: Synthesis of compound 11

[0152] To 5b (3.1 g, 5 mmol) in tetrahydrofuran was added acetonitrile, 1a (2.5 g, 6 mmol), potassium carbonate (2.75 g, 20 mmol) and potassium iodide (1.66 g, 10 mmol) and the reaction stirred at 40-50 °C for 16-20 h. The reaction was cooled to room temperature, filtered and the filtrate was washed with saturated sodium bicarbonate solution and extracted with dichloromethane twice. The combined organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The product was purified by column chromatography on silica gel (DCM / MeOH 40:1-30:1) to give compound 13 (2.2 g, pale yellow oil) in 46% yield.

[0153] MS m / z (ESI): 964.8 [M+1] + .

[0154] Example 6 Synthesis of compound 14

[0155] Step 1: Synthesis of compound 6a

[0156] Compound 6a (11.9 g, yellowish oil) was obtained in 70% yield. MS m / z (ESI): 339.3 [M+1]

[0157] MS m / z (ESI): 339.3 [M+1] + .

[0158] Step 2: Synthesis of compound 6b

[0159] Compound 6b (9.6 g, yellowish oil) was obtained in 90% yield. MS m / z (ESI): 355.3 [M+1]

[0160] MS m / z (ESI): 339.3 [M+1] + .

[0161] Step 3: Synthesis of compound 6c

[0162] Compound 6b (8.9 g, 25 mmol), was added to 1-(3-aminopropyl)imidazole (12.5 g, 100 mmol) at room temperature, warmed to 70-80 °C, stirred for 16 h, monitored the reaction progress, compound 6b was consumed completely, cooled to room temperature, dissolved in dichloromethane, washed with saturated brine twice, dried the organic phase over anhydrous sodium sulfate, concentrated to get the crude product, separated by silica gel column chromatography (DCM / MeOH 40:1-20:1), compound 6c (6.0 g, light yellow oil), yield 50%.

[0163] MS m / z (ESI): 480.4 [M+1] + .

[0164] Step 4: Synthesis of compound 14

[0165] Compound 6c (6.0 g, 12.5 mmol), compound 1c (6.7 g, 15 mmol) was added to 10 ml tetrahydrofuran, 20 ml acetonitrile mixed solution at room temperature, warmed to 70-80 °C, stirred for 16 h, removed the solvent at 45 °C, dissolved in dichloromethane, washed with saturated brine twice, dried the organic phase over anhydrous sodium sulfate, concentrated to get the crude product, separated by silica gel column chromatography (DCM / MeOH 120:1-100:1), compound 16 (3.9 g, light yellow oil), yield 34%.

[0166] MS m / z (ESI): 920.9 [M+1] + ;

[0167] 1 H NMR (300 MHz, CDC13): δ 7.52 (s, 1H), 7.07 (s, 1H), 6.94 (s, 1H), 5.33-5.35 (m, 1H), 4.58-4.69 (m, 1H), 4.08 (t, 2H, J = 6.6 Hz), 3.95-4.03 (m, 2H), 2.90-3.77 (m, 3H), 2.32-2.77 (m, 10H), 1.82-1.94 (m, 9H), 0.87-1.64 (m, 67H), 0.67 (s, 3H).

[0168] Example 7 Synthesis of compound 16

[0169] Step 1: Synthesis of compound 7a

[0170] Compound 7a (14.51 g, yellowish oil) was obtained in 83% yield by dissolving 9-bromononanoic acid (8.85 g, 40 mmol), 1-heptanol (13.94 g, 120 mmol) in 100 mL of dichloromethane, adding p-toluenesulfonic acid (7.8 mmol), anhydrous sodium sulfate (120 mmol), stirring at room temperature overnight, removing the solvent by rotary evaporation after monitoring the reaction completion by TLC, and then purifying by silica gel column chromatography (DCM / MeOH 120:1-100:1).

[0171] 1 H NMR (300 MHz, CDC13) δ: 0.86-0.90 (m, 6H), 1.27-1.48 (m, 24H), 1.52-1.64 (m, 6H), 1.85-1.88 (m, 2H), 3.37-3.53 (m, 4H), 3.54-3.59 (m, 2H), 4.44-4.47 (m, 1H).

[0172] Step 2: Synthesis of compound 7b

[0173] Compound 7a (11.7 g, 26.8 mmol) was added to a mixture of 25 mL of tetrahydrofuran and 50 mL of acetonitrile at room temperature, followed by 1-(3-aminopropyl)imidazole (13.4 g, 108 mmol), potassium carbonate (7.3 g, 53 mmol), and potassium iodide (0.5 g, 3 mmol), and the mixture was warmed to 40-50°C, stirred for 16 h, and then cooled to room temperature after the complete consumption of compound 3a was confirmed. The solvent was removed at 45°C, and the crude product was dissolved in dichloromethane, washed with saturated brine twice, dried over anhydrous sodium sulfate, concentrated, and then purified by silica gel column chromatography (DCM / MeOH 60:1-40:1) to obtain compound 7b (6.1 g, yellowish oil) in 47% yield.

[0174] MS m / z (ESI): 480.5 [M+1] + .

[0175] Step 3: Synthesis of compound 16

[0176] Compound 7b (7.2 g, 15 mmol), compound 1c (8.0 g, 18 mmol) were added to a mixture of 15 mL of tetrahydrofuran and 30 mL of acetonitrile at room temperature, and the mixture was stirred at 70-80 °C for 16 h. The solvent was removed at 45 °C, and the crude product was dissolved in dichloromethane and washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product, which was separated by silica gel column chromatography (DCM / MeOH 100:1-50:1) to give compound 18 (8.4 g, yellowish oil) in 61% yield.

[0177] MS m / z (ESI): 920.8 [M+1] + ;

[0178] 1 H NMR (300 MHz, CDC13): 7.57 (dd, 1 H, J = 1.9, 1.1 Hz), 7.18 (dd, 1 H, J = 3.4, 1.1 Hz), 6.85 (dd, 1 H, J = 3.4, 1.9 Hz), 5.23 (dd, 1 H, J = 7.1, 6.9 Hz), 4.75-4.96 (m, 2 H), 3.72-3.83 (m, 2 H), 3.50-3.61 (m, 4 H), 2.42-2.90 (m, 10 H), 1.18-2.20 (m, 61 H), 1.04 (d, 3 H, J = 6.8 Hz), 0.73-0.93 (m, 17 H), δ 0.66 (q, 2 H, J = 7.2 Hz).

[0179] Example 8 Synthesis of compound 17

[0180] Step 1: Synthesis of compound 8a

[0181] 1-bromo-nonadeca-7,10-diene (cis-7,10) (10.3 g, 30 mmol) was added to a mixture of 25 mL of tetrahydrofuran and 50 mL of acetonitrile at room temperature, and then 1-(3-aminopropyl)imidazole (15 g, 120 mmol), potassium carbonate (8.3 g, 60 mmol), and potassium iodide (0.5 g, 3 mmol) were sequentially added. The mixture was stirred at 40-50 °C for 16 h. After the consumption of 1-bromo-nonadeca-7,10-diene was confirmed, the mixture was cooled to room temperature, and the solvent was removed at 45 °C. The crude product was dissolved in dichloromethane and washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product, which was separated by silica gel column chromatography (DCM / MeOH 60:1-40:1) to give compound 8a (7.6 g, yellowish oil) in 65% yield.

[0182] MS m / z (ESI): 388.4 [M+1]+ .

[0183] Step 2: Synthesis of compound 17

[0184] Compound 8a (5.8 g, 15 mmol), compound 1c (8.0 g, 18 mmol) were added into 15 mL tetrahydrofuran, 30 mL acetonitrile mixed solution at room temperature, heated to 70-80 °C, stirred for 16 h, 45 °C to remove the solvent, dissolved in dichloromethane, washed with saturated brine twice, the organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product, then separated and purified by silica gel column chromatography (DCM / MeOH 100:1-50:1) to give compound 19 (7.0 g, yellowish oil), yield 57%.

[0185] MS m / z (ESI): 814.7 [M+1] + ;

[0186] 1 H NMR (300 MHz, CDC13): 7.67 (t, 1H, J = 2.0 Hz), 7.22 (dd, 1H, J = 4.8, 2.0 Hz), 6.94 (dd, 1H, J = 4.8, 2.0 Hz), 5.52-5.74 (m, 3H), 5.23-5.47 (m, 3H), 3.81-3.92 (m, 2H), 2.58-3.01 (m, 12H), 1.11-2.23 (m, 52H), 80.73-1.02 (m, 16H).

[0187] Example 9 Synthesis of compound 18

[0188] Step 1: Synthesis of compound 9a

[0189] 2-hexyl decanoic acid (1.28 g, 5.0 mmol) was dissolved in 20 mL dichloromethane, bis(2-hydroxyethyl) disulfide (1.54 g, 10.0 mmol), DMAP (0.21 g, 2.0 mmol), triethylamine (0.62 g, 6.0 mmol) were added and stirred to dissolve. A solution of EDC.HCL (1.10 g, 6.0 mmol) in dichloromethane was added dropwise, and after the addition was completed, it was stirred at room temperature for 16 hours. Quench with water, add dilute hydrochloric acid, adjust the pH to 1-3, separate the organic phase. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated, and column chromatography gave compound 9a (1.00 g, yellowish oil), yield 51%.

[0190] MS m / z (ESI): 393.2 [M+1] +

[0191] Step 2: Synthesis of compound 9b

[0192] Compound 9a (1.00 g, 2.5 mmol) was dissolved in 20 mL of dichloromethane, triethylamine (0.76 g, 7.5 mmol) was added, and the solution was cooled to 0 °C in an ice water bath. Methylsulfonyl chloride (0.72 g, 6.2 mmol) was added, and the solution was stirred at room temperature for 16-20 h. The reaction was complete, and the solution was washed twice with saturated sodium bicarbonate and twice with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 9b (1.00 g, light yellow oil) in 85% yield.

[0193] MS m / z (ESI): 471.2 [M+1] + .

[0194] Step 3: Synthesis of compound 9c

[0195] Compound 9b (0.94 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 solution was stirred at room temperature for 16-20 h. The reaction was complete, and the solution was filtered over celite. The filtrate was diluted with dichloromethane, washed twice with saturated sodium bicarbonate and twice with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 9c (758 mg, light yellow oil) in 76% yield.

[0196] MS m / z (ESI): 500.3 [M+1] + .

[0197] Step 4: Synthesis of compound 18

[0198] Compound 9c (0.75 g, 1.5 mmol) and compound 1c (0.8 g, 1.8 mmol) were added to a mixture of 5 mL of tetrahydrofuran and 30 mL of acetonitrile at room temperature, and the solution was warmed to 70-80 °C and stirred for 16 h. The solvent was removed at 45 °C, and the crude product was dissolved in dichloromethane, washed twice with saturated brine, and dried over anhydrous sodium sulfate. The crude product was separated by silica gel column chromatography (DCM / MeOH 100:1-50:1) to give compound 20 (917 mg, light yellow oil) in 65% yield.

[0199] MS m / z (ESI): 940.7 [M+1] + ;

[0200] 1H NMR (300 MHz, CDC13): 7.57 (dd, 1 H, J = 1.9, 1.1 Hz) 7.18 (dd, 1 H, J = 3.4, 1.1 Hz), 6.85 (dd, 1 H, J = 3.4, 1.9 Hz), 5.23 (dd, 1 H, J = 7.1, 6.9 Hz), 4.89 (m, 1 H), 4.36-4.47 (t, 2 H, J = 5.2 Hz), 3.72-3.83 (m, 2 H), 3.26-3.48 (m, 4 H), 3.08 (t, 2 H, J = 7.1 Hz), 2.34-2.89 (m, 9 H), 1.03-2.17 (m, 54 H), 0.73-0.93 (m, 17 H), δ 0.66 (q, 2 H, J = 7.2 Hz).

[0201] Example 10 Synthesis of compound 21

[0202] Step 1: Synthesis of compound 10a

[0203] Cholesterol (11.6 g, 30 mmol) was added into 60 mL of dichloromethane, followed by the addition of 5-bromo-1-pentanoic acid (5.5 g, 30 mmol), DMAP (1.5 g, 12 mmol), and a solution of EDC.HCL (6.9 g, 36 mmol) in dichloromethane dropwise. After the addition was completed, the mixture was stirred at room temperature for 16 h. The reaction was quenched by the addition of water, and diluted with dilute hydrochloric acid to adjust the pH value to 1-3. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to give compound 10a (10.5 g, yellowish oil) in 64% yield.

[0204] MS m / z (ESI): 549.3 [M+1] + .

[0205] Step 2: Synthesis of compound 21

[0206] Compound 1b (7.0 g, 15 mmol) and compound 10a (9.9 g, 18 mmol) were added into 15 mL of tetrahydrofuran and 30 mL of acetonitrile, and the mixture was heated to 70-80 °C and stirred for 16 h. The solvent was removed at 45 °C, and the crude product was dissolved in dichloromethane and washed with saturated brine twice. The organic phase was dried over anhydrous sodium sulfate, concentrated to give the crude product, which was further purified by silica gel column chromatography (DCM / MeOH 100:1-50:1) to give compound 23 (6.5 g, yellowish oil) in 46% yield.

[0207] MS m / z (ESI): 933.2 [M+1]+ ;

[0208] 1 H NMR (300 MHz, CDC13): δ 7.53 (s, 1H), 7.10 (s, 1H), 6.97 (s, 1H), 5.33-5.35 (m, 1H), 4.58-4.69 (m, 1H), 4.08 (t, 4H, J = 6.6 Hz), 2.31-2.56 (m, 11H), 1.86-2.01 (m, 8H), 0.87-1.65 (m, 74H), 0.70 (s, 3H).

[0209] Example 11: mRNA encapsulation by three-component LNP composition and LNP-mRNA characterization

[0210] For example, using Luc mRNA, the construction of three-component steroid-cationic lipid LNP-mRNA vaccine was performed: the lipid compound, auxiliary lipid, and polyethylene glycol lipid of the present application were dissolved in ethanol solution according to the molar ratio in Table 1 to obtain a lipid mixture, and the mRNA was dissolved in an acetic acid buffer solution at pH 4.0. The lipid mixture and mRNA were prepared into mRNA-LNP compositions by microfluidic equipment Precision Nanosystems Ignite nanoparticle preparation instrument at a flow rate ratio of 1:3 (see Table 1 for specific formulations). The packaged mRNA-LNP was dialyzed and ultrafiltrated and concentrated into a 10.7 mM Tris 20 mM NaAc pH 7.5 buffer solution, and after sterile filtration, samples for subsequent animal experiments were obtained. The samples were detected for encapsulation efficiency, average particle size, PDI, and zeta potential.

[0211] Table 1 Lipid formulation molar percentage and nitrogen-phosphorus ratio of three-component LNP formed by steroid-cationic lipid compounds

[0212] Compound (3) of patent CN112424214A

[0213] Compound (ClinDMA) of patent US7514099B2:

[0214] Compound 5 of patent CN117069785A

[0215] As shown in Table 2, the results indicate that the compositions of the steroid-cationic lipids, auxiliary phosphate esters, polyethylene glycol lipids, and mRNA of the present invention exhibit good physicochemical properties. For example, the average particle size is between 60-100 nm, the PDI is less than 0.15, indicating good polydispersity; the zeta potential is between ±10 mV, indicating low electroweakness; and the encapsulation efficiency is greater than 85%. The PDI and mRNA encapsulation efficiency of the three-component lipid nanoparticle formulation provided by the present invention are superior to the lipid nanoparticle formulations made from lipid compounds provided in CN112424214A and US7514099B2. This demonstrates that the three-component LNPs made from the steroid-cationic lipid compounds provided by the present invention can achieve efficient encapsulation of mRNA.

[0216] Table 2 Characterization parameters of the three-component Luc-mRNA-LNP composition formed by steroid-cationic lipid compounds.

[0217] Example 12: Cellular experiments with a composition of mRNA-LNPs encoding Luc

[0218] The cell density was 6×10 5 Hep3B cells (cells / mL) were seeded at a rate of 200 μL / well in 96-well cell culture plates. After 24 hours, each well was transfected with 100 ng of Luc mRNA-LNP (prepared in Example 11). The cell culture plates were then incubated at 37°C in a 5% CO2 cell culture incubator. The negative control group was transfected with an equal volume of physiological saline. After 24 hours, a Luciferase detection kit was added, and the fluorescence value was detected using a multi-mode microplate reader. The results are shown in Figure 1. The results indicate that the three-component LNP composition formed using the steroid-cationic lipid compound described in this invention can achieve high expression of Luc-mRNA in cells, with a significantly higher expression level than the control group.

[0219] Example 13: In vivo expression and distribution of mRNA-LNPs encoding Luc in mice

[0220] LNP encapsulating 5 pg of Lucifease mRNA was injected intramuscularly into 6-8 weeks old female BALB / c mice (3 per group), and 24 hours after administration, the mice were anesthetized, intraperitoneally injected with D-Luciferin luciferase substrate, and imaged. The mice were dissected, and the heart, liver, spleen, lung, kidney, and pancreas were imaged ex vivo. As shown in FIG. 2, after intramuscular injection of the three-component LNP constructed using the cholesterol cationic lipid compound provided in the present application, the mRNA was mainly expressed at the injection site, but not in the liver, spleen, and other organs. In contrast, a large amount of mRNA entered the liver and spleen in addition to the injection site in the four-component LNP of ALC-0315, indicating that the three-component LNP formed using the cholesterol cationic lipid compound provided in the present application can greatly reduce the exposure of mRNA in other internal organs, thereby improving the safety of the drug.

[0221] Example 14: Animal immunization test of new crown antigen mRNA-LNP composition

[0222] The preparation of the new crown antigen mRNA-LNP was performed according to the lipid formulation in Table 1 in Example 11 using the new crown S protein antigen mRNA. 6-8 weeks old female BALB / c mice were randomly divided into groups of 5, and immunized by intramuscular injection of the hind leg. Immunization was performed on day 0 and day 14, and the immunization dose was 3 pg mRNA-LNP. The negative control group was injected with the same volume of normal saline. On day 28, blood was collected and serum was separated, and the specific antibody titer against the SARS-CoV-2 virus S protein antigen was detected by ELISA. The mice were sacrificed and the spleen tissue was taken, and ICS was performed for antigen-specific (IFN-γ, TNF-a, and IL-2 secreting CD8+T cell immune detection. The results are shown in FIGS. 3, 4a, 4b, and 4c. The three-component LNP (LNP1-LNP11) constructed using the cationic lipid compound provided in the present application can induce better humoral immunity, and the geometric mean antibody titer (GMT) is significantly higher than that of the control groups 1, 2, and 3, and is basically consistent with that induced by the commercial ALC-0315 four-component LNP. However, the CD8+T cell immune effect induced by the LNP (LNP1-LNP11) provided in the present application is significantly better than that of the three-component LNP constructed using the control compound, and is significantly better than that of the ALC-0315 four-component LNP. Among them, LNP1 constructed using compound 5 induced significantly better CD8+T cell immunity than other groups. The above results show that the three-component LNP constructed using the compound provided in the present application can induce high humoral immunity when delivering vaccine mRNA, and at the same time, the delivery system can induce higher CD8+T cell immunity, thereby having greater advantages for vaccines that need to induce high CD8+T cell immune response, especially tumor vaccines.

[0223] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the same. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent replacements for some or all of the technical features therein. Such modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A compound of formula (I) ###0001### (I) or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, wherein R1, R2are each independently selected from H, OR', wherein R' is selected from H, C1-C9alkyl, C2-C9alkenyl, C2-C9alkynyl, C 3-8 cycloalkyl, C 3-8 cycloalkenyl or C 3-8 cycloalkynyl; L1, L2are each independently selected from a chemical bond (-), -0-, -S-, -0(C=0)0-, -(C=0)NR a -, -NR a (C=0)-, -0(C=0)-, -(C=0)0-, -S-S-, -S(0) x -, -OS(O) x O-, -C(=0)S-, -SC(=0)-, -NR a C(=0)NR b -, -OC(=0)NR a -, -NR a C(=0)0-, -OC(=0)S-, -SC(=0)0-, -P(0)(OR a )0-, -OP(0)(OR a )0-, or C 1-12 alkylene; x is selected from 0, 1 or 2; R a , R b are each independently selected from H, C 1-12 alkyl, C 2-12 alkenyl or C 2-12 alkynyl; R3 is selected from a naturally occurring or non-naturally occurring steroid; R4is selected from optionally substituted C 6-24 alkyl, C 6-24 alkenyl, C 6-24 alkyne or C 6-24 alkoxy; m, n, o, p, q are each independently selected from any integer between 0-12; with the proviso that the compound:

2. The compound according to claim 1, characterized in that, R1, R2 are each independently selected from H, OH, C1-C9 alkoxy; L1, L2are each independently selected from the group consisting of a chemical bond (-), -0-, -S-, -0(C=0)0-, -(C=0)NR a -, -NR a (C=0)-, -0(C=0)-, -(C=0)0-, -S-S-, -S(0) x -, -OS(O) x O-, -C(=0)S-, -SC(=0)-, -NR a C(=0)NR b -, -OC(=0)NR a -, -NR a C(=0)0-, -OC(=0)S-, -SC(=0)0-; preferably, L1, L2are each independently selected from the group consisting of -0-, -S-, -0(C=0)0-, -(C=0)NR a -, -NR a (C=0)-, -0(C=0)-, -(C=0)0-, -S-S-, -S(0) x -, -OS(O) x O-, -C(=0)S-, -SC(=0)-; more preferably, L1, L2are each independently selected from the group consisting of -0-, -S-, -0(C=0)0-, -0(C=0)-, -(C=0)0-, -S-S-; x is selected from 0, 1 or 2; R a , R b are each independently selected from H, C 1-12 alkyl, C 2-12 alkenyl or C 2-12 alkynyl; R3 is selected from a naturally occurring or non-naturally occurring steroid; R4is selected from the group consisting of optionally substituted C 6-24 alkyl, C 6-24 alkenyl; preferably, R4is selected from the group consisting of optionally substituted C 6-24 alkyl; m, n, o, p, q are each independently selected from any integer between 0-12.

3. The compound of claim 1, wherein said R3 is a sterol; Preferably, said sterol is an animal sterol, or an oxidized or reduced form thereof; and / or, said sterol is a plant sterol, or an oxidized or reduced form thereof; and / or, said sterol is a synthetic sterol, or an oxidized or reduced form thereof; More preferably, said sterol is selected from cholestanol, an oxidized form of cholestanol, a reduced form of cholestanol, alkyl lithocholates, stigmasterol, stigmastanol, campesterol, ergosterol or sitosterol; More preferably, said sterol is selected from cholestanol, an oxidized form of cholestanol, a reduced form of cholestanol, alkyl lithocholates, stigmasterol, stigmastanol, campesterol, ergosterol or sitosterol; More preferably, said sterol is selected from avenasterol, beta-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprostanol, desmosterol, chalinosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, dinosterol, epicholesterol, ergosterol, fucosterol, hexahydro-lumisterol, hydroxycholesterol, lanosterol, lumisterol, mycocerotic acid, mycosterol, sitostanol, sitosterol, stigmastanol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid or lithocholic acid; More preferably, the sterol has the structural formula: wherein R" is C 1-20 alkyl.

4. The compound according to any one of claims 1 to 3, wherein said R4 is selected from: Preferably, said R4 is selected from:

5. The compound according to any one of the preceding claims, selected from or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof; wherein L1, L2, R4, m, p and q are as defined in any one of claims 1-4.

6. The compound according to claim 5 or its stereoisomers, tautomers, and pharmaceutically acceptable salts; wherein L1is selected from any one of -0-, -0(C=0)0-, -0(C=0)-, -(C=0)0-; L2is selected from any one of -0-, -0(C=0)0-, -0(C=0)-, -(C=0)0-, -S-S-; R4is selected from m is selected from 2 or 3 or 4; p and q are each independently selected from 1, 2, 3, 4, 5, 6, 7, or 8.

7. The compound of claim 5, which is a compound represented by Formula II-1 or a stereoisomer, a tautomer, and pharmaceutically acceptable salts thereof; wherein L1is selected from -(C=0)0-; L2is selected from -0(C=0)-; R4is selected from m is selected from 3; p is selected from 2; q is selected from any integer between 4-8.

8. The compound of any one of claims 1-7, wherein, The compound is selected from: or stereoisomers, tautomers, and pharmaceutically acceptable salts thereof.

9. A lipid nanoparticle characterized in that, The use of a compound according to any one of claims 1-8 or a stereoisomer, tautomer, and pharmaceutically acceptable salt thereof.

10. The lipid nanoparticle of claim 9, wherein, Also included are polyethylene glycol lipids and at least one helper lipid; wherein the helper lipid is selected from a neutral lipid, a zwitterionic lipid or an anionic lipid.

11. The lipid nanoparticle of claim 10, wherein, The polyethylene glycol lipid is selected from 2-[(polyethylene glycol)-2000]-N,N-tetracosanoylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycero-methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[ammonium(polyethylene glycol)] (PEG-DSPE), PEG-disterosidylglycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycerol amide (PEG-DAG), PEG-dipalmitoyl phosphatidyl ethanolamine (PEG-DPPE) or PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA); The helper lipid is selected from 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), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), palmitoyloleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidyl ethanolamine (POPE), phosphocholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidyl ethanolamine (PE), egg phosphatidylcholine (EPC), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), 1-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1,2-eicosenoyl-sn-glycero-3-phosphocholine (DEPC), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, distearoyl phosphatidyl ethanolamine (DSPE), or lysophosphatidyl ethanolamine.

12. The lipid nanoparticle of claim 10, wherein, The compound according to any one of claims 1 to 8 has a molar ratio of helper lipid of 1:2 to 5:1, and the molar proportion of polyethylene glycol lipid in the total lipid is 0.5% to 20%; preferably, the compound according to any one of claims 1 to 8 has a molar ratio of helper lipid of 1:1 to 4:1, and the molar proportion of polyethylene glycol lipid in the total lipid is 0.5% to 5%.

13. The lipid nanoparticle of claim 10, wherein, It has a diameter of 15 nm to 300 nm.

14. Use of the compound according to claims 1 to 8 and / or the lipid nanoparticle according to claims 9 to 13 for the manufacture of a biological active substance delivery system.

15. Use according to claim 14, characterized in that, The biological active substance is DNA or RNA, wherein the RNA is selected from the group consisting of antisense RNA, saRNA, mRNA, IncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, circRNA and self-replicating mRNA; Preferably, the biological active substance delivery system is an mRNA drug. Preferably, the mRNA drug is a vaccine for the prevention and treatment of cancer, viral infection, bacterial infection, fungal infection. Preferably, the virus is selected from the group consisting of norovirus, Ebola virus, coronavirus, cytomegalovirus, dengue virus, Zika virus, coxsackie virus, enterovirus, hepatitis virus, herpes simplex virus, human papillomavirus, influenza virus, Marburg virus, measles virus, poliovirus, rabies virus, rotavirus, and / or rhabdovirus.

16. The lipid nanoparticle of any one of claims 9-13, wherein, Further comprising a biologically active substance.

17. The lipid nanoparticle of claim 16, wherein, The biologically active substance is DNA or RNA, wherein the RNA is preferably selected from the group consisting of antisense RNA, saRNA, mRNA, IncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, circRNA, and self-replicating mRNA.

18. The lipid nanoparticle of claim 16 or 17, wherein, The lipid nanoparticle has a nitrogen to phosphorus ratio of (1-15):

1.

19. A medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The medicament comprises the lipid nanoparticle according to any one of claims 16-18, and a pharmaceutically acceptable excipient.

20. The medicament according to claim 19, characterized in that, The medicament is a liquid formulation, a lyophilized powder.

21. The medicament according to claim 19, characterized in that, The medicament is an oral formulation, a muscle injection formulation, a subcutaneous injection formulation, an intravenous injection formulation, an aerosol inhalation formulation, and / or a dry powder inhalation formulation, a microneedle patch.

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