Aminolipid compounds, methods for their preparation, compositions thereof and uses thereof

Abstract

The present disclosure relates to amino lipid compounds, their preparation methods, compositions thereof and their uses.Specifically, the present disclosure relates to an amino lipid compound represented by formula (I) or its pharma-ceutically acceptable salt or stereoisomer, and its use in the formulation of lipid nanoparticles for delivering active ingredients.The present disclosure also relates to the composition containing said amino lipid compound, in particular lipid nanoparticles and its uses. JPEG2025508877000185.jpg38170

Description

[Technical field]

[0001] The present disclosure relates to amino lipid compounds that can be used to prepare lipid nanoparticles for delivering active ingredients, and methods for preparing the same. The present disclosure also relates to compositions, particularly lipid nanoparticles, comprising said amino lipid compounds, and uses thereof. [Background technology]

[0002] Gene therapy drugs introduce genes with specific genetic information into target cells by artificial means, and the expressed target proteins exert regulatory, therapeutic, and even curative effects on diseases caused by congenital or acquired gene defects, or the gene series interferes with or regulates the expression of related genes to exert clinical therapeutic effects. However, gene therapy drugs still face several challenges, such as the difficulty of directly introducing nucleic acids into cells and the fact that they are highly susceptible to degradation by nucleases in the cytoplasm. Therefore, in order to facilitate the achievement of the therapeutic and / or preventive purposes of gene therapy drugs, it is necessary to develop more amino lipid compounds that can be used to deliver nucleic acids as active ingredients, as well as related preparation methods and applications. Summary of the Invention

[0003] Summary of the Invention In one aspect of the disclosure, there is provided an amino lipid compound represented by formula (I): [ka]

[0004] In the formula, R1, R2, R3, R4, R5, Z1, Z2, Z3, Z4, Z5, Z6, A1, A2, A3, A4, A5, A6 and A7 are each defined as described below. In another aspect of the present disclosure, a method for preparing the amino lipid compounds is provided. In another aspect of the present disclosure, there is provided the use of the amino lipid compounds described above in the manufacture of a vehicle for an active ingredient. In another aspect of the present disclosure, there is provided a lipid nanoparticle comprising the amino lipid compound described above. In another aspect of the disclosure, a composition is provided comprising the amino lipid compound. In another aspect of the present disclosure, there is provided a use of the amino lipid compound, lipid nanoparticle, or composition described above in the manufacture of a medicament. In another aspect of the present disclosure, there is provided a use of the amino lipid compound, lipid nanoparticle, or composition in the manufacture of a pharmaceutical for nucleic acid transfer. [Brief description of the drawings]

[0005] [Figure 1] 1 shows the results of an ALT enzyme activity test 12 hours after in vivo delivery of lipid nanoparticles in Experimental Example 4. [Diagram 2] 1 shows the results of an ELISPOT cell immunoassay for mouse splenic lymphocytes using a representative amino lipid compound in Experimental Example 5. [Diagram 3] 1 shows the results of an ELISPOT cell immunoassay for mouse splenic lymphocytes using a representative amino lipid compound in Experimental Example 5. [Figure 4] 1 shows the results of an ELISPOT cell immunoassay for splenic lymphocytes of mice treated with a representative amino lipid compound in Experimental Example 5. [Diagram 5] 1 shows the IgG titer of serum from mice immunized for 14 days with LNPs containing IN002.5.1 mRNA encapsulated in a representative amino lipid compound in Experimental Example 6. [Figure 6] 1 shows the IgG titer of serum from mice immunized for 14 days with LNPs containing N002.5.1 mRNA encapsulated in a representative amino lipid compound in Experimental Example 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Detailed Description of the Invention definition Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art. References to technology used herein are intended to mean technology commonly understood in the art, including variations or equivalent technology substitutes that are obvious to those skilled in the art. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are provided to better explain this disclosure.

[0007] As used herein, the terms "comprising," "including," "having," "containing," or "involving," and other variations thereof, are inclusive or open-ended and do not exclude other, non-recommended elements or method steps.

[0008] As used herein, the term "hydrocarbyl" refers to the group remaining after losing one hydrogen atom from an aliphatic hydrocarbon, and includes linear or branched, saturated or unsaturated hydrocarbyl. Hydrocarbyl groups include, but are not limited to, alkyl, alkenyl, and alkynyl. Preferably, the hydrocarbyl group contains 1 to 24 carbon atoms (C1-C 24 hydrocarbyl), for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms (C1, C2, C3, ... C 17 , C 18 , C 19 , or C 20 Examples of hydrocarbyl groups include C1-C 24 Hydrocarbyl, C1-C 20 Hydrocarbyl, C1-C 18 Hydrocarbyl, C1-C 16 Hydrocarbyl, C1-C 12 Hydrocarbyl, C1-C 10Hydrocarbyl includes, but is not limited to, hydrocarbyl, C1-C8 hydrocarbyl, C1-C7 hydrocarbyl, C1-C6 hydrocarbyl, C1-C4 hydrocarbyl, C1-C3 hydrocarbyl, C1-C2 hydrocarbyl, C2-C8 hydrocarbyl, C2-C4 hydrocarbyl, C4-C8 hydrocarbyl, C4-C9 hydrocarbyl, C5-C8 hydrocarbyl, C1-C4 hydrocarbyl, C2-C8 hydrocarbyl, C3 hydrocarbyl, C4 hydrocarbyl, C5 hydrocarbyl, C6 hydrocarbyl, C7 hydrocarbyl, and C8 hydrocarbyl. Unless otherwise expressly stated herein, hydrocarbyl is optionally substituted, and the substituents refer to the definition of "optionally substituted" below. In certain embodiments, hydrocarbyl has no branching (i.e., is linear) or has one branch, two branches, or multiple branches.

[0009] As used herein, the term "hydrocarbylene" refers to the divalent group remaining after the loss of one hydrogen atom from hydrocarbyl, as defined above. Unless otherwise expressly stated in the specification, hydrocarbylene is also optionally substituted.

[0010] As used herein, the term "alkyl" refers to a linear or branched, saturated monovalent hydrocarbyl. Preferably, an alkyl group has 1 to 24 carbon atoms (C1-C 24 alkyl), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms (C1, C2, C3, ... C 17 , C 18 , C 19 , or C 20 Examples of alkyl groups include C1-C 24 Alkyl, C1-C 20 Alkyl, C1-C 18 Alkyl, C1-C 16 Alkyl, C1-C 12 Alkyl, C1-C 10Examples of alkyl include, but are not limited to, alkyl, C1-C8 alkyl, C1-C7 alkyl, C1-C6 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C8 alkyl, C2-C4 alkyl, C4-C8 alkyl, C4-C9 alkyl, C5-C8 alkyl, C1-C4 alkyl, C2-C8 alkyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, and tridecane-7-yl. Unless otherwise expressly stated herein, alkyl is optionally substituted.

[0011] As used herein, the term "alkylene" refers to the divalent group remaining after losing one additional hydrogen atom from an alkyl, as defined above. Unless otherwise expressly stated in the specification, alkylene is also optionally substituted.

[0012] As used herein, the term "alkenyl" refers to a linear or branched monovalent hydrocarbyl containing one or more double bonds (C=C). Preferably, the alkenyl group contains 2 to 24 carbon atoms (C2-C 24 alkenyl), for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms (C2, C3, ... C 17 , C 18 , C 19 , or C 20 alkenyl) having one, two, three, four or more double bonds. Examples of alkenyl groups include C2-C 24 Alkenyl, C2-C 20 Alkenyl, C2-C 18 Alkenyl, C2-C 16 Alkenyl, C2-C 12 Alkenyl, C2-C 10Alkenyl includes, but is not limited to, alkenyl, C2-C8 alkenyl, C2-C7 alkenyl, C2-C6 alkenyl, C2-C4 alkenyl, C2-C3 alkenyl, C4-C8 alkenyl, C4-C9 alkenyl, and C5-C8 alkenyl. Some more specific examples include, but are not limited to, ethenyl, propenyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-2-enyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, hept-1-enyl, hept-2-enyl, hept-3-enyl, oct-1-enyl, oct-2-enyl, oct-3-enyl, non-1-enyl, non-2-enyl, and non-3-enyl. In some preferred embodiments, the alkenyl group has one double bond. Unless stated otherwise expressly in the specification, the alkenyl is optionally substituted.

[0013] As used herein, the term "alkenylene" refers to the divalent group remaining after losing one additional hydrogen atom from the alkenyl defined above. Unless expressly stated otherwise in the specification, alkenylene is optionally substituted.

[0014] As used herein, the term "alkynyl" refers to a linear or branched monovalent alkynyl group containing one or more triple bonds (C≡C). Preferably, the alkynyl group is an alkyl group having 2 to 24 carbon atoms (C2-C 24 alkynyl), for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms (C2, C3, ... C 17 , C 18 , C 19 , or C 20 Alkynyl groups include C2-C alkynyl groups having 1, 2, 3, 4 or more triple bonds. 24 Alkynyl, C2-C 20 Alkynyl, C2-C 18 Alkynyl, C2-C 16 Alkynyl, C2-C 12 Alkynyl, C2-C 10Examples include, but are not limited to, alkynyl, C2-C8 alkynyl, C2-C7 alkynyl, C2-C6 alkynyl, C2-C4 alkynyl, C2-C3 alkynyl, C4-C8 alkynyl, C4-C9 alkynyl, and C5-C8 alkynyl. Some more specific examples include, but are not limited to, ethynyl, propynyl, but-1-ynyl, but-2-ynyl, pent-1-ynyl, pent-2-ynyl, hex-1-ynyl, hex-2-ynyl, hex-3-ynyl, hept-1-ynyl, hept-2-ynyl, hept-3-ynyl, oct-1-ynyl, oct-2-ynyl, oct-3-ynyl, non-1-ynyl, non-2-alkynyl, and non-3-alkynyl. In some preferred embodiments, the alkynyl group has one triple bond. Unless stated otherwise specifically in the specification, an alkynyl is optionally substituted.

[0015] As used herein, the term "alkynylene" refers to the divalent group remaining after losing one additional hydrogen atom from the alkynyl defined above. Unless otherwise expressly stated in the specification, the alkynylene is optionally substituted.

[0016] As used herein, the terms "cyclohydrocarbyl", "cyclohydrocarbylene", and "hydrocarbon ring" refer to a monocyclic or polycyclic hydrocarbon ring that is saturated (i.e., "cycloalkyl" and "cycloalkylene") or unsaturated (i.e., having one or more double bonds (cycloalkenyl) and / or triple bonds (cycloalkynyl) in the ring) having ring carbon atoms. In certain embodiments, "cyclohydrocarbyl", "cyclohydrocarbylene", and "hydrocarbon ring" have, for example, 3 to 10, suitably 3 to 8, more suitably 3 to 6, e.g., 5 to 6, or 5 to 7 ring carbon atoms. "Cyclohydrocarbyl", "cyclohydrocarbylene", and "hydrocarbon ring" include, but are not limited to, cyclopropyl(ren)(ring), cyclobutyl(ren)(ring), cyclopentyl(ren)(ring), cyclohexyl(ren)(ring), cycloheptyl(ren)(ring), cyclooctyl(ren)(ring), cyclononyl(ren)(ring), cyclohexenyl(ren)(ring), and the like. Unless expressly stated otherwise in the specification, cyclohydrocarbyl, cyclohydrocarbylene, and hydrocarbon rings are optionally substituted.

[0017] As used herein, the term "cycloalkyl" refers to a saturated monocyclic or polycyclic (e.g., bicyclic) hydrocarbon ring (e.g., monocyclic such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or bicyclo[1.1.1]pentyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, or bicyclo[5.2.0]nonyl, spirocyclic such as decalin, bicyclic including fused or bridged systems, etc.). In certain embodiments, cycloalkyl has, for example, 3 to 10, e.g., 3 to 7, 5 to 6, or 5 to 7 carbon atoms. Unless expressly stated otherwise in the specification, cycloalkyl is optionally substituted.

[0018] As used herein, the term "heterohydrocarbyl" or subclasses thereof (e.g., heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, etc.) refer to a stable linear, branched, or cyclic hydrocarbon radical or combinations thereof consisting of a specified number of carbon atoms and at least one heteroatom. Heteroatom refers to an atom other than carbon and hydrogen. In certain embodiments, heterohydrocarbyls contain one, two, three, or more heteroatoms. In certain embodiments, heterohydrocarbyls contain one or more (e.g., two or three) identical heteroatoms or multiple (e.g., two or three) different heteroatoms. Preferably, the heteroatoms are selected from O, N, and S. Examples of heterohydrocarbyl include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-CH2-O-CH2-CH3, -CH2-(CH2)3-O-(CH2)5-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -CH=CH-O-CH3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, and -CH2-NH-OCH3. Unless expressly stated otherwise in the specification, heterohydrocarbyl or subclass thereof (e.g., heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, etc.) is optionally substituted.

[0019] As used herein, the term "heterohydrocarbylene" or sub-concepts thereof (heteroalkylene, heteroalkenylene, heteroalkynylene, heteroarylene, etc.) refers to the divalent group remaining after losing one additional hydrogen atom from the heterohydrocarbyl defined above. Unless otherwise expressly stated in the specification, "heterohydrocarbylene" or sub-concepts thereof (heteroalkylene, heteroalkenylene, heteroalkynylene, heteroarylene, etc.) are optionally substituted.

[0020] As used herein, the term "heterocycle", "heterocyclyl" or "heterocyclylene" refers to a cyclic group having a ring structure and containing one or more heteroatoms among the ring-forming atoms. In certain embodiments, the ring-forming atoms contain one or more heteroatoms that are the same or different. In certain embodiments, the one or more heteroatoms contained in the ring-forming atoms are selected from N, O and S. The "heterocycle", "heterocyclyl" or "heterocyclylene" disclosed herein is saturated or unsaturated. In certain embodiments, the "heterocycle", "heterocyclyl" or "heterocyclylene" consists of a monocyclic ring, a bicyclic ring, or a polycyclic ring. In certain embodiments, the "heterocycle", "heterocyclyl" or "heterocyclylene" is a 4-10 membered heterocycle, for example, a 4-7 membered heterocycle, a 5-7 membered heterocycle. Preferably, in certain embodiments, the heterocycle is an optionally substituted 4- to 10-membered saturated heterocycle, where the ring-forming atoms include 1, 2, 3, 4, 5, or 6 heteroatoms selected from N, O, and S. More preferably, the heterocycle is an optionally substituted 4- to 7-membered saturated heterocycle, where the ring-forming atoms include 1, 2, 3, or 4 heteroatoms selected from N, O, and S; more preferably, the heterocycle is an optionally substituted 5- to 7-membered (e.g., 5- to 6-membered) saturated heterocycle, where the ring-forming atoms include 1, 2, or 3 heteroatoms selected from N, O, and S. Examples of heterocycles include, but are not limited to, azetidine, oxetanyl, tetrahydrofuran, pyrrolidine, imidazolidine, pyrazolidine, tetrahydropyran, piperidine, morpholine, thiomorpholine, piperazine, preferably pyrrolidine, piperidine, piperazine, morpholine. The heterocycle may be optionally substituted with one or more substituents, the substituents being referred to below in the definition of "optionally substituted." Unless expressly stated otherwise in the specification, the heterocycle, heterocyclyl, or heterocyclylene is optionally substituted.

[0021] As used herein, the terms "aryl" and "aromatic ring" refer to an all-carbon monocyclic or fused polycyclic aromatic group having a conjugated pi-electron system. 6-10 Aryl(ene)" and "C 6-10 The term "aromatic ring" means an aromatic group containing 6 to 10 carbon atoms, such as phenyl (benzene ring) or naphthyl (naphthalene ring). Unless expressly stated otherwise in the specification, aryl and aromatic rings are optionally substituted.

[0022] As used herein, the term "heteroaryl" or "heteroaryl ring" refers to a monocyclic, bicyclic or tricyclic aromatic ring system containing at least one heteroatom selected from N, O and S, e.g. having 5, 6, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular containing 1 or 2 or 3 or 4 or 5 or 6 or 9 or 10 carbon atoms, which in each case may further be benzo-fused. For example, the heteroaryl or heteroaryl ring can be selected from thienyl (ring), furyl (ring), pyrrolyl (ring), oxazolyl (ring), thiazolyl (ring), imidazolyl (ring), pyrazolyl (ring), isoxazolyl (ring), isothiazolyl (ring), oxadiazolyl (ring), triazolyl (ring), thiadiazolyl (ring), and the like, and benzo derivatives thereof; or pyridyl (ring), pyridazinyl (ring), pyrimidinyl (ring), pyrazinyl (ring), triazinyl (ring), and the like, and benzo derivatives thereof. Unless expressly stated otherwise herein, the heteroaryl or heteroaryl ring is optionally substituted.

[0023] As used herein, the term "optionally substituted" means that one or more hydrogen atoms bonded to an atom or group are independently unsubstituted or substituted with one or more (e.g., 1, 2, 3, or 4) substituents. Substituents include, independently, deuterium (D), tritium (T), halogen, -OH, mercapto, cyano, -CD3, C1-C6 alkyl (preferably C1-C3 alkyl), C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl (preferably C3-C8 cycloalkyl), aryl, heterocyclyl (preferably 3- to 8-membered heterocyclyl), heteroaryl, aryl C1-C6 alkyl-, heteroaryl C1-C6 alkyl, C1-C6 haloalkyl, -OC1-C6 alkyl (preferably -OC1-C3 alkyl), -OC2-C6 alkenyl, OC1-C6 alkylphenyl, C1-C6 alkyl-OH (preferably C1-C4 alkyl-OH), C1-C6 alkyl-SH, C1-C6 alkyl-O-C1-C6 alkyl, OC1-C6 haloalkyl, NH2, C1-C6 alkyl-NH2 (preferably C1-C3 alkyl-NH2), -N(C1-C6 alkyl)2 (preferably -N(C1-C3 alkyl)2), -NH(C1-C6 alkyl) (preferably -NH(C1-C3 alkyl)), -N(C1-C6 alkyl)(C1-C6 alkylphenyl), -NH(C1-C6 alkylphenyl), nitro, -C(O)-OH, -C(O)OC1-C6 alkyl (preferably -C(O)OC1-C3 alkyl), -CONRiRii (wherein Ri and Ri are H, D and C1-C6 alkyl, preferably C1-C3 alkyl).), -NHC(O)(C1-C6 alkyl), -NHC(O)(phenyl), -N(C1-C6 alkyl)C(O)(C1-C6 alkyl), -N(C1-C6 alkyl)C(O)(phenyl), -C(O)C1-C6 alkyl, -C(O)heteroaryl (preferably -C(O)-5- to 7-membered heteroaryl), -C(O)C1-C6 alkylphenyl, -C(O)C1-C6 haloalkyl, -OC(O)C1-C6 alkyl (preferably -OC(O)C1-C3 alkyl), -S(O)2-C1-C6 alkyl, -S(O)-C1-C6 alkyl, -S(O)2-phenyl, -S(O)2-C1-C6 haloalkyl, -S(O)2NH2, -S(O)2NH(C1-C6 alkyl), -S(O)2NH(phenyl), -NHS(O)2(C1-C6 alkyl), -NHS(O)2(phenyl) and -NHS(O)2(C1-C6 haloalkyl), wherein alkyl, cycloalkyl, phenyl, aryl, heterocyclyl and heteroaryl are each independently selected from the group consisting of aryl, cycloalkyl, phenyl, aryl, heterocyclyl and heteroaryl. Each of these further comprises halogen, -OH, -NH2, cycloalkyl, 3- to 8-membered heterocyclyl, C1-C4 alkyl, C1-C4 haloalkyl-, -OC1-C4 alkyl, -C1-C4 alkyl-OH, -C1-C4 alkyl-O-C1-C4 alkyl, -OC1-C4 haloalkyl, cyano, nitro, -C(O)-OH, -C(O)OC1-C6 alkyl, -CON(C1-C6 alkyl)2, -CONH(C1-C6 alkyl), -CONH2, -NHC(O)(C1-C6 alkyl) ), -NH(C1-C6 alkyl)C(O)(C1-C6 alkyl), -SO2(C1-C6 alkyl), -SO2(phenyl), -SO2(C1-C6 haloalkyl), -SON2NH2, -SON2NH(C1-C6 alkyl), -SON2NH(phenyl), -NHSO2(C1-C6 alkyl), -NHSO2(phenyl), and -NHSO2(C1-C6 haloalkyl). When an atom or group is substituted with multiple substituents, the multiple substituents may be the same or different.

[0024] In certain embodiments, the substituents are independently selected from halogen (e.g., chlorine, bromine, fluorine, iodine), carboxylic acid (e.g., -C(=O)-OH), oxygen (e.g., =O), sulfur (e.g., =S), hydroxyl (e.g., -OH), ester group (e.g., -C(=O)ORiii or -OC(=O)Riii), aldehyde group (e.g., -C(=O)H), carbonyl (e.g., represented by -C(=O)Riii, or C=O), acyl halide (e.g., -C(=O)X, where X is bromine, fluorine, chlorine, or iodine), carboxylic ester group (e.g., -OC(=O)ORiii), alkoxy group (e.g., -ORiii), acetal (e.g., -C(ORiii)2Riii, where each ORiii is the same or different and is an alkoxy group), phosphate (e.g., P(=O)4 3- ), thiols (e.g. -SH), sulfoxides (e.g. -S(=O)Riii), sulfinic acids (e.g. -S(=O)OH), sulfonic acids (e.g. -S(=O)2OH), thioaldehydes (e.g. -C(=S)H), sulfates (e.g. S(=O)4 2-), sulfonyl (e.g., -S(=O)2Riii), sulfinyl (e.g., -S(=O)Riii), amido (e.g., -C(=O)N(Riii)2 or -N(Riii)C(=O)Riii), azido (e.g., -N3), nitro (e.g., -NO2), cyano (e.g., -CN), isocyano (e.g., -NC), acyloxy (e.g., -OC(=O)Riii), amino (e.g., -N(Riii)2, -N(Riii)H, or -NH2), carbamoyl (e.g., -OC(=O)N(Riii)2, -OC(=O)N(Riii)H or -OC(=O)NH), sulfonamide (e.g., -S(=O)2N(Riii)2, -S(=O)2N(Riii)H, -S(=O)2NH2, -N(Riii)S(=O)2Riii, -N(H)S(=O)2Riii, -N(Riii)S(=O)2H, or -N(H)S(=O)2H), alkyl, alkenyl, alkynyl, cyclohydrocarbyl (e.g., cycloalkyl, cycloalkenyl, or cycloalkynyl), heterocyclohydrocarbyl (e.g., a heterocyclo containing one or more heteroatoms selected from S, N, and O), or heterocyclenyl containing one or more heteroatoms selected from S, N, and O), aryl (e.g., phenyl or fused ring groups), heteroaryl (e.g., 8- to 10-membered bicyclic heteroaryl containing 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur), -C(=O)SRiii, -C(=N-CN)N(Riii)2, -C(=NO-CH3)N(Riii)2, -C(=N-SO2-NH2)N(Riii)2, -C(=CH-NO2)N(Riii)2, -OC(=O)N(Riii)2, -CHN(R iii)N(Riii)2, -C(=O)N(Riii)ORiii, -N(Riii)2C(=O)ORiii, -OP(=O)(ORiii)2, -P(=O)(ORiii)2, -N(ORiii)C(=O)Riii, -N(ORiii)S(=O)2Riii, -N( ORiii)C(=O)ORiii, -N(ORiii)C(=O)N(Riii)2, -N(ORiii)C(=S)N(Riii)2, -N(ORiii)C(NRiii)N(Riii)2, -N(ORiii)C(CHRiii)N(Riii)2,In any of the above, Riii is hydrogen, or alkyl, or alkenyl, or alkynyl, or heteroalkyl, or heteroalkenyl, or heteroalkynyl, as defined herein. In some embodiments, Riii is hydrogen, or C1-C, 12 Alkyl, or C1-C 12 Alkenyl, or C1-C 12 Alkynyl, or C1-C 12 Heteroalkyl, or C1-C 12 Heteroalkenyl, or C1-C 12 It is heteroalkynyl.

[0025] In certain embodiments, the substituents themselves may be further substituted, for example, with one or more substituents as defined herein. For example, C1-C6 alkyl as a substituent may be further substituted with one or more substituents as defined herein.

[0026] As used herein, the term "pharmaceutically acceptable salt" refers to a base salt of an organic or inorganic acid, including, but not limited to, hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate, trifluoroacetate, thiocyanate, maleate, hydroxymaleate, glutarate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, benzoate, salicylate, phenylacetate, cinnamate, lactate, malonate, pivalate, succinate, fumarate, malate, mandelate, tartrate, gallate, gluconate, laurate, palmitate, pectinate, picrate, citrate, or combinations thereof.

[0027] As used herein, the term "halo" or "halogen" group is defined to include F, Cl, Br, or I.

[0028] Numerical ranges described herein should be understood to encompass the boundaries and any subranges contained therein. For example, a range of "1-10" should be understood to include not only the values ​​1 and 10 explicitly mentioned, but also the individual values ​​(e.g., 2, 3, 4, 5, 6, 7, 8, and 9) and subranges (e.g., 1-2, 1.5-2.5, 1-3, 1.5-3.5, 2.5-4, 3-4.5, etc.) within the range of 1-10. This principle also applies to ranges that use only one value as a minimum or maximum value.

[0029] As used herein, the term "isomers" refers to different compounds that have the same molecular formula. "Stereoisomers" are isomers that differ only in the arrangement of atoms in space. "Atropisomers" are stereoisomers that result from hindered rotation about a single bond. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of a pair of enantiomers in any ratio may be called a "racemic" mixture. "Diastereoisomers" are stereoisomers that have at least two asymmetric atoms and are not mirror images of each other. "Tautomers" are isomers of a compound that are in equilibrium with each other. The concentration of an isomer depends on the environment in which the compound is found, for example, whether the compound is a solid, in an organic solution, or in an aqueous solution.

[0030] In certain embodiments, "stereoisomer" also includes E isomers, Z isomers, or mixtures thereof, cis isomers, trans isomers, or mixtures thereof.

[0031] The nucleic acids and / or polynucleotides useful in the present disclosure include a coding region encoding a polypeptide of interest, a 5'-UTR at the 5'-end of the coding region, and a 3'-UTR at the 3'-end of the coding region. In some embodiments, the nucleic acid or polynucleotide further includes at least one of a polyadenylation region and a Kozak sequence. In some embodiments, the nucleic acid or polynucleotide (e.g., mRNA) may include a 5' cap structure. Any region of the nucleic acid may include one or more alternative nucleosides, such as 5-substituted uridine (e.g., 5-methoxyuridine), 1-substituted pseudouridine (e.g., 1-methyl-pseudouridine or 1-ethyl-pseudouridine), and / or 5-substituted cytidine (e.g., 5-methyl-cytidine).

[0032] The term "5'-UTR" or "5'-untranslated region" can be an RNA sequence in an mRNA that is located upstream of a coding sequence and is not translated into a protein. The 5'-UTR of a gene typically begins at a transcription initiation site and ends at a nucleotide upstream of the translation initiation codon of the coding sequence. The 5'-UTR can contain elements that control gene expression, such as a ribosome binding site, a 5' terminal oligopyrimidine tract, and a translation initiation signal, such as a Kozak sequence. An mRNA can be post-transcriptionally modified by the addition of a 5' cap. Thus, the 5'-UTR in a mature mRNA can also refer to the RNA sequence between the 5' cap and the initiation codon. As used herein, the term "3' untranslated region" or "3'-UTR" can be an RNA sequence in an mRNA that is located upstream of a coding sequence and is not translated into a protein. The 3'-UTR in an mRNA is located between the stop codon of the coding sequence and the poly(A) sequence, for example, beginning at a nucleotide downstream of the stop codon and ending at a nucleotide upstream of the poly(A) sequence. The sequences of the 5'-UTR and / or 3'-UTR may be homologous or heterologous to the sequence of the coding region. The 3'-UTR may consist of a 3'-UTR derived from at least one of the following genes: albumin gene, α-globin gene, β-globin gene, tyrosine hydroxylase gene, lipoxygenase gene, and collagen α gene.

[0033] In the present specification, the terms "polyadenylation region", "poly(A) sequence" and "poly(A) tail" are used interchangeably. Naturally occurring poly(A) sequences are typically composed of adenine ribonucleotides. Preferably, "polyadenylation region" refers to a poly(A) sequence that includes nucleotides or nucleotide segments other than adenine ribonucleotides. Poly(A) sequences are usually located at the 3' end of an mRNA, for example, at the 3' end (downstream) of the 3'-UTR. The length of the polyA region may vary. In particular, in some embodiments, the length of the polyA region of the nucleic acid molecule of the present disclosure is at least 30 nucleotides; in some embodiments, the length of the polyA region of the nucleic acid molecule of the present disclosure is at least 80 nucleotides; in some embodiments, the length of the polyA region of the nucleic acid molecule of the present disclosure is at least 100 nucleotides.

[0034] As used herein, the term "5' cap structure" refers to a 5' cap structure typically located at the 5' end of a mature mRNA. In some embodiments, the 5' cap structure is linked to the 5' end of an mRNA with a 5'-5'-triphosphate bond. The 5' cap structure is typically formed from modified (e.g., methylated) ribonucleotides (especially from guanine nucleotide derivatives). For example, m7GpppN (cap 0, or "cap 0" is a cap structure formed by the 5'-phosphate group of hnRNA interacting with the 5'-phosphate group of m7GTP under the action of guanylate transferase to form a 5',5'-phosphodiester bond), where N is the terminal 5' nucleotide of the nucleic acid having the 5'-cap structure. In some embodiments, the 5' cap structure includes, but is not limited to, cap 0, cap 1 (a cap structure formed from cap 0 by further methylating the 2'-OH of the ribose on the first nucleotide of an hnRNA, or "cap 1"), cap 2 (a cap structure formed from cap 1 by further methylating the 2'-OH of the ribose on the second nucleotide of an hnRNA, or "cap 2"), cap 4, a cap 0 analog, a cap 1 analog, a cap 2 analog, or a cap 4 analog.

[0035] Amino lipid compounds In one aspect, the present disclosure provides an amino lipid compound represented by formula (I) or a pharma- ceutically acceptable salt or stereoisomer thereof: [ka]

[0036] During the ceremony, Z1, Z2, Z3, Z4, Z5, and Z6 are each independently -CH(OH)-, -C=C-, -C≡C-, -O-, -C(=O)O-, -OC(=O)-, -N(R6)C(=O)-, -C(=O)N(R6)-, -N(R6)C(=O)N(R6)-, -OC(=O)N(R6)-, -N(R6)C(=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -SS-, or a bond; A1, A2, A3, A4, A5, A6, and A7 are each independently C1-C 12 is hydrocarbylene, cyclohydrocarbyl, phenyl, benzyl, a heterocycle, or a bond; R1 and R2 are each independently H or C1-C 18 hydrocarbyl, or cyclohydrocarbyl, phenyl, benzyl, or a heterocycle; or R1 and R2 together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocycle; R3 is H or C1-C 18 hydrocarbyl, cyclohydrocarbyl, phenyl, benzyl, heterocycle; R4 and R5 are each independently C1-C 18 is hydrocarbyl, or cyclohydrocarbyl, phenyl, benzyl, or a heterocycle; R6 is H or C1-C 18 Hydrocarbyl or -OH, -O- optionally substituted C2-C 18 Hydrocarbyl, or -C=C-, or -C≡C- optionally substituted C4-C 18 is a hydrocarbyl; Preferably, Z5 and Z6 are each independently -OC(=O)-, -C(=O)O-, or a bond.

[0037] In some embodiments, the present disclosure provides an amino lipid compound of formula (I) as described above, or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein either Z5 or Z6 is -(C=O)O-; or both are -(C=O)O-. In some embodiments, the present disclosure provides an amino lipid compound of formula (I) as described above, or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein either Z5 or Z6 is -O(C=O)-; or both are -O(C=O)-. In some embodiments, the present disclosure provides an amino lipid compound of formula (I) above, or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein either Z1 or Z2 is a bond; or both Z1 and Z2 are bonds. In some embodiments, the present disclosure provides an amino lipid compound of formula (I) above, or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein either A1 or A2 is a bond; and both are bonds. In some embodiments, the present disclosure provides that Z3 is -(C=O)O- or -O(C=O)-; and further, R4 and R5 are branched C3-C 18 Provided is an amino lipid compound of formula (I) as above, or a pharma- ceutically acceptable salt or stereoisomer thereof, which is hydrocarbyl.

[0038] In another aspect, the present disclosure provides an amino lipid compound represented by formula (I) or a pharma- ceutically acceptable salt or stereoisomer thereof: [ka]

[0039] During the ceremony: Z1, Z2, Z3, Z4, Z5, and Z6 are each independently -CH(OR7)-, -C=C-, -C≡C-, -O-, -C(=O)O-, -OC(=O)-, -N(R6)C(=O)-, -C(=O)N(R6)-, -N(R6)C(=O)N(R6)-, -OC(=O)N(R6)-, -N(R6)C(=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -SS-, or a bond; A1, A2, A3, A4, A5, A6, and A7 are each independently C1-C 12is hydrocarbylene, C3-C7 cyclohydrocarbyl, phenyl, benzyl, a 4-7 membered heterocycle, or a bond; R1 and R2 are each independently H or C1-C 18 hydrocarbyl, or C3-C7 cyclohydrocarbyl, phenyl, benzyl, or a 4- to 7-membered heterocycle; or R1 and R2 together with the nitrogen atom to which they are attached form a 4- to 7-membered heterocycle having said nitrogen atom and 0, 1, 2, or 3 additional heteroatoms in the ring independently selected from N, O, and S, said heterocycle being optionally substituted with 1, 2, 3, or more substituents independently selected from C1-C8 alkyl, C1-C8 haloalkyl, -O-C1-C8 alkyl, -O-C1-C8 haloalkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2; R3 is H or C1-C 18 hydrocarbyl, or C3-C7 cyclohydrocarbyl, phenyl, benzyl, 4-7 membered heterocycle; R4 and R5 are each independently C1-C 18 hydrocarbyl, or C3-C7 cyclohydrocarbyl, phenyl, benzyl, or a 4- to 7-membered heterocycle; R6 is H or C1-C 18 Hydrocarbyl or -OH, -O- optionally substituted C2-C 18 Hydrocarbyl, or -C=C-, or -C≡C- optionally substituted C4-C 18 Hydrocarbyl, or C1-C 18 is heterohydrocarbyl; and R7 is H or C1-C 12 It is a hydrocarbyl.

[0040] In some embodiments, R6 is H or C1-C 18 Hydrocarbyl or -OH, -O- optionally substituted C2-C 18 Hydrocarbyl, or -C=C-, or -C≡C- optionally substituted C4-C 18It is a hydrocarbyl.

[0041] In some embodiments, the present disclosure provides: A1, A2, A3, A4, A5, A6, and A7 are each independently C1-C 12 Alkylene, C2-C with 1, 2, 3, 4 or more double bonds 12 Alkenylene, C3-C6 cycloalkyl, phenyl, benzyl, or a 5- to 6-membered heterocycle or a bond. Preferably, A1, A2, A3, A4, A5, A6, and A7 are each independently C1-C 12 Alkylene, C2-C with 1, 2, 3, 4 or more double bonds 12 Alkenylene or a bond. Preferably, A1 and A2 are each independently C1-C6 alkylene or a bond, more preferably C1, C2, C3, C4, or C5 alkylene or a bond, even more preferably C1 or C2 alkylene or a bond. Preferably, A3 is C1-C6 alkylene or a bond, more preferably C2, C3, C4, or C5 alkylene, even more preferably C2, C3, or C4 alkylene. Preferably, A4 is C1-C6 alkylene or a bond, more preferably C1, C2, C3, C4, or C5 alkylene or a bond, even more preferably C1, C2, C3, or C4 alkylene or a bond. Preferably, A5 is C1-C8 alkylene or a bond, more preferably C1, C2, C3, C4, or C5 alkylene or a bond, even more preferably C1, C2, C3, or C4 alkylene or a bond. Preferably, A6 and A7 are each independently C5-C 12 Alkylene, more preferably C-C 11 Alkylene, more preferably C7, C8, C9 or C 10 Provided is an amino lipid compound of formula (I) as above, or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein: R is an alkylene;

[0042] In some embodiments described above, R1 and R2 are each independently H or C1-C 18Alkyl, C2-C with 1, 2, 3, 4 or more double bonds 18 Alkenyl or C3-C6 cycloalkyl, phenyl, benzyl, or 5- to 6-membered heterocycle, preferably C1-C 18 Alkyl or C2-C with 1, 2, 3, 4 or more double bonds 18 alkenyl; or R1 and R2, together with the nitrogen atom to which they are attached, form a 4-7 membered heterocycle having said nitrogen atom and 0, 1, 2 or 3 additional heteroatoms independently selected from N, O and S in the ring, said heterocycle being optionally substituted with 1, 2, 3 or more substituents independently selected from C1-C8 alkyl, C1-C8 haloalkyl, -O-C1-C8 alkyl, -O-C1-C8 haloalkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl). In some preferred embodiments, R1 and R2 are each independently selected from C1-C8 alkyl, C1-C8 haloalkyl, -O-C1-C8 alkyl, -O-C1-C8 haloalkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl). 18 It is alkyl or a bond, preferably C1, C2, C3, C4, or C5 alkyl or a bond, more preferably methyl, ethyl, n-propyl, or isopropyl. In another preferred embodiment, R1 and R2 together with the nitrogen atom to which they are bonded form a 4- to 7-membered heterocycle, preferably a 5- to 6-membered heterocycle, having the nitrogen atom in the ring.

[0043] In some embodiments described above, R is H, C 18 Alkyl, C2-C with 1, 2, 3, 4 or more double bonds 18 alkenyl, or C3-C6 cycloalkyl, phenyl, benzyl, or a 5-6 membered heterocycle. 18 Alkyl or C2-C with 1, 2, 3, 4 or more double bonds 18 alkenyl. Preferably, R3 is C1-C 16 Alkyl, more preferably C-C 14Alkyl, more preferably C1, C2, C3, C4, C5, C6, C7, C8, C9 or C 10 Alkyl

[0044] In some embodiments of the above, R and R are each independently C-C 18 Alkyl, C2-C with 1, 2, 3, 4 or more double bonds 18 alkenyl or C3-C6 cycloalkyl, phenyl, benzyl, or a 5- to 6-membered heterocycle. 18 Alkyl or C2-C with 1, 2, 3, 4 or more double bonds 18 Preferably, R and R are each independently a branched or linear C-C alkenyl. 18 Alkyl, more preferably branched or straight chain C8, C9, C 10 , C 11 , C 12 , C 13 or C 14 Alkyl, more preferably branched C 12 or C 13 It is an alkyl.

[0045] In some embodiments described above, R6 is H, C1-C 18 Alkyl, C2-C with 1, 2, 3, 4 or more double bonds 18 Alkenyl, -OH, -O-optionally substituted C2-C 18 Alkyl, -O-C2-C with 1, 2, 3, 4 or more double bonds, optionally substituted 18 Alkenyl, or -C=C-, -C≡C- optionally substituted C4-C 18 Alkyl, or -C≡C-C4-C with 1, 2, 3, 4 or more double bonds, optionally substituted 18 Alkyl, or C1-C containing O, N or S 18 Heteroalkyl, or C2-C containing O, N or S 18 Heteroalkenyl, or C2-C containing O, N or S 18Preferably, R6 is H, C1-C8 alkyl, C2-C8 alkyl with 1, 2, or 3 double bonds. 12 Alkenyl, -OH, -O-optionally substituted C2-C 12 Alkyl, -O-C2-C with 1, 2, or 3 optionally substituted double bonds 12 Alkenyl, -C=C-, -C≡C- optionally substituted C4-C 12 Alkyl, or -C≡C-C4-C with 1, 2, or 3 optionally substituted double bonds 12 Alkyl, or C1-C8 heteroalkyl containing O, N or S, or C2-C 12 Heteroalkenyl, or C2-C containing O, N or S 12 Heteroalkynyl

[0046] In some embodiments described above, R6 is H, C1-C 18 Alkyl, C2-C with 1, 2, 3, 4 or more double bonds 18 Alkenyl, -OH, -O-optionally substituted C2-C 18 Alkyl, -O-C2-C with 1, 2, 3, 4 or more double bonds, optionally substituted 18 Alkenyl, or -C=C-, -C≡C- optionally substituted C4-C 18 Alkyl, or -C≡C-C4-C with 1, 2, 3, 4 or more double bonds, optionally substituted 18 Preferably, R6 is H, C1-C8 alkyl, C2-C6 alkyl with 1, 2, or 3 double bonds. 12 Alkenyl, -OH, -O-optionally substituted C2-C 12 Alkyl, -O-C2-C with 1, 2, or 3 optionally substituted double bonds 12 Alkenyl, -C=C-, -C≡C- optionally substituted C4-C 12 Alkyl, or -C≡C-C4-C with 1, 2, or 3 optionally substituted double bonds 12 It is an alkyl.

[0047] In some embodiments described above, R7 is H, C1-C 12 Alkyl or C2-C with 1, 2, or 3 double bonds 12 alkenyl. Preferably, R7 is H or C1-C 12 It is an alkyl. In some embodiments described above, Z5 and Z6 are each independently -OC(=O)-, -C(=O)O-, or a bond. In some embodiments described above, at least one of Z1 and Z2 is a bond, and preferably both Z1 and Z2 are bonds. In some embodiments described above, at least one of A1 and A2 is a bond, and preferably both A1 and A2 are bonds. In some embodiments described above, A5 is a bond. In some embodiments described above, at least one of A6 and A7 is a bond, and preferably both A6 and A7 are bonds. In some embodiments described above, Z4 is a bond. In some embodiments described above, Z3 is -CH(OR7)-, -(C=O)O-, -O(C=O)-, or a bond.

[0048] In some preferred embodiments, the present disclosure provides: Z1, Z2, and Z4 each independently represent a bond; Z3 is -CH(OR7)-, -C(=O)O-, -OC(=O)-, or a bond; Z5 and Z6 are each independently -C(=O)O- or -OC(=O)-; A1, A2, and A5 are 、 each independently is a bond; A3, A6, and A7 are each independently selected from C1-C 12 Alkylene, C2-C with 1, 2, 3, 4 or more double bonds 12 alkenylene; A4 is C1-C 12Alkylene, C2-C with 1, 2, 3, 4 or more double bonds 12 alkenylene or a bond; R1 and R2 are each independently C1-C 18 Alkyl or C2-C with 1, 2, 3, 4 or more double bonds 18 alkenyl; or R1 and R2 together with the nitrogen atom to which they are attached form a 4-7 membered heterocycle having said nitrogen atom and 0, 1, 2 or 3 additional heteroatoms independently selected from N, O and S in the ring, said heterocycle being optionally substituted with 1, 2, 3 or more substituents independently selected from C1-C8 alkyl, C1-C8 haloalkyl, -O-C1-C8 alkyl, -O-C1-C8 haloalkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2; R3 is H, C1-C 18 Alkyl or C2-C with 1, 2, 3, 4 or more double bonds 18 alkenyl; R4 and R5 are each independently C1-C 18 Alkyl or C2-C with 1, 2, 3, 4 or more double bonds 18 alkenyl; and R7 is H, C1-C 12 Alkyl or C2-C with 1, 2, or 3 double bonds 12 Alkenyl, preferably H, or C1-C 12 is alkyl, Provided is an amino lipid compound of formula (I) above, or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0049] In some such embodiments, the present disclosure provides: Z3 is a bond; A4 is a bond; R1 and R2 are each independently C1-C 18 Alkyl or C2-C with 1, 2, 3, 4 or more double bonds 18alkenyl; or R1 and R2 together with the nitrogen atom to which they are attached form a 4-7 membered heterocycle having said nitrogen atom and 0, 1, or 2 additional heteroatoms independently selected from N, O, and S in the ring, said heterocycle being optionally substituted with 1, 2, or 3 substituents independently selected from C1-C8 alkyl, C1-C8 haloalkyl, -O-C1-C8 alkyl, -O-C1-C8 haloalkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2; R3 is C1-C 18 Alkyl or C2-C with 1, 2, 3, 4 or more double bonds 18 Alkenyl, preferably C1-C 18 is alkyl, Provided is an amino lipid compound of formula (I) above, or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0050] In other such embodiments, the present disclosure provides: Z3 is -CH(OR7)-; R1 and R2 are each independently C1-C 18 Alkyl or C2-C with 1, 2, 3, 4 or more double bonds 18 Alkenyl, preferably C1-C 18 is alkyl, Provided is an amino lipid compound of formula (I) above, or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0051] In some of the above-described embodiments, R7 is H. In some of such embodiments, A4 is a bond. In other embodiments, A4 is a C1-C 12 Alkylene, C2-C with 1, 2, 3, 4 or more double bonds 12 Alkenylene, preferably C1-C 10 alkenylene, more preferably C1-C8 alkylene, more preferably n4=1, 2, 3, 4, 5, 6, 7 or 8 -(CH2) n4In some embodiments described above, R3 is C1-C 18 Alkyl or C2-C with 1, 2, 3, 4 or more double bonds 18 Alkenyl, preferably C1-C 18 It is an alkyl.

[0052] In other embodiments of the above, R7 is H, C1-C 12 Alkyl or C2-C with 1, 2, or 3 double bonds 12 alkenyl. Preferably, R7 is C1-C 12 In some such embodiments, R3 is H. In other embodiments, R3 is C1-C 18 Alkyl or C2-C with 1, 2, 3, 4 or more double bonds 18 alkenyl. Preferably, R3 is C1-C 18 In some embodiments of the above, A4 is a bond. In other embodiments, A4 is a C1-C 12 Alkylene, C2-C with 1, 2, 3, 4 or more double bonds 12 Alkenylene. Preferably, A4 is C1-C 10 Alkylene, more preferably C1-C8 alkylene, more preferably n4=1, 2, 3, 4, 5, 6, 7 or 8 -(CH2) n4 -It is.

[0053] In other such embodiments, the present disclosure provides: Provided is an amino lipid compound of formula (I) as above, or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein Z3 is -C(=O)O- or -OC(=O)-.

[0054] In some embodiments described above, Z3 is -C(=O)O-, where the C(=O) moiety is attached to A4; and R3 is C1-C 18 Alkyl or C2-C with 1, 2, 3, 4 or more double bonds 18 It is alkenyl.

[0055] In some such embodiments, A4 is a bond. In other embodiments, A4 is a C1-C 12 Alkylene, C2-C with 1, 2, 3, 4 or more double bonds 12 Alkenylene. Preferably, A4 is C1-C 10 Alkylene, more preferably C1-C8 alkylene, more preferably n4=1, 2, 3, 4, 5, 6, 7 or 8 -(CH2) n4 -It is.

[0056] In some embodiments described above, each hydrocarbylene or alkylene defined for A1, A2, A3, A4, A5, A6, or A7 is independently C1-C 10 In other embodiments, the hydrocarbylene or alkylene defined for A1, A2, A3, A4, A5, A6, or A7 is independently a C2-C 10 It is alkenylene.

[0057] In some embodiments described above, A3 is C1-C8 alkylene or C2-C8 alkenylene having 1 or 2 double bonds. Preferably, A3 is C1-C6 alkylene or C2-C6 alkenylene having 1 double bond, more preferably C2-C4 alkylene or C2-C4 alkenylene having 1 double bond, and more preferably -(CH2)2-, -(CH2)3- or -(CH2)4-.

[0058] In some embodiments of the above, A6 and A7 are each independently C1-C 10 Alkylene, C2-C with 1, 2, 3, 4 or more double bonds 10 Preferably, A6 and A7 are each independently C5-C 10 Alkylene, C5-C with 1, 2, 3, 4 or more double bonds 10Alkenylene, more preferably C6-C9 alkylene, C6-C9 alkenylene having 1, 2, 3, 4 or more double bonds, more preferably C7-C8 alkylene, C7-C8 alkenylene having 1, 2 or more double bonds.

[0059] In some embodiments described above, the hydrocarbyl or alkyl defined for R1 or R2 is C1-C8 alkyl, preferably C1-C6 alkyl, more preferably C1-C4 alkyl, and more preferably C1-C2 alkyl.

[0060] In another embodiment of the above, the heterocycle formed by R1 and R2 together with the nitrogen atom to which they are attached is a 5-7 membered heterocycle having the nitrogen atom and 0, 1, or 2 additional heteroatoms independently selected from N, O, and S in the ring, and the heterocycle is optionally substituted with 1, 2, or 3 substituents independently selected from C1-C6 alkyl, C1-C6 haloalkyl, -O-C1-C6 alkyl, -O-C1-C6 haloalkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2. Preferably, the heterocycle is a 5-6 membered heterocycle having the nitrogen atom and no additional heteroatoms in the ring, and the heterocycle is optionally substituted with 1, 2, or 3 substituents independently selected from C1-C4 alkyl, preferably C1-C3 alkyl. More preferably, the heterocycle is [ka] It is.

[0061] In some embodiments described above, the hydrocarbyl or alkyl defined for R is C1-C 12 Alkyl, preferably C-C 10 Alkyl, and more preferably -(CH2) n31 -CH3, or -CH((CH2) n32 -CH3)-(CH2) n33-CH3, where n31 is 0, 1, 2, 3, 4, 5, 6, 7, or 8, n32 is 0, 1, 2, 3, 4, 5, or 6, preferably 0, 1, 2, or 3; and n33 is 0, 1, 2, 3, 4, 5, 6, 7, or 8, preferably 0, 1, 2, 3, 4, 5, or 6.

[0062] In some embodiments described above, the hydrocarbyl or alkenyl defined for R3 is a C2-C 12 Alkenyl, preferably C-C with one double bond 10 It is alkenyl.

[0063] In some embodiments described above, the hydrocarbyl or alkyl defined for R is C1-C 10 Alkyl, preferably C1-C8 alkyl, more preferably -(CH2) n7 -CH3, where n7 is 0, 1, 2, 3, 4, 5, 6, or 7.

[0064] In some embodiments described above, the hydrocarbyl or alkenyl defined for R7 is a C2-C 10 Alkenyl, preferably C2-C8 alkenyl having one or two double bonds.

[0065] In some of the above-mentioned embodiments, at least one of Z5 and Z6 is -C(=O)O-, preferably both Z5 and Z6 are -C(=O)O-, more preferably wherein the C(=O) moiety is bonded to A6 or A7.

[0066] In some embodiments described above, the hydrocarbyl or alkyl defined for R4 or R5 is branched, preferably a branched C3-C 18 Alkyl, more preferably branched C-C 18 Alkyl, e.g., branched C 11 -C 18 Alkyl or branched C 13 -C 15is alkyl, and preferably [ka] It is.

[0067] In some embodiments described above, the hydrocarbyl or alkenyl defined for R4 or R5 is branched, preferably a branched C3-C 18 Alkenyl, more preferably branched C-C 18 Alkenyl, for example, branched C 11 -C 18 Alkenyl or branched C 13 -C 15 alkenyl, where said alkenyl group has 1, 2, or 3 double bonds.

[0068] In some embodiments, the amino lipid compound of formula (I) or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein the amino lipid compound has a structure of formula (II): [ka]

[0069] During the ceremony, Z3, Z5 and Z6 are each independently -C(=O)O- or -OC(=O)-; A3 is a C2-C5 alkylene; A4 is a C1-C5 alkylene or a bond; R1, R2 are each independently H or C1-C3 alkyl; or R1 and R2 together with the nitrogen atom to which they are attached form a 4-7 membered heterocycle having said nitrogen atom in the ring, said heterocycle being optionally substituted with one, two, three or more substituents independently selected from C1-C8 alkyl, C1-C8 haloalkyl, -O-C1-C8 alkyl, -O-C1-C8 haloalkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl)2; R3 is C1-C10 is alkyl; and R4 and R5 are each independently a branch of C1-C 18 It is an alkyl.

[0070] In some embodiments, the present disclosure provides amino lipid compounds represented by formula (II) as described herein above, or pharma- ceutically acceptable salts or stereoisomers thereof, which, if applicable, include one or more of the following characteristics: In some embodiments, Z3 is -C(=O)O-. In some embodiments, Z5 is -C(=O)O-. In some embodiments, Z6 is -C(=O)O-. In some embodiments, A3 is C3-C4 alkylene. In some embodiments, A4 is C1-C2 alkylene, C3-C4 alkylene, or a bond. In some embodiments, R1 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R2 is methyl, ethyl, n-propyl, or isopropyl.

[0071] In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 5-6 membered heterocycle having said nitrogen atom in the ring, said heterocycle being optionally substituted with one, two, three or more substituents independently selected from C1-C8 alkyl, C1-C8 haloalkyl, -O-C1-C8 alkyl, -O-C1-C8 haloalkyl, halogen, OH, CN, nitro, NH2, -NH(C1-C6 alkyl), and -N(C1-C6 alkyl), e.g., methyl.

[0072] In some embodiments, the heterocycle is [ka] It is.

[0073] In some embodiments, R3 is C1, C2, C3, C4, C5, C6, C7, or C8 alkyl, wherein the alkyl is linear or branched.

[0074] In some embodiments, R4 is a branched C 13 -C 15 In some embodiments, R4 is [ka] It is.

[0075] In some embodiments, R5 is a branched C 13 -C 15 In some embodiments, R5 is [ka] It is.

[0076] In various embodiments, the present disclosure provides an amino lipid compound of formula (I) or (II) as described above, or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein the amino lipid compound has any of the structures shown in Table 1 below.

[0077] [Table 1] JPEG2025508877000012.jpg239170JPEG2025508877000013.jpg240170JPEG2025508877000014.jpg243170 JPEG2025508877000015.jpg235170JPEG2025508877000016.jpg232170JPEG2025508877000017.jpg237170 JPEG2025508877000018.jpg232170JPEG2025508877000019.jpg231170JPEG2025508877000020.jpg234170 JPEG2025508877000021.jpg222170JPEG2025508877000022.jpg215170JPEG2025508877000023.jpg234170 JPEG2025508877000024.jpg226170JPEG2025508877000025.jpg232170JPEG2025508877000026.jpg238170 JPEG2025508877000027.jpg232170JPEG2025508877000028.jpg226170JPEG2025508877000029.jpg221170 JPEG2025508877000030.jpg221170JPEG2025508877000031.jpg231170JPEG2025508877000032.jpg219170 JPEG2025508877000033.jpg233170JPEG2025508877000034.jpg226170JPEG2025508877000035.jpg172170

[0078] All of the amino lipid compounds of the present disclosure have hydrophobic properties due to the presence of long non-polar residues, and at the same time hydrophilic properties due to the amino groups. Due to this amphiphilic property, the amino lipid compounds of the present disclosure can be used to form lipid nanoparticles such as lipid bilayers, micelles, and liposomes.

[0079] In the context of the present disclosure, the term "lipid nanoparticles" refers to nanometer-sized materials produced by introducing amino lipid compounds into an aqueous solution. The particles are in particular lipid nanoparticles, lipid bilayer vesicles (liposomes), multilamellar vesicles or micelles.

[0080] In some preferred embodiments, the lipid nanoparticles are liposomes comprising the amino lipid compounds of the present disclosure. Within the scope of the present disclosure, liposomes are small microparticles consisting of a bilayer of lipid amphiphilic molecules encapsulating an aqueous compartment.

[0081] The formation of liposomes is not a spontaneous process. When lipids are introduced into water, lipid vesicles first form, which then form a bilayer or a series of bilayers, each separated by water molecules. Liposomes are formed by sonicating lipid vesicles in water.

[0082] In the context of this disclosure, the term "lipid bilayer" refers to a thin film formed by two layers of lipid molecules. The term "micelle" refers to an aggregate of surfactant molecules dispersed in a liquid colloid. Typical micelles in aqueous solutions form aggregates upon contact with water with hydrophilic head regions that chelate the hydrophobic single tail region in the center of the micelle.

[0083] In one aspect, the present disclosure provides the use of the amino lipid compounds of the present disclosure for the manufacture of a vehicle for an active ingredient. In some embodiments, the vehicle is in the form of a lipid nanoparticle, such as a lipid bilayer, a micelle, or a liposome.

[0084] Lipid Nanoparticles (LNPs) In another aspect, the present disclosure provides lipid nanoparticles comprising an amino lipid compound of the present disclosure and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0085] In some embodiments, the lipid nanoparticles further comprise one or more of a helper lipid, a structural lipid, and a PEG-lipid (polyethylene glycol-lipid).

[0086] In some further embodiments, the lipid nanoparticle further comprises a helper lipid, a structural lipid, and a PEG lipid.

[0087] In some embodiments, the lipid nanoparticles comprise an amino lipid compound in an amount of about 25.0% to 75.0%, e.g., about 25.0%-28.0%, 28.0%-32.0%, 32.0%-35.0%, 35.0%-40.0%, 40.0%-42.0%, 42.0%-45.0%, 45.0%-46.3%, 46.3%-48.0%, 48.0%-49.5%, 49.5%-50.0%, 50.0%-55.0%, 55.0%-60.0%, 60.0%-65.0%, or 65.0%-75.0%, based on the total amount of amino lipid compound, helper lipid, structural lipid, and PEG-lipid.

[0088] In some embodiments, the lipid nanoparticles comprise helper lipids in an amount of about 5.0% to 45.0%, e.g., about 5.0%-9.0%, 9.0%-9.4%, 9.4%-10.0%, 10.0%-10.5%, 10.5%-11.0%, 11.0%-15.0%, 15.0%-16.0%, 16.0%-18.0%, 18.0%-20.0%, 20.0%-25.0%, 25.0%-33.5%, 33.5%-37.0%, 37.0%-40.0%, 40.0%-42.0%, or 42.0%-45.0%, based on the total amount of amino lipid compound, helper lipid, structural lipid, and PEG-lipid.

[0089] In some embodiments, the lipid nanoparticles comprise from about 0.0% to 50.0%, for example, about 0.0%-10.0%, 10.0%-15.5%, 15.5%-18.5%, 18.5%-22.5%, 22.5%-23.5%, 23.5%-28.5%, 28.5%-33.5%, 33.5%-35.0%, 35.0%-36.5%, 36.5%-37.5%, 38.5%-40.5%, 40.5%-42.5%, 42.5%-44.5%, 44.5%-46.5%, 46.5%-48.5%, 48.5%-50.5%, 50.5%-52.5%, 52.5%-54.5%, 54.5%-56.5%, 56.5%-58.5%, 58.5%-60.5%, 58.5%-60.5%, 58.5%-60.5%, 58.5%-70.5%, 58.5%-70.5%, 58.5%-80.5%, 58.5%-80.5%, 58.5%-90.5%, 58.5%-90.5%, 58.5%-10 ... and comprising structural lipids in an amount (mol percent) of 5%-38.0%, 38.0%-38.5%, 38.5%-39.0%, 39.0%-39.5%, 39.5%-40.5%, 40.5%-41.5%, 41.5%-42.5%, 42.5%-42.7%, 42.7%-43.0%, 43.0%-43.5%, 43.5%-45.0%, 45.0%-46.5%, 46.5%-48.5%, or 46.5%-50.0%.

[0090] In some embodiments, the lipid nanoparticles comprise PEG-lipids in an amount (mol percent) of about 0.5% to 5.0%, e.g., about 0.5%-1.0%, 1.0%-1.5%, 1.5%-1.6%, 1.6%-2.0%, 2.0%-2.5%, 2.5%-3.0%, 3.0%-3.5%, 3.5%-4.0%, 4.0%-4.5%, or 4.5%-5.0%, based on the total amount of amino lipid compound, helper lipid, structural lipid, and PEG-lipid.

[0091] In the context of the present disclosure, the helper lipid is a phospholipid. The phospholipid is generally semi-synthetic and may be naturally derived or chemically modified. The phospholipid may include DSPC (distearoylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOPC (dioleoyllecithin), DOPS (dioleoylphosphatidylserine), DSPG (1,2-octacosyl-sn-glycero-3-phospho-(1'-rac-glycerol)), DPPG (dipalmitoylphosphatidylglycerol), DPPC (dipalmitoylphosphatidylcholine), DGTS (1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)homoserine), lysophospholipid, etc. Preferably, the helper lipid is one or more selected from the group consisting of DSPC, DOPE, DOPC, and DOPS. In some embodiments, the helper lipid is DSPC and / or DOPE.

[0092] In the context of the present disclosure, the structured lipid is a sterol, including but not limited to cholesterol, cholesterol ester, steroid hormone, steroid vitamin, bile acid, cholesterin, ergosterol, β-sitosterol, oxidized cholesterol derivatives, etc. Preferably, the structured lipid is at least one selected from cholesterol, cholesteryl ester, steroid hormone, steroid vitamin, and bile acid. In some embodiments, the structured lipid is cholesterol, preferably high-purity cholesterol, especially high-purity cholesterol for injection such as CHO-HP.

[0093] In the context of the present disclosure, PEG-lipid (polyethylene glycol-lipid) is a conjugate of polyethylene glycol and a lipid structure. Preferably, the PEG-lipid is selected from PEG-DMG and PEG-distearoylphosphatidylethanolamine (PEG-DSPE), preferably PEG-DMG. Preferably, the PEG-DMG is a polyethylene glycol (PEG) derivative of 1,2-dimyristoyl-sn-glycerol. Preferably, the PEG has an average molecular weight of about 2,000 to 5,000, preferably about 2,000.

[0094] In some embodiments described above, the lipid nanoparticles have a molar ratio of amino lipid compounds of the present disclosure:helper lipid:structural lipid:PEG-lipid of about 45:10:42.5:2.5, or 45:11:41.5:2.5, or 42.0:10.5:45.0:2.5, or 42.0:16.0:39.5:2.5, or 40.0:16.0:41.5:2.5, or 40.0:18.0:3 9.5:2.5, or 35.0:16.0:46.5:2.5, or 35.0:25.0:36.5:3.5, or 28.0:33.5:35.0:3.5, or 32.0:37.0:40.5:0.5, or 35.0:40.0:22.5:2.5, or 40.0:42.0:15.5:2.5, or 40.0:20.0:38.5:1.5, or 45.0:15.0:38. 5:1.5, or 55.0:5.0:38.5:1.5, or 60.0:5.0:33.5:1.5, or 45.0:20.0:33.5:1.5, or 50.0:20.0:28.5:1.5, or 55.0:20.0:23.5:1.5, or 60.0:20.0:18.5:1.5, or 40.0:15.0:43.5:1.5, or 50.0:15.0:33.5:1. 5, or 55.0:15.0:28.5:1.5, or 60.0:15.0:23.5:1.5, or 40.0:10.0:48.5:1.5, or 45.0:10.0:43.5:1.5, or 55.0:10.0:33.5:1.5, or 40.0:5.0:53.5:1.5, or 45.0:5.0:48.5:1.5, or 50.0:5.0:43.5:1.5. In some such embodiments, the helper lipid is DOPE and the structural lipid is CHO-HP.

[0095] In other embodiments described above, the lipid nanoparticles have a molar ratio of amino lipid compounds of the present disclosure:helper lipid:structural lipid:PEG-lipid of about 50.0:10.0:38.5:1.5, or 50.0:9.0:38.0:3.0, or 49.5:10.0:39.0:1.5, or 48.0:10.0:40.5:1.5, or 46.3:9.4:42.7:1.6, or 45.0:9.0 :43.0:3.0, or 45.0:11.0:41.5:2.5, or 42.0:10.5:45.0:2.5, or 42.0:16.0:39.5:2.5, or 40.0:16.0:41.5:2.5, or 40.0:18.0:39.5:2.5, or 35.0:40.0:22.5:2.5, or 40.0:20.0:38.5:1.5, or 45.0:15.0:38 .5:1.5, or 55.0:5.0:38.5:1.5, or 60.0:5.0:33.5:1.5, or 45.0:20.0:33.5:1.5, or 50.0:20.0:28.5:1.5, or 55.0:20.0:23.5:1.5, or 60.0:20.0:18.5:1.5, or 40.0:15.0:43.5:1.5, or 50.0:15.0:33.5:1 0.5, or 55.0:15.0:28.5:1.5, or 60.0:15.0:23.5:1.5, or 40.0:10.0:48.5:1.5, or 45.0:10.0:43.5:1.5, or 55.0:10.0:33.5:1.5, or 40.0:5.0:53.5:1.5, or 45.0:5.0:48.5:1.5, or 50.0:5.0:43.5:1.5. In some such embodiments, the helper lipid is DSPC and the structural lipid is CHO-HP.

[0096] In some embodiments, the lipid nanoparticles have the amino lipid compounds of the present disclosure, the helper lipids, the structural lipids, and the PEG-lipids in mole percent (%) as shown in Table 2, Nos. 1-24, below, based on the total amount of the amino lipid compounds, the helper lipids, the structural lipids, and the PEG-lipids:

[0097] [Table 2]

[0098] In some embodiments, the lipid nanoparticles have the amino lipid compounds of the present disclosure, the helper lipids, the structural lipids, and the PEG-lipids in mole percent (%) as shown in Table 3 below, Nos. 25-42, based on the total amount of the amino lipid compounds, the helper lipids, the structural lipids, and the PEG-lipids:

[0099] [Table 3]

[0100] As mentioned above, the lipid nanoparticles of the present disclosure can be used for delivery of active ingredients.

[0101] In some embodiments, the active ingredient comprises a therapeutic and / or prophylactic agent.

[0102] The term "therapeutic agent" or "prophylactic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject.

[0103] An "effective amount" or a "therapeutically effective amount" refers to an amount of the amino lipid compound of the present disclosure or lipid nanoparticles containing the amino lipid compound of the present disclosure that is sufficient to effectively effect treatment in a mammal (preferably a human) when administered to the mammal (preferably a human). The amount of lipid nanoparticles of the present disclosure that constitutes a "therapeutically effective amount" depends on the amino lipid compound, the condition and its severity, the mode of administration, and the age of the mammal being treated, but can be routinely determined by one of ordinary skill in the art in light of his or her knowledge and the present disclosure.

[0104] Preferably, the pharma- ceutical active ingredient is a biologically active ingredient, which is a substance that, when introduced into a cell or host, has a biological effect, e.g., by stimulating an immune or inflammatory response, by exerting an enzymatic activity, by complementing a mutation, etc. Biologically active ingredients include, but are not limited to, nucleic acids, proteins, peptides, antibodies, small molecules, and mixtures thereof.

[0105] Preferably, the biologically active moiety is a nucleic acid.

[0106] In another preferred embodiment, the biologically active ingredient is an anti-neoplastic agent, an antibiotic, an immunomodulatory agent, an anti-inflammatory agent, an agent acting on the central nervous system, a polypeptide, a polypeptoid, or a mixture thereof.

[0107] When lipid nanoparticles encapsulate an active ingredient in their internal aqueous space, they are sometimes called "lipid nanoparticle pharmaceuticals."

[0108] In the context of this disclosure, the term "cell" is a generic term and includes individual cells, tissues, organs, insect cells, avian cells, fish cells, amphibian cells, mammalian cells, primary cells, continuous cell lines, stem cells, and / or cultures of genetically engineered cells (e.g., recombinant cells expressing heterologous polypeptides or proteins, such as growth factors or blood factors).

[0109] In some embodiments described above, the lipid nanoparticles of the present disclosure further comprise a nucleic acid.

[0110] In some embodiments, the mass ratio of amino lipid compounds of the present disclosure to nucleic acid in the lipid nanoparticle is about (5-30):1, e.g., about (5-10):1, (10-15):1, (15-20):1, (20-25):1, or (25-30):1, preferably about 10:1.

[0111] In some embodiments, the nucleic acid is selected from the group consisting of RNA, antisense oligonucleotides, and DNA.

[0112] In some embodiments, the RNA is selected from the group consisting of messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), short interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single guide RNA (sgRNA), Cas9 mRNA, or a mixture thereof.

[0113] In some embodiments, the messenger RNA (mRNA) encodes a polypeptide and / or protein of interest. Any naturally occurring or non-naturally occurring or otherwise modified polypeptide is included. In some embodiments, the polypeptide and / or protein encoded by the mRNA can have a therapeutic and / or prophylactic effect when expressed in a cell.

[0114] In some embodiments, the RNA is an siRNA that can selectively reduce expression of a gene of interest or downregulate expression of a gene. For example, the siRNA can be selected such that a gene associated with a particular disease, disorder, or condition is silenced upon administration of lipid nanoparticles containing the siRNA to a subject in need thereof. The siRNA may comprise a sequence complementary to an mRNA sequence that codes for a gene or protein of interest. In some embodiments, the siRNA can be an immunomodulatory siRNA.

[0115] In certain embodiments, RNA is sgRNA and / or cas9 mRNA.sgRNA and / or cas9 mRNA can be used as gene editing tool.For example, sgRNA-cas9 complex can affect the mRNA translation of cellular genes.

[0116] In some embodiments, RNA is shRNA or its encoding vector or plasmid.shRNA can be produced in target cell after appropriate construct is delivered into nucleus.The construct and mechanism related to shRNA are well known in the relevant technical field.

[0117] In some embodiments, the DNA is a plasmid.

[0118] In some embodiments, lipid nanoparticles are used to deliver nucleic acids. In some embodiments, lipid nanoparticles can be used for, for example, gene therapy, gene vaccination, protein replacement therapy, antisense therapy, or interfering RNA therapy.

[0119] Pharmaceutical Compositions In another aspect, the present disclosure provides a pharmaceutical composition comprising a lipid nanoparticle as described above and a pharma- ceutically acceptable carrier, diluent, or excipient.

[0120] In some embodiments, the pharmaceutical composition further comprises a buffer. In some such embodiments, the buffer is selected from phosphate buffer and Tris buffer, preferably phosphate buffer. In some embodiments, the buffer has a concentration of about 5 mmol / L to about 30 mmol / L, preferably about 10 mmol / L. In some embodiments, the buffer has a pH of about 6 to 8, preferably about 7 to 8, more preferably about 7 to 7.5.

[0121] In some embodiments, the pharmaceutical composition further comprises a cryoprotectant. In some such embodiments, the cryoprotectant is selected from sucrose and trehalose, preferably sucrose. In some embodiments, the cryoprotectant has a concentration of about 50 mg / ml to 100 mg / ml.

[0122] In some embodiments described above, the pharmaceutical composition further comprises a cryoprotectant. In some such embodiments, the cryoprotectant is selected from sucrose and trehalose, preferably sucrose. In some embodiments, the cryoprotectant has a concentration of about 50 mg / ml to 100 mg / ml.

[0123] Purpose The lipid nanoparticles of the present disclosure have excellent properties of encapsulating biologically active components. The lipid nanoparticles containing biologically active components can be used to deliver any of a variety of therapeutic agents into cells. The present disclosure includes the use of lipid nanoparticles as described above to deliver biologically active components into cells. The present disclosure also provides a method for delivering biologically active components to cells, tissues or organs, comprising contacting the lipid nanoparticles of the present disclosure containing biologically active components with cells, tissues or organs. This provides new therapeutic possibilities for subjects.

[0124] In some embodiments, the tissue or organ is selected from the group consisting of spleen, liver, kidney, lung, femur, ocular tissue, vascular endothelium, lymph, and tumor tissue.

[0125] Preferably, the cell is a mammalian cell; more preferably, the mammalian cell is in a mammal. As used herein, the subject may be any mammal, and is preferably selected from the group consisting of mice, rats, pigs, cats, dogs, horses, goats, cows, and monkeys, etc. In some preferred embodiments, the subject is a human.

[0126] The present disclosure provides a method for producing a polypeptide and / or protein of interest in a mammalian cell, comprising contacting the cell with a lipid nanoparticle comprising mRNA encoding the polypeptide and / or protein of interest, wherein upon contacting the cell with the lipid nanoparticle, the mRNA can be taken up into the cell and translated to produce the polypeptide and / or protein of interest.

[0127] In yet another aspect, the present disclosure provides the use of an amino lipid compound, lipid nanoparticle, or pharmaceutical composition of the present disclosure in the manufacture of a medicament. Preferably, the pharmaceutical composition is used for the treatment and / or prevention of a disease.

[0128] In some embodiments, the disease is selected from the group consisting of a rare disease, an infectious disease, a cancer, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, and a metabolic disease.

[0129] The medicines are used, for example, in gene therapy, protein replacement therapy, antisense therapy or therapy with interfering RNA, genetic vaccination, etc.

[0130] In some embodiments, the cancer is selected from one or more of lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer, or prostate cancer. In some embodiments, the genetic disease is selected from one or more of hemophilia, thalassemia, and Gaucher disease.

[0131] In some embodiments, genetic vaccination is preferably used to treat and / or prevent cancer, allergy, toxicity, and pathogen infection. In some embodiments, the pathogen is selected from one or more of a virus, a bacterium, or a fungus.

[0132] The present disclosure provides a method of treating a disease or condition in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of lipid nanoparticles as described above.

[0133] Preferably, the disease or disorder is selected from the group consisting of a rare disease, an infectious disease, a cancer, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, and a metabolic disease.

[0134] In yet another aspect, the present disclosure provides the use of the amino lipid compound, lipid nanoparticle, or pharmaceutical composition of the present disclosure in the manufacture of a medicament for nucleic acid transfer.In some embodiments, the nucleic acid is selected from the group consisting of RNA, antisense oligonucleotide, and DNA.In some embodiments, the RNA is selected from the group consisting of messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), short interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single guide RNA (sgRNA), Cas9 mRNA, or a mixture thereof.In some embodiments, the DNA is a plasmid.

[0135] Preparation method In yet another aspect, the present disclosure also provides a general synthetic process for preparing the amino lipid compounds of formula (I) of the present disclosure, as follows: [ka]

[0136] During the ceremony, Z1, Z2, Z3, Z4, Z5, Z6, A1, A2, A3, A4, A5, A6, A7, R1, R2, R3, R4, R5 and R6 have the same meanings as defined above for the amino lipid compounds of formula (I). Specifically, the method includes: (1) M1 and M2-a are subjected to a reductive amination reaction to obtain M3, or M1 and M2-b are subjected to an alkylation reaction to obtain M3; (2) reacting M4 with an acylating agent (or triphosgene, chloroformate, DIC, DSC) to give M5; and (3) M5 is reacted with M3 in the presence of a base (e.g., triethylamine or pyridine, DMAP) to give the amino lipid compound of formula (I).

[0137] In yet another embodiment, the lipid nanoparticles or compositions of the present disclosure can be prepared according to methods known in the art. For example, the method may include the steps of: (1) Formulation: Formulating a suitable aqueous phase; and formulating an organic phase containing an amino lipid compound of the present disclosure, and optionally a helper lipid, a structural lipid, and / or a PEG lipid; (2) Encapsulation: mixing an appropriate amount of the aqueous phase with the organic phase; (3) Dialysis: Optionally, dialyzing the mixture of step (2); and (4) Sterilization: Optionally, the product of step (3) is sterilized by a sterilizing filter, such as a 0.22 μm microporous membrane.

[0138] In some embodiments, lipid nanoparticles or compositions of the present disclosure comprising nucleic acids, particularly mRNA, may be prepared by a method comprising the steps of: (1) Formulation: Formulating an aqueous phase containing nucleic acid; and formulating an organic phase (e.g., an ethanol phase) containing an amino lipid compound of the present disclosure, and optionally a helper lipid, a structural lipid, and / or a PEG lipid; (2) Encapsulation: mixing an appropriate amount of the aqueous phase with the organic phase; (3) Dialysis: Optionally, dialyzing the mixture of step (2); (4) Sterilization: Optionally, the product of step (3) is sterilized by a sterilizing filter, such as a 0.22 μm microporous membrane.

[0139] The present disclosure also includes the following embodiments: Embodiment 1. An amino lipid compound having the structure of formula (I), or a pharma- ceutically acceptable salt or stereoisomer thereof: [ka]

[0140] During the ceremony, Z1, Z2, Z3, Z4, Z5, and Z6 are each independently -CH(OH)-, -C=C-, -C≡C-, -O-, -C(=O)O-, -OC(=O)-, -N(R6)C(=O)-, -C(=O)N(R6)-, -N(R6)C(=O)N(R6)-, -OC(=O)N(R6)-, -N(R6)C(=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -SS-, or a bond; Z3 is -CH(OH)-, -C=C-, -C≡C-, -O-, -C(=O)O-, -OC(=O)-, -N(R6)C(=O)-, -C(=O)N(R6)-, -N(R6)C(=O)N(R6)-, -OC(=O)N(R6)-, -N(R6)C(=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -SS- or a bond; A1, A2, A3, A4, A5, A6, and A7 are each independently C1-C 12 is hydrocarbylene, cyclohydrocarbyl, phenyl, benzyl, a heterocycle, or a bond; R1 and R2 are each independently H or C1-C 18 hydrocarbyl, or cyclohydrocarbyl, phenyl, benzyl, or a heterocycle; or R1 and R2 together with the nitrogen atom to which they are attached form a 5- to 7-membered heterocycle; R3 is H or C1-C 18 is hydrocarbyl or cyclohydrocarbyl, phenyl, benzyl, heterocycle; R4 and R5 are each independently C1-C 18 hydrocarbyl, or cyclohydrocarbyl, phenyl, benzyl, heterocycle; R6 is H or C1-C 18 Hydrocarbyl or -OH, -O- optionally substituted C2-C 18 Hydrocarbyl, or -C=C-, -C≡C- optionally substituted C4-C 18 It is a hydrocarbyl.

[0141] Embodiment 2. The amino lipid compound of embodiment 1, wherein Z5 and Z6 are each independently -OC(=O)-, -C(=O)O-, or a bond. Embodiment 3. The amino lipid compound of any one of embodiments 1-2, wherein one of Z5 and Z6 is -C(=O)O-. Embodiment 4. The amino lipid compound of embodiments 1-2, wherein both Z5 and Z6 are -C(=O)O-. Embodiment 5. The amino lipid compound of any one of embodiments 1-2, wherein one of Z5 and Z6 is -OC(=O)-. Embodiment 6. The amino lipid compound of embodiments 1-2, wherein both Z5 and Z6 are -OC(=O)-. Embodiment 7 The amino lipid compound of any one of embodiments 1-2, wherein one of Z1 and Z2 is a bond. Embodiment 8 The amino lipid compound of any one of embodiments 1-2, wherein both Z1 and Z2 are a bond. Embodiment 9. The amino lipid compound of any one of embodiments 1-2, wherein one of A1 and A2 is a bond. Embodiment 10. The amino lipid compound of any one of embodiments 1-2, wherein both A1 and A2 are bonds. Embodiment 11. The amino lipid compound of any one of embodiments 1-2, wherein Z3 is -C(=O)O- or -OC(=O)-. Embodiment 12. R4 and R5 are branched C3-C 18 The amino lipid compound of embodiment 11, wherein the group is a hydrocarbyl group. Embodiment 13. A lipid nanoparticle comprising an amino lipid compound of any one of embodiments 1 to 12 and a pharma- ceutically acceptable carrier, diluent or excipient. Embodiment 14. A pharmaceutical composition comprising an amino lipid compound of any one of embodiments 1-12 and a pharma- ceutically acceptable carrier, diluent or excipient.

[0142] Embodiment 15. Use of an amino lipid compound according to any one of embodiments 1 to 12, a lipid nanoparticle according to embodiment 13, or a pharmaceutical composition according to embodiment 14 in the manufacture of a medicament for gene therapy, gene vaccination, antisense therapy, or therapy with interfering RNA. Embodiment 16 The use according to embodiment 15, wherein the gene therapy is useful for the treatment of cancer and genetic diseases. Embodiment 17. The use according to embodiment 16, wherein the cancer is selected from one or more of lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphatic system cancer, blood cancer, or prostate cancer; and the genetic disease is selected from one or more of hemophilia, thalassemia, and Gaucher disease. Embodiment 18. The use according to embodiment 17, wherein the genetic vaccination is useful for the treatment of cancer, allergies, toxicity and pathogen infections. Embodiment 19. The use according to embodiment 18, wherein the pathogen is selected from one or more of a virus, a bacterium, or a fungus. Embodiment 20. Use of the lipid nanoparticles according to embodiment 13 or the amino lipid compound according to any one of embodiments 1 to 12 in the manufacture of a medicament for nucleic acid transfer, wherein the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), short interfering RNA (siRNA), and small nuclear RNA (snRNA).

[0143] Beneficial effects The amino lipid compounds of the present disclosure can form vehicles such as lipid nanoparticles that have excellent properties for encapsulating biologically active ingredients and can be used to deliver biologically active ingredients, particularly water-insoluble drugs or active ingredients (such as nucleic acids) that are prone to degradation or degradation, and can improve their bioavailability and efficacy, or transfection efficiency (in the case of nucleic acids), or safety, or preference for specific organs or tissues. EXAMPLES

[0144] In order to make the objectives, technical solutions and advantages of the present disclosure clearer, the present disclosure is described below with reference to specific examples. The following examples are merely illustrative of the present disclosure and are not intended to be limiting.

[0145] example The following examples are offered by way of illustration and not by way of limitation. Experimental methods for which specific conditions are not specified in the examples are typically under conventional conditions or those recommended by the raw material or commercial product manufacturers, and reagents of unspecified origin are typically conventional reagents that are commercially available. The abbreviations used in the examples have the following meanings: DSC N,N'-disuccinimidyl carbonate; TLC thin layer chromatography; EA Ethyl acetate DCM Dichloromethane TEA Triethylamine; PMA Phosphomolybdic Acid THF Tetrahydrofuran DMF N,N-Dimethylformamide; EDCl 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DMAP 4-Dimethylaminopyridine; TEA-3HF Triethylamine trifluoride; M-DMG-2000 MethoxyPEG dimyristoyl-rac-glycero; h time min

[0146] Example 1: Synthesis of amino lipid compound 108 (1) Synthesis of compound 108-M3 [ka]

[0147] N,N-Dimethylaminopropylamine (108-M1) (3.2 g, 31.53 mmol), heptanal (108-M2) (3 g, 26.26 mmol), ethanol (15 mL), and palladium on carbon (0.15 g) were added to a 25 mL single-neck flask. The flask was transferred to a 500 mL autoclave and purged with nitrogen three times, then filled with hydrogen to 2.0 MPa, evacuated to 0.5 MPa, repeated three times, and finally filled with hydrogen to 1.0 MPa. The mixture was allowed to react at room temperature for approximately 3 h.

[0148] TLC using EA:n-hexane=1:5 as the developing solvent was used for monitoring, and the raw material heptanal was detected at Rf=0.8. After developing with methanol:DCM=1:1 and fumigating with iodine, the product (108-M3) was detected at Rf=0.2, and the starting material N,N-dimethylaminopropylamine (108-M1) was detected at Rf=0.1. After the reaction was completed, the reaction mixture was suction filtered, the palladium on activated carbon was recovered, and the filtrate was concentrated to obtain 4.6 g of crude product 108-M3. This crude product 108-M3 was purified by silica gel column chromatography and eluted with EA:methanol=3:1 to obtain 3.30 g of 108-M3 in a yield of 52%.

[0149] (2) Synthesis of compound M5 [ka] [ka]

[0150] 1) Synthesis of compound M4-1 Dimethyl sebacate (M4-0) (170 g, 738.2 mmol) was added to a 2 L one-neck round-bottom flask, 850 mL of DMF was added, and the mixture was cooled to -10°C. 46 g of potassium hydroxide powder was added while stirring, and the mixture was reacted at -10°C for 20 hours. The reaction solution was diluted with 1000 mL of water, extracted with 500 mL of EA, and the solvent was removed by evaporation to obtain a crude product, which was purified by silica gel column chromatography eluted with EA:n-hexane = 1:5 to obtain 80.0 g of monomethyl sebacate (M4-1) in a 50% yield.

[0151] 2) Synthesis of compound M4-2 M4-1 (120 g, 555 mmol), dimethylamine hydrochloride (59 g), EDCl (149 g) and DMAP (6.8 g) were added sequentially to a 2 L single-neck round-bottom flask, dissolved in 1.2 L DCM, stirred well, and finally pyridine (123 g) was added and stirred at room temperature overnight. 1 L of water was added to dilute the mixture, and the pH was adjusted to 3 with dilute hydrochloric acid. The organic phase was separated and the aqueous phase was extracted with 500 mL of DCM. The organic phases were combined and the solvent was removed by evaporation to give 126 g of crude methyl 10-(dimethylamino)-10-oxodecanoate (M4-2-1), which was used directly in the next reaction.

[0152] Crude M4-2-1 (126 g) was added to a 1 L four-neck flask, dissolved in DCM (600 mL), and cooled to 0° C. under nitrogen protection. Titanium tetrachloride (118 g) was added dropwise, followed by TEA (73.4 g), and the reaction was allowed to proceed at 0° C. for 2 h. 600 mL of water was added slowly and quenched with stirring at 0° C. The organic phase was separated and the aqueous phase was extracted with 300 mL of DCM. The organic phases were combined and the solvent was removed by evaporation to give 133 g of methyl 12-(dimethylamino)-2-(8-dimethylamino)-8-oxooctyl)-3,12-dioxodecanoate (M4-2-2), which was used directly in the next reaction without further purification.

[0153] M4-2-2 (133 g) was placed in a 1 L single-neck flask, hydrobromic acid (500 ml, 48% aqueous solution) was added, and the mixture was refluxed at 120° C. overnight. The mixture was cooled to room temperature with stirring, stirred at −10° C. for 20 minutes, and then suction filtered. The filter cake was washed with about 100 mL of water to obtain 120 g of crude 10-oxononadecanedioic acid (M4-2).

[0154] Crude M4-2 (120 g) and acetonitrile (1 L) were added to a 2 L one-neck flask and heated to 85 °C and refluxed for 0.5 h. After cooling to room temperature, the mixture was stirred at -10 °C for 20 min and suction filtered. The filter cake was washed with approximately 100 mL of cold acetonitrile, transferred to a flask, and the residual solvent was evaporated to give 45 g of M4-2 as an off-white solid. 1 H NMR (600 MHz, DMSO-d6) δ 11.94 (brs, 1H), 2.37 (t, J = 7.3, 4H), 2.18 (t, J = 7.5, 4H), 1.50-11.41 (m, 8H), 1.24-1.19 (m, 16H). LC-MS (ESI): (M-1) calculated 341.23, found 341.4.

[0155] 3) Synthesis of compound M4-3 EDCl (16.8 g), DCM (100 ml), triethylamine (8.86 g), DMAP (1.78 g), 7-tridecanol (10.5 g), and M4-2 (10.0 g, 29.2 mol) were added in sequence to a 500 mL round-bottom flask and stirred overnight at room temperature. The conversion of 7-tridecanol was almost complete by TLC monitoring (developed with EA:n-hexane = 1:25, stained with PMA, starting material 7-tridecanol Rf = 0.4, product DTN-T Rf = 0.5), and the reaction was terminated. 200 mL of DCM and 300 mL of water were added for extraction, the liquid was separated, the organic phase was collected, and the solvent was removed by evaporation to obtain 30 g of yellow oily crude product, which was purified by silica gel column chromatography, eluted with EA:n-hexane = 1:25, to obtain 15.72 g of M4-3 in 85% yield. 1H NMR (600 MHz, CDCl3) δ 4.88-4.84 (m, 2H), 2.37 (t, J = 7.5, 4H), 2.27 (t, J = 7.5, 4H), 1.64-1.48 (m, 16H), 1.31-1.26 (m, 48H), 0.87 (t, J = 6.9, 12H).

[0156] 4) Synthesis of compound M4 M4-3 (30.0 g, 42.4 mmol) was added to a 1 L one-neck flask, dissolved in methanol (300 ml), and cooled to 0°C. Sodium borohydride (1.7 g) was slowly added with stirring, and the mixture was reacted at 0°C for 1 hour after the addition, and then adjusted to pH 6 with dilute hydrochloric acid. The solvent was removed by evaporation. Extraction was performed by adding 300 mL of DCM and 300 mL of water, and the organic phase was collected and the solvent was removed by evaporation to obtain 42.7 g of crude product, which was purified by silica gel column chromatography and eluted with EA:n-hexane=1:15 to obtain 24.6 g of M4. 1 H NMR (600 MHz, CDCl3) δ 4.89-4.84 (m, 2H), 3.58-3.56 (m, 1H), 2.27 (t, J = 7.5, 4H), 1.62-1.59 (m, 4H), 1.50-1.26 (m, 64H), 0.87 (t, J = 6.9, 12H).

[0157] 5) Synthesis of compound M5 M4 (24 g, 33.8 mmol), toluene (240 ml), and TEA (5.1 g) were added sequentially to a 500 ml one-neck flask, stirring was started at room temperature, and triphosgene (15 g) was slowly added. After the addition, the temperature was raised to 70° C. and the reaction was allowed to proceed for 2 hours. Samples were taken for monitoring (TLC, EA:n-hexane=1:9, stained with PMA, Rf=0.2 for starting material, Rf=0.8 for product) until the reaction was complete.

[0158] The reaction mixture was cooled to room temperature and filtered under suction. The filter cake was washed with toluene. The filtrate was collected and the solvent was removed by evaporation. The residue was purified by silica gel column chromatography, eluted with EA:n-hexane=1:5-25 to give 21.6 g of M5 as a pale yellow transparent oil in 83% yield. 1 H NMR (600 MHz, CDCl3) δ 4.90-4.85 (m, 3H), 2.28 (t, J = 7.5, 4H), 1.62-1.59 (m, 4H), 1.65-1.59 (m, 8H), 1.51-1.49 (m, 8H), 1.37-1.22 (m, 52H), 0.88 (t, J = 7.1, 12H).

[0159] (3) Synthesis of amino lipid compound 108 [ka]

[0160] 108-M3 (266 mg, 1.55 mmol), THF (10 ml) and potassium carbonate (178.3 mg) were added in sequence to a 50 ml one-neck flask, stirred, and then cooled to 0°C. M5 (1.0 g, 1.29 mmol) and THF (10 ml) were added from a constant pressure dropping funnel, and M5 was slowly dropped into the one-neck flask over about 10 minutes. After the dropwise addition, the mixture was stirred at 0°C for 5 minutes and then reacted at room temperature. After 1.5 hours, the reaction was monitored by TLC (EA: n-hexane = 30: 1, developed with PMA, the product and starting material 108-M3 were at the origin, and compound 108-M3 was Rf = about 0.5) until the reaction was completed. The reaction solution was extracted by adding 100 ml of water and 100 ml of EA, the organic phase was collected, and the aqueous phase was extracted three times with 100 ml of EA. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated to give 1.1 g of crude product, which was purified by silica gel column chromatography eluting with EA:n-hexane=1:5 to give 630 mg of amino lipid compound 108 as a colorless oil in 53.8% yield and 97.41% purity. 1H NMR (600 MHz, CDCl3) δ 4.88-4.84 (m, 2H), 4.74 (m, 1H), 3.25-3.17 (m, 4H), 2.26 (t, J = 7.5, 6H), 2.21 (s, 6H), 1.70 (m, 2H), 1.62-1.58 (m, 4H), 1.50-1.49 (m, 14H), 1.30-1.25 (m, 56H), 0.90-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 907.8, found 908.2.

[0161] Example 2: Synthesis of Aminolipid Compound 109 [ka]

[0162] Following the general synthetic process, 190 mg of amino lipid compound 109 was prepared as a colorless oil with a yield of 21% and a purity of 94.94% from compound M5 (1.0 g, 1.29 mmol) and 109-M3 (1.55 mmol). 1 H NMR (600 MHz, CDCl3) δ 4.88-4.84 (m,2H), 4.74 (m,1H), 3.25-3.17 (m,4H), 2.27 (t,J=7.5,6H), 2.22(s,6H), 1.70 (m,2H), 1.72-1.69 (m,4H), 1.63-1.58 (m,14H), 1.33-1.26 (m,58H), 0.87 (t,J=7.0,15H). LC-MS (ESI): (M + H) calculated 921.9, found 922.2.

[0163] Example 3: Synthesis of Aminolipid Compound 110 [ka]

[0164] Following the general synthetic process, 640 mg of amino lipid compound 110 was prepared as a colorless oil with a yield of 60% and a purity of 97.71% from compound M5 (1.0 g, 1.29 mmol) and 110-M3 (1.55 mmol). 1 H NMR (600 MHz, CDCl3) δ 4.88-4.84 (m,2H), 4.74 (m,1H), 3.25-3.17 (m,4H), 2.27 (t,J=7.5,6H), 2.22 (s,6H), 1.72-1.70 (m,2H), 1.63-1.58 (m,4H), 1.51-1.50 (m,14H), 1.28-1.26 (m,60H), 0.88-0.86 (m,15H). LC-MS (ESI): (M + H) calculated 935.9, found 936.3.

[0165] Example 4: Synthesis of Aminolipid Compound 111 [ka]

[0166] Following the general synthetic process, 510 mg of compound 111 was prepared as a colorless oil with a yield of 47% and a purity of 97.18% from compound M5 (1.0 g, 1.29 mmol) and 111-M3 (1.55 mmol). 1 H NMR (600 MHz, CDCl3) δ 4.88-4.84 (m,2H), 4.73 (m,1H), 3.25-3.16 (m,4H), 2.26 (t,J=7.5,6H), 2.21 (s,6H), 1.70 (m,2H), 1.62-1.57 (m,4H), 1.50-1.49 (m,14H), 1.27-1.25 (m,62H), 0.88-0.86 (m,15H). LC-MS (ESI): (M + H) calculated 949.9, found 950.3.

[0167] Example 5: Synthesis of Aminolipid Compound 112 [ka]

[0168] Following the general synthetic process, 760 mg of amino lipid compound 112 was prepared from compound M5 (1.0 g, 1.29 mmol) and 112-M3 (1.55 mmol) as a colorless oil in 69% yield and 94.69% purity. 1 H NMR (600 MHz, CDCl3) δ 4.88-4.84 (m,2H), 4.73 (m,1H), 3.25-3.16 (m,4H), 2.26 (t,J=7.5,6H), 2.21 (s,6H), 1.71-1.69 (m,2H), 1.62-1.58 (m,4H), 1.50-1.49 (m,14H), 1.27-1.25 (m,64H), 0.88-0.86 (m,15H). LC-MS (ESI): (M + H) calculated 963.9, found 964.3.

[0169] Example 6: Synthesis of Aminolipid Compound 113 [ka]

[0170] Following the general synthetic process, 640 mg of amino lipid compound 113 was prepared from compound M5 (1.0 g, 1.29 mmol) and 113-M3 (1.55 mmol) as a colorless oil in 58% yield and 98.04% purity. 1 H NMR (600 MHz, CDCl3) δ 4.89-4.85 (m,2H), 4.74 (m,1H), 3.26-3.17 (m,4H), 2.27 (t,J=7.5,6H), 2.22 (s,6H), 1.71-1.70 (m,2H), 1.63-1.58 (m,4H), 1.51-1.50 (m,14H), 1.28-1.25 (m,66H), 0.89-0.86 (m,15H). LC-MS (ESI): (M + H) calculated 977.9, found 978.4.

[0171] Example 7: Synthesis of Aminolipid Compound 114 [ka]

[0172] Following the general synthetic process, 270 mg of amino lipid compound 114 was prepared from compound M5 (1.0 g, 1.29 mmol) and 114-M3 (1.55 mmol) as a colorless oil in 22% yield and 75.59% purity. 1 H NMR (600 MHz, CDCl3) δ4.89-4.84 (m, 2H),4.74-4.72 (m, 1H), 3.40-3.33 (m, 2H), 3.24-3.19 (m, 2H), 2.63-2.53 (m, 6H), 2.27 (t, J=7.5, 4H), 1.78-1.74 (m, 4H), 1.63-1.58 (m, 4H), 1.51-1.50 (m, 14H), 1.33-1.26 (m, 58H), 0.89-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 933.86, found 934.3.

[0173] Example 8: Synthesis of Aminolipid Compound 115 [ka]

[0174] Following the general synthetic process, 468 mg of amino lipid compound 115 was prepared from compound M5 (1.0 g, 1.29 mmol) and 115-M3 (1.55 mmol) as a colorless oil in 38% yield and 77.72% purity. 1H NMR (600 MHz, CDCl3) δ4.88-4.84 (m, 2H),4.74-4.72 (m, 1H), 3.28-3.16 (m, 4H), 2.48-2.43 (m, 6H), 2.27 (t, J = 7.5, 4H),1.77-1.74 (m, 6H), 1.63-1.58 (m, 4H), 1.50-1.49 (m, 14H), 1.33-1.26 (m, 58H), 0.88-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 947.87, found 948.3.

[0175] Example 9: Synthesis of Aminolipid Compound 117 [ka]

[0176] Following the general synthetic process, 140 mg of amino lipid compound 117 was prepared from compound M5 (1.0 g, 1.29 mmol) and 117-M3 (1.55 mmol) as a colorless oil in 13% yield and 97.21% purity. 1 H NMR (600 MHz, CDCl3) δ4.88-4.84 (m, 2H),4.73 (m, 1H), 3.21-3.16 (m, 4H), 2.84 (m, 1H), 2.65 (m, 1H), 2.31-2.25 (m, 6H), 2.11 (m, 1H), 1.69-1.58 (m, 9H), 1.50-1.49 (m, 15H), 1.33-1.26 (m, 60H), 1.04 (d, J = 6.1, 3H), 0.88-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 975.9, found 976.4.

[0177] Example 10: Synthesis of amino lipid compound 137 [ka]

[0178] Following the general synthetic process, 200 mg of amino lipid compound 137 was prepared as a colorless oil in 20% yield and 97.46% purity from compound M5 (1.0 g, 1.29 mmol) and 137-M3 (1.55 mmol). 1 H NMR (600 MHz, CDCl3) δ 5.00-4.83 (m, 2H), 4.77 (s, 1H), 3.38-3.33 (m, 2H), 3.29-3.17 (m, 2H), 2.60-2.44 (m, 2H), 2.32-2.29 (m, 10H), 1.69-1.60 (m, 4H), 1.56-1.54 (m, 14H), 1.34-1.30 (m, 56H), 0.94-0.90 (m, 15H). LC-MS (ESI): (M + H) calculated 893.82, found 894.3.

[0179] Example 11: Synthesis of Aminolipid Compound 138 [ka]

[0180] Following the general synthetic process, 490 mg of amino lipid compound 138 was prepared as a colorless oil with a yield of 42% and a purity of 77.73% from compound M5 (1.0 g, 1.29 mmol) and 138-M3 (1.55 mmol). 1 H NMR (600 MHz, CDCl3) δ4.88-4.84 (m, 2H), 4.73 (m, 1H), 3.35-3.28 (m, 2H), 3.23-3.19 (m, 2H), 2.45-2.40 (m, 2H), 2.28-2.25 (m, 10H), 1.63-1.58 (m, 4H), 1.51-1.50 (m, 14H), 1.33-1.26 (m, 58H), 0.90-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 907.84, found 908.3.

[0181] Example 12: Synthesis of Aminolipid Compound 139 [ka]

[0182] Following the general synthetic process, 470 mg of amino lipid compound 139 was prepared as a colorless oil with a yield of 45% and a purity of 95.87% from compound M5 (1.0 g, 1.29 mmol) and 139-M3 (1.55 mmol). 1 H NMR (600 MHz, CDCl3) δ4.88-4.84 (m, 2H), 4.73 (m, 1H), 3.35-3.28 (m, 2H), 3.23-3.19 (m, 2H), 2.46-2.40 (m, 2H), 2.28-2.26 (m, 10H), 1.63-1.58 (m, 4H), 1.51-1.50 (m, 14H), 1.33-1.26 (m, 60H), 0.88-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 921.86, found 922.3.

[0183] Example 13: Synthesis of amino lipid compound 140 [ka]

[0184] Following the general synthetic process, 470 mg of amino lipid compound 140 was prepared from compound M5 (1.0 g, 1.29 mmol) and 140-M3 (1.55 mmol) as a colorless oil in 44% yield and 96.31% purity. 1H NMR (600 MHz, CDCl3) δ4.87-4.84 (m, 2H), 4.73 (m, 1H), 3.35-3.28 (m, 2H), 3.23-3.19 (m, 2H), 2.46-2.40 (m, 2H), 2.28-2.26 (m, 10H), 1.63-1.58 (m, 4H), 1.51-1.50 (m, 14H), 1.33-1.26 (m, 62H), 0.88-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 935.87, found 936.3.

[0185] Example 14: Synthesis of Aminolipid Compound 141 [ka]

[0186] Following the general synthetic process, 330 mg of amino lipid compound 141 was prepared from compound M5 (1.0 g, 1.29 mmol) and 141-M3 (1.55 mmol) as a colorless oil in 31% yield and 95.98% purity. 1 H NMR (600 MHz, CDCl3) δ4.89-4.84 (m, 2H), 4.73 (m, 1H), 3.35-3.28 (m, 2H), 3.23-3.19 (m, 2H), 2.46-2.40 (m, 2H), 2.28-2.26 (m, 10H), 1.63-1.58 (m, 4H), 1.51-1.50 (m, 14H), 1.33-1.26 (m, 62H), 0.88-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 949.89, found 950.3.

[0187] Example 15: Synthesis of Aminolipid Compound 142 [ka]

[0188] Following the general synthetic process, 580 mg of amino lipid compound 142 was prepared from compound M5 (1.0 g, 1.29 mmol) and 142-M3 (1.55 mmol) as a colorless oil in 53% yield and 98.23% purity. 1 H NMR (600 MHz, CDCl3) δ4.89-4.84 (m, 2H), 4.73 (m, 1H), 3.35-3.28 (m, 2H), 3.23-3.19 (m, 2H), 2.46-2.40 (m, 2H), 2.28-2.26 (m, 10H), 1.63-1.58 (m, 4H), 1.51-1.50 (m, 14H), 1.28-1.26 (m, 66H), 0.89-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 963.9, found 964.3

[0189] Example 16: Synthesis of Aminolipid Compound 255 (1) Synthesis of compound 255-B [ka]

[0190] 1) Synthesis of compound 255-A 5-Bromopentanoic acid (20.0 g, 110.5 mmol), DMF (269 mg, 3.7 mmol), and DCM (50 ml) were added to a 500 ml one-neck flask, cooled to 0° C. with stirring, and thionyl chloride (16.29 g, 221 mmol) was slowly added dropwise at 0° C. After maintaining the temperature for 30 minutes, the mixture was transferred to room temperature and reacted overnight. The solvent and excess thionyl chloride were removed by evaporation under reduced pressure to obtain 24 g of acyl chloride compound.

[0191] N-butanol (5.46 g, 73.7 mmol) was weighed, dissolved in DCM (50 ml) and cooled to 0° C. with stirring. Freshly prepared acyl chloride was added dropwise at 0° C. After the addition was completed, the reaction was warmed to room temperature and reacted for 5 h, after which 200 mL of water was added and extracted twice with 200 mL of DCM. The organic phases were combined, washed with 100 mL of brine, dried over anhydrous sodium sulfate, and the solvent was evaporated to give 28 g of crude 255-A.

[0192] The crude product was purified by silica gel column chromatography eluting with EA:n-heptane=1:25 to give 15.2 g of 255-A as a pale yellow oil in 83% yield.

[0193] 2) Synthesis of compound 255-B 3-Dimethylaminopropylamine (9.8g, 96.2mmol) and acetonitrile (75ml) were added to a 500ml one-neck flask, stirred to dissolve, potassium carbonate (6.64g, 48.1mmol) was added, and cooled to -10℃. 255-A obtained in step 1) was dissolved in acetonitrile (75ml) and slowly added dropwise to the reaction system. After the dropwise addition was completed, the mixture was reacted at -10℃ for 5 hours, then transferred to room temperature and reacted overnight. The solvent was removed by evaporation. The residue was extracted three times with water (100ml) and ethyl acetate (100ml). The organic phases were combined, washed with 50ml of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain 10.74g of crude 255-B, which was used directly in the next reaction.

[0194] (2) Synthesis of amino lipid compound 255 [ka]

[0195] Crude 255-B (2.5 g, 9.72 mmol) and THF (50 ml) were added to a 250 mL flask, dissolved under stirring, and cooled to 0 ° C. M5 (5 g, 6.48 mmol) was weighed, dissolved in THF (50 ml), and slowly added dropwise to the reaction system. After the dropwise addition was completed, the mixture was reacted at 0 ° C for 1 hour, and extracted three times with water (100 ml) and EA (100 ml). The organic phases were combined, washed with water (50 ml) and saturated aqueous sodium chloride solution (50 ml) in sequence, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to obtain 8 g of crude 225-B, which was purified by silica gel column chromatography and eluted with EA: n-heptane = 1: 25 to obtain 3.25 g of amino lipid compound 225 as a pale yellow oil with a purity of 94.68% and a yield of 50%. 1 H NMR (600 MHz, CDCl3) δ 4.96-4.85 (m, 2H), 4.83-4.72 (m, 1H), 4.06 (t, J = 6.7 Hz, 2H), 3.28 (m, 4H), 2.49-2.18 (m, 14H), 1.88-1.48 (m, 26H), 1.37-1.17 (m, 52H), 0.97 (t, J = 7.4 Hz, 3H), 0.91 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 993.88, found 994.5.

[0196] Example 17: Synthesis of Aminolipid Compound 250 [ka]

[0197] Following the general synthetic process, 600 mg of amino lipid compound 250 was prepared from M5 (1.0 g, 1.29 mmol) and 250-B (0.47 g, 1.94 mmol) as a pale yellow oil with a purity of 95.54% and a yield of 47%. 1H NMR (600 MHz, CDCl3) δ 4.94-4.86 (m, 2H), 4.77 (m, 1H), 4.16 (q, J = 6.8 Hz, 2H), 3.34-3.14 (m, 4H), 2.32 (m, 8H), 2.25 (s, 4H), 1.54 (m, 25H), 1.38-1.24 (m, 56H), 0.91 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 979.86, found 980.4.

[0198] Example 18: Synthesis of Aminolipid Compound 251 [ka]

[0199] Following the general synthetic process, 1000 mg of amino lipid compound 251 was prepared from M5 (1.5 g, 1.94 mmol) and 251-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 95.54% and a yield of 52%. 1 H NMR (600 MHz, CDCl3) δ 4.94-4.86 (m, 2H), 4.77 (m, 1H), 4.16 (q, J = 6.8 Hz, 2H), 3.34-3.14 (m, 4H), 2.32 (m, 8H), 2.25 (s, 4H), 1.54 (m, 27H), 1.38-1.24 (m, 53H), 0.97 (t, J = 7.4 Hz, 3H), 0.91 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 993.88, found 994.5.

[0200] Example 19: Synthesis of Aminolipid Compound 252 [ka]

[0201] Following the general synthetic process, 650 mg of amino lipid compound 252 was prepared from M5 (1.0 g, 1.29 mmol) and 252-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 95.88% and a yield of 56%. 1 H NMR (600 MHz, CDCl3) δ 4.94-4.86 (m, 2H), 4.77 (m, 1H), 4.16 (q, J = 6.8 Hz, 2H), 3.34-3.14 (m, 4H), 2.32 (m, 8H), 2.25 (s, 4H), 1.54 (m, 29H), 1.38-1.24 (m, 53H), 4.77 (t, J = 12.3, 6.1 Hz, 1H), 0.98 (t, J = 7.4 Hz, 3H), 0.92 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 1007.89, found 1008.5.

[0202] Example 20: Synthesis of amino lipid compound 253 [ka]

[0203] Following the general synthetic process, 300 mg of amino lipid compound 253 was prepared from M5 (1.0 g, 1.29 mmol) and 253-B (0.45 g, 1.94 mmol) as a pale yellow oil with a purity of 96.09% and a yield of 27%. 1 H NMR (600 MHz, CDCl3) δ 4.96-4.86 (m, 2H), 4.82-4.70 (m, 1H), 4.16 (q, J = 7.1 Hz, 2H), 3.36-3.18 (m, 4H), 2.42-2.15 (m, 14H), 1.86-1.46 (m, 22H), 1.43-1.20 (m, 55H), 0.92 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 965.85, found 966.5.

[0204] Example 21: Synthesis of Aminolipid Compound 254 [ka]

[0205] Following the general synthetic process, 100 mg of amino lipid compound 254 was prepared from M5 (1.0 g, 1.29 mmol) and 254-B (0.47 g, 1.94 mmol) as a pale yellow oil with a purity of 91.94% and a yield of 8.9%. 1 H NMR (600 MHz, CDCl3) δ 4.96-4.85 (m, 2H), 4.83-4.72 (m, 1H), 4.06 (t, J = 6.7 Hz, 2H), 3.28 (m, 4H), 2.49-2.18 (m, 14H), 1.88-1.48 (m, 24H), 1.37-1.17 (m, 52H), 0.97 (t, J = 7.4 Hz, 3H), 0.91 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 979.86, found 980.5.

[0206] Example 22: Synthesis of Aminolipid Compound 256 [ka]

[0207] Following the general synthetic process, 480 mg of amino lipid compound 256 was prepared from M5 (1.0 g, 1.29 mmol) and 256-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 92.94% and a yield of 42%. 1H NMR (600 MHz, CDCl3) δ 4.96-4.85 (m, 2H), 4.83-4.72 (m, 1H), 4.06 (t, J = 6.7 Hz, 2H), 3.28 (m, 4H), 2.49-2.18 (m, 14H), 1.88-1.48 (m, 26H), 1.37-1.17 (m, 54H), 0.91 (m, 15H). LC-MS (ESI): (M + H) calculated 1007.89, found 1008.3

[0208] Example 23: Synthesis of Aminolipid Compound 257 [ka]

[0209] Following the general synthetic process, 290 mg of amino lipid compound 257 was prepared from M5 (1.0 g, 1.29 mmol) and 257-B (0.42 g, 1.94 mmol) as a pale yellow oil with a purity of 95.16% and a yield of 27%. 1 H NMR (600 MHz, CDCl3) δ 4.96-4.84 (m, 2H), 4.81-4.70 (m, 1H), 4.17 (q, 7.1 Hz, 2H), 3.37-3.18 (m, 4H), 2.31 (m, 8H), 2.25 (s, 6H), 1.94-1.82 (m, 2H), 1.72 (m, 2H), 1.68-1.60 (m, 4H), 1.54 (m, 12H), 1.40-1.22 (m, 55H), 0.91 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 951.83, found 952.4.

[0210] Example 24: Synthesis of Aminolipid Compound 258 [ka]

[0211] Following the general synthetic process, 340 mg of amino lipid compound 258 was prepared from M5 (1.0 g, 1.29 mmol) and 258-B (0.45 g, 1.94 mmol) as a pale yellow oil with a purity of 92.94% and a yield of 31%. 1 H NMR (600 MHz, CDCl3) δ 4.96-4.84 (m, 2H), 4.81-4.70 (m, 1H), 4.06 (t, J = 6.7 Hz, 2H), 3.37-3.18 (m, 4H), 2.31 (m, 8H), 2.25 (s, 6H), 1.94-1.82 (m, 2H), 1.72 (m, 2H), 1.68-1.60 (m, 6H), 1.54 (m, 12H), 1.40-1.22 (m, 52H), 0.97 (t, J = 7.4 Hz, 3H), 0.91 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 965.85, found 966.2.

[0212] Example 25: Synthesis of Aminolipid Compound 259 [ka]

[0213] Following the general synthetic process, 320 mg of amino lipid compound 259 was prepared from M5 (1.0 g, 1.29 mmol) and 259-B (0.47 g, 1.94 mmol) as a pale yellow oil with a purity of 93.85% and a yield of 29%. 1H NMR (600 MHz, CDCl3) δ 4.96-4.84 (m, 2H), 4.81-4.70 (m, 1H), 4.10 (t, J = 6.7 Hz, 2H), 3.37-3.18 (m, 4H), 2.31 (m, 8H), 2.25 (s, 6H), 1.94-1.82 (m, 2H), 1.72 (m, 2H), 1.68-1.60 (m, 6H), 1.54 (m, 12H), 1.40-1.22 (m, 54H), 0.97 (t, J = 7.4 Hz, 3H), 0.91 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 979.86, found 980.3.

[0214] Example 26: Synthesis of Aminolipid Compound 260 [ka]

[0215] Following the general synthetic process, 440 mg of amino lipid compound 260 was prepared from M5 (1.0 g, 1.29 mmol) and 260-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 95.54% and a yield of 40%. 1 H NMR (600 MHz, CDCl3) δ 4.95-4.86 (m, 2H), 4.82-4.71 (m, 1H), 4.09 (t, J = 6.8 Hz, 2H), 3.29 (m, 4H), 2.31 (m, 8H), 2.24 (s, 6H), 1.88 (m, 2H), 1.73 (m, 2H), 1.65 (m, 6H), 1.54 (m, 12H), 1.41-1.20 (m, 56H), 0.92 (m, 15H). LC-MS (ESI): (M + H) calculated 993.88, found 994.2.

[0216] Example 27: Synthesis of Aminolipid Compound 261 [ka]

[0217] Following the general synthetic process, 140 mg of amino lipid compound 261 was prepared from M5 (1.0 g, 1.29 mmol) and 261-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 92.13% and a yield of 12%. 1 H NMR (600 MHz, CDCl3) δ 4.94-4.85 (m, 2H), 4.80-4.73 (m, 1H), 4.09 (t, J = 6.8 Hz, 2H), 3.35-3.20 (m, 4H), 2.38-2.19 (m, 14H), 1.88 (m, 2H), 1.74 (m, 2H), 1.68-1.61 (m, 6H), 1.54 (m, 12H), 1.42-1.22 (m, 58H), 0.92 (m, 15H). LC-MS (ESI): (M + H) calculated 1007.89, found 1008.3.

[0218] Example 28: Synthesis of Aminolipid Compound 262 [ka]

[0219] Following the general synthetic process, 500 mg of amino lipid compound 262 was prepared from M5 (1.0 g, 1.29 mmol) and 262-B (0.39 g, 1.94 mmol) as a pale yellow oil with a purity of 88.60% and a yield of 47%. 1H NMR (600 MHz, CDCl3) δ 4.97-4.84 (m, 2H), 4.84-4.72 (m, 1H), 4.07 (t, J = 5.8 Hz, 2H), 3.54 (m, 2H), 3.37-3.23 (m, 2H), 2.60 (m, 2H), 2.30 (m, 6H), 2.25 (s, 6H), 1.73 (m, 2H), 1.68-1.60 (m, 4H), 1.54 (m, 12H), 1.39-1.21 (m, 55H), 0.91 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 937.81, found 938.2.

[0220] Example 29: Synthesis of Aminolipid Compound 263 [ka]

[0221] Following the general synthetic process, 580 mg of amino lipid compound 263 was prepared from M5 (1.0 g, 1.29 mmol) and 263-B (0.42 g, 1.94 mmol) as a pale yellow oil with a purity of 95.37% and a yield of 55%. 1H NMR (600 MHz, CDCl3) δ 4.95-4.83 (m, 2H), 4.81-4.72 (m, 1H), 4.07 (t, J = 5.8 Hz, 2H), 3.55 (m, 2H), 3.29 (m, 2H), 2.61 (m, 2H), 2.30 (m, 6H), 2.25 (s, 6H), 1.80-1.62 (m, 8H), 1.54 (m, 12H), 1.30 (m, 52H), 0.96 (t, J = 7.2 Hz, 3H), 0.91 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 951.83, found 952.2.

[0222] Example 30: Synthesis of amino lipid compound 264 [ka]

[0223] Following the general synthetic process, 220 mg of amino lipid compound 264 was prepared from M5 (1.0 g, 1.29 mmol) and 264-B (0.45 g, 1.94 mmol) as a pale yellow oil with a purity of 98.65% and a yield of 20%. 1 H NMR (600 MHz, CDCl3) δ 4.95-4.83 (m, 2H), 4.81-4.72 (m, 1H), 4.07 (t, J = 5.8 Hz, 2H), 3.55 (m, 2H), 3.29 (m, 2H), 2.61 (m, 2H), 2.30 (m, 6H), 2.25 (s, 6H), 1.80-1.62 (m, 8H), 1.54 (m, 12H), 1.30 (m, 54H), 0.96 (t, J = 7.2 Hz, 3H), 0.91 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 965.85, found 966.2.

[0224] Example 31: Synthesis of amino lipid compound 265 [ka]

[0225] Following the general synthetic process, 450 mg of amino lipid compound 265 was prepared from M5 (1.0 g, 1.29 mmol) and 265-B (0.47 g, 1.94 mmol) as a pale yellow oil with a purity of 98.47% and a yield of 40%. 1H NMR (600 MHz, CDCl3) δ 4.95-4.83 (m, 2H), 4.81-4.72 (m, 1H), 4.07 (t, J = 5.8 Hz, 2H), 3.55 (m, 2H), 3.29 (m, 2H), 2.61 (m, 2H), 2.30 (m, 6H), 2.25 (s, 6H), 1.80-1.62 (m, 8H), 1.54 (m, 12H), 1.42-1.16 (m, 56H), 0.98-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 979.86, found 980.2.

[0226] Example 32: Synthesis of Aminolipid Compound 266 [ka]

[0227] Following the general synthetic process, 487 mg of amino lipid compound 266 was prepared from M5 (1.0 g, 1.29 mmol) and 266-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 94.01% and a yield of 42%. 1 H NMR (600 MHz, CDCl3) δ 4.95-4.83 (m, 2H), 4.81-.72 (m, 1H), 4.07 (t, J = 5.8 Hz, 2H), 3.55 (m, 2H), 3.29 (m, 2H), 2.61 (m, 2H), 2.30 (m, 6H), 2.25 (s, 6H), 1.80-1.62 (m, 8H), 1.54 (m, 12H), 1.42-1.16 (m, 58H), 0.98-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 993.88, found 994.3.

[0228] Example 33: Synthesis of Aminolipid Compound 267 [ka]

[0229] Following the general synthetic process, 360 mg of amino lipid compound 267 was prepared from M5 (1.0 g, 1.29 mmol) and 267-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 98.16% and a yield of 31%. 1 H NMR (600 MHz, CDCl3) δ 4.95-4.83 (m, 2H), 4.81-4.72 (m, 1H), 4.07 (t, J = 5.8 Hz, 2H), 3.55 (m, 2H), 3.29 (m, 2H), 2.61 (m, 2H), 2.30 (m, 6H), 2.25 (s, 6H), 1.80-1.62 (m, 8H), 1.54 (m, 12H), 1.42-1.16 (m, 60H), 0.98-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 1007.89, found 1008.2.

[0230] Example 34: Synthesis of Aminolipid Compound 268 [ka]

[0231] Following the general synthetic process, 500 mg of amino lipid compound 268 was prepared from M5 (1.0 g, 1.29 mmol) and 268-B (0.37 g, 1.94 mmol) as a pale yellow oil with a purity of 90.61% and a yield of 47%. 1 H NMR (600 MHz, CDCl3) 4.97-4.80 (m,2H), 4.68-4.79 (m, 1H), 4.11-4.07 (m, 2H), 4.00 (s, 1H), 3.93 (s, 1H), 3.34 (dt, J = 31.0, 7.2 Hz, 2H), 2.50-2.10 (m, 12H), 1.66-1.09 (m, 70H), 0.96-0.81 (m, 15H). LC-MS (ESI): (M + H) calculated 923.80, found 924.1.

[0232] Example 35: Synthesis of Aminolipid Compound 269 [ka]

[0233] Following the general synthetic process, 510 mg of amino lipid compound 269 was prepared from M5 (1.0 g, 1.29 mmol) and 269-B (0.39 g, 1.94 mmol) as a pale yellow oil with a purity of 96.02% and a yield of 48%. 1H NMR (600 MHz, CDCl3) 4.97-4.80 (m,2H), 4.68-4.79 (m, 1H), 4.11-4.07 (m, 2H), 4.00 (s, 1H), 3.93 (s, 1H), 3.34 (dt, J = 31.0, 7.2 Hz, 2H), 2.50-2.10 (m, 12H), 1.66-1.09 (m, 72H), 0.96-0.81 (m, 15H). LC-MS (ESI): (M + H) calculated 937.81, found 938.1.

[0234] Example 36: Synthesis of amino lipid compound 270 [ka]

[0235] Following the general synthetic process, 550 mg of amino lipid compound 270 was prepared from M5 (1.0 g, 1.29 mmol) and 270-B (0.42 g, 1.94 mmol) as a pale yellow oil with a purity of 97.66% and a yield of 51%. 1H NMR (600 MHz, CDCl3) 4.97-4.80 (m,2H), 4.68-4.79 (m, 1H), 4.11-4.07 (m, 2H), 4.00 (s, 1H), 3.93 (s, 1H), 3.34 (dt, J = 31.0, 7.2 Hz, 2H), 2.50-2.10 (m, 12H), 1.66-1.09 (m, 74H), 0.96-0.81 (m, 15H). LC-MS (ESI): (M + H) calculated 951.83, found 952.2.

[0236] Example 37: Synthesis of Aminolipid Compound 271 [ka]

[0237] Following the general synthetic process, 230 mg of amino lipid compound 271 was prepared from M5 (1.0 g, 1.29 mmol) and 271-B (0.45 g, 1.94 mmol) as a pale yellow oil with a purity of 94.22% and a yield of 21%. 1 H NMR (600 MHz, CDCl3) 4.90-4.82 (m, 2H), 4.80-4.65 (m, 1H), 4.14-4.04 (m, 2H), 4.01 (s, 1H), 3.93 (s, 1H), 3.34 (dt, J = 31.0, 7.2 Hz, 2H), 1.56-1.44 (m, 12H), 1.39-1.18 (m, 76H), 0.95-0.82 (m, 15H). LC-MS (ESI): (M + H) calculated 965.85, found 966.2.

[0238] Example 38: Synthesis of amino lipid compound 272 [ka]

[0239] Following the general synthetic process, 450 mg of amino lipid compound 272 was prepared from M5 (1.0 g, 1.29 mmol) and 272-B (0.47 g, 1.94 mmol) as a pale yellow oil with a purity of 98.47% and a yield of 40%. 1 H NMR (600 MHz, CDCl3) 4.90-4.82 (m, 2H), 4.80-4.65 (m, 1H), 4.14-4.04 (m, 2H), 4.01 (s, 1H), 3.93 (s, 1H), 3.34 (dt, J = 31.0, 7.2 Hz, 2H), 1.56-1.44 (m, 12H), 1.39-1.18 (m, 78H), 0.95-0.82 (m, 15H). LC-MS (ESI): (M + H) calculated 979.86, found 980.2.

[0240] Example 39: Synthesis of amino lipid compound 273 [ka]

[0241] Following the general synthetic process, 460 mg of amino lipid compound 273 was prepared from M5 (1.0 g, 1.29 mmol) and 273-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 96.41% and a yield of 40%. 1 H NMR (600 MHz, CDCl3) 4.90-4.82 (m, 2H), 4.80-4.65 (m, 1H), 4.14-4.04 (m, 2H), 4.01 (s, 1H), 3.93 (s, 1H), 3.34 (dt, J = 31.0, 7.2 Hz, 2H), 1.56-1.44 (m, 12H), 1.39-1.18 (m, 80H), 0.95-0.82 (m, 15H). LC-MS (ESI): (M + H) calculated 993.88, found 994.2.

[0242] Example 40: Synthesis of amino lipid compound 274 [ka]

[0243] Following the general synthetic process, 420 mg of amino lipid compound 274 was prepared from M5 (1.0 g, 1.29 mmol) and 274-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 95.60% and a yield of 36%. 1 H NMR (600 MHz, CDCl3) 4.90-4.82 (m, 2H), 4.80-4.65 (m, 1H), 4.14-4.04 (m, 2H), 4.01 (s, 1H), 3.93 (s, 1H), 3.34 (dt, J = 31.0, 7.2 Hz, 2H), 1.56-1.44 (m, 12H), 1.39-1.18 (m, 82H), 0.95-0.82 (m, 15H). LC-MS (ESI): (M + H) calculated 1007.89, found 1008.3.

[0244] Example 41: Synthesis of amino lipid compound 275 [ka]

[0245] Following the general synthetic process, 280 mg of amino lipid compound 275 was prepared from M5 (1.0 g, 1.29 mmol) and 275-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 90.83% and a yield of 25%. 1H NMR (600 MHz, CDCl3) δ 5.66 (t, J = 8.8 Hz, 1H), 5.56 (t, J = 8.8 Hz, 1H)4.90 (p, J = 6.2 Hz, 2H), 4.79-4.74 (m, 1H), 4.66 (d, J = 6.9 Hz, 2H), 3.34-3.23 (m, 4H), 2.31 (dd, J = 20.5, 13.1 Hz, 8H), 2.24 (s, 6H), 2.18-2.12 (m, 2H), 1.89 (s, 2H), 1.73 (s, 2H), 1.68-1.61 (m, 4H), 1.54 (d, J = 5.4 Hz, 12H), 1.38-1.23 (m, 54H), 1.05-0.99 (m, 3H), 0.91 (m, 12H). LC-MS (ESI): (M + H) calculated 991.86, found 992.1.

[0246] Example 42: Synthesis of Aminolipid Compound 276 [ka]

[0247] Following the general synthetic process, 110 mg of amino lipid compound 276 was prepared from M5 (1.0 g, 1.29 mmol) and 276-B (0.52 g, 1.94 mmol) as a pale yellow oil with a purity of 93.19% and a yield of 10%. 1 H NMR (600 MHz, CDCl3) 5.67 (m, 1H), 5.61-5.52 (m, 1H), 4.94-4.86 (m, 2H), 4.79-4.74 (m, 1H), 4.66 (d, J = 6.8 Hz, 2H), 3.29 (m, 4H), 2.38-2.08 (m, 16H), 1.93-1.23 (m, 74H), 0.93 (m, 15H). LC-MS (ESI): (M + H) calculated 1005.88, found 1006.3.

[0248] Example 43: Synthesis of amino lipid compound 277 [ka]

[0249] Following the general synthetic process, 350 mg of amino lipid compound 277 was prepared from M5 (1.0 g, 1.29 mmol) and 277-B (0.52 g, 1.94 mmol) as a pale yellow oil with a purity of 96.47% and a yield of 33%. 1H NMR (600 MHz, CDCl3) 5.54 (m, 1H), 5.37-5.30 (m, 1H), 4.94-4.85 (m, 2H), 4.80-4.74 (m, 1H), 4.10 (m, 2H), 3.34-3.21 (m, 4H), 2.44-2.04 (m, 18H), 1.93-1.22 (m, 72H), 1.00 (m, 3H), 0.91 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 1005.88, found 1006.3.

[0250] Example 44: Synthesis of amino lipid compound 279 [ka]

[0251] Following the general synthetic process, 250 mg of amino lipid compound 279 was prepared from M5 (1.0 g, 1.29 mmol) and 279-B (0.58 g, 1.94 mmol) as a pale yellow oil with a purity of 98.62% and a yield of 21%. 1 H NMR (600 MHz, CDCl3) δ 5.50 (m, 1H), 5.36-5.30 (m, 1H), 4.89-4.84 (m, 2H), 4.73 (m, 1H), 4.06 (m, 2H), 3.31-3.18 (m, 4H), 2.30 (m, 16H), 2.08-1.21 (m, 78H), 0.94-0.82 (m, 15H). LC-MS (ESI): (M + H) calculated 1033.91, found 1034.4.

[0252] Example 45: Synthesis of Aminolipid Compound 280 [ka]

[0253] Following the general synthetic process, 190 mg of amino lipid compound 280 was prepared from M5 (1.0 g, 1.29 mmol) and 280-B (0.61 g, 1.94 mmol) as a pale yellow oil with a purity of 91.43% and a yield of 16%. 1 H NMR (600 MHz, CDCl3) 5.66-5.60 (m, 1H), 5.54-5.48 (m, 1H), 4.89-4.83 (m, 2H), 4.76-4.71 (m, 1H), 4.62 (d, J = 6.8 Hz, 2H), 3.33-3.19 (m, 4H), 2.34-2.06 (m, 16H), 1.89-1.23 (m, 80H), 0.88 (td, J = 7.0, 4.8 Hz, 15H). LC-MS (ESI): (M + H) calculated 1047.92, found 1048.4

[0254] Example 46: Synthesis of Aminolipid Compound 281 [ka]

[0255] Following the general synthetic process, 480 mg of amino lipid compound 281 was prepared from M5 (1.0 g, 1.29 mmol) and 281-B (0.55 g, 1.94 mmol) as a pale yellow oil with a purity of 96.03% and a yield of 41%. 1H NMR (600 MHz, CDCl3) 5.68-5.59 (m, 1H), 5.50 (d, J = 11.6, 6.7 Hz, 1H), 4.91-4.83 (m, 2H), 4.80-4.70 (m, 1H), 4.66 (m, 2H), 4.01(s, 1H), 3.94(s, 1H), 3.34 (m, 2H), 2.29-2.06 (m, 14H), 1.78-1.22 (m, 78H), 0.92-0.83 (m, 15H). LC-MS (ESI): (M + H) calculated 1019.89, found 1020.3.

[0256] Example 47: Synthesis of Aminolipid Compound 282 [ka]

[0257] Following the general synthetic process, 300 mg of amino lipid compound 282 was prepared from M5 (1.0 g, 1.29 mmol) and 282-B (0.44 g, 1.94 mmol) as a pale yellow oil with a purity of 94.87% and a yield of 27%. 1 H NMR (600 MHz, CDCl3) δ 5.71-5.63 (m, 1H), 5.55-5.47 (m, 1H), 4.93-4.87 (m, 2H), 4.83-4.73 (m, 1H), 4.73-4.67 (m, 2H), 4.06 (s, 1H), 3.98 (s, 1H), 3.38 (dt, J = 29.1, 7.2 Hz, 2H), 2.36-2.10 (m, 14H), 1.80-1.23 (m, 70H), 1.03 (m, 3H), 0.91 m, 12H). LC-MS (ESI): (M + H) calculated 963.83, found 964.2.

[0258] Example 48: Synthesis of amino lipid compound 284 [ka]

[0259] Following the general synthetic process, 530 mg of amino lipid compound 284 was prepared from M5 (1.0 g, 1.29 mmol) and 284-B (0.55 g, 1.94 mmol) as a pale yellow oil with a purity of 94.29% and a yield of 45%. 1 H NMR (600 MHz, CDCl3) 5.64 (m, 1H), 5.52-5.45 (m, 1H), 4.90-4.82 (m, 2H), 4.77-4.70 (m, 1H), 4.62 (d, J = 6.7 Hz, 2H), 3.29-3.13 (m, 4H), 2.32-2.09 (m, 16H), 1.75-1.20 (m, 78H), 1.02-0.97 (m, 3H), 0.87 (m, 12H). LC-MS (ESI): (M + H) calculated 1019.89, found 1020.2.

[0260] Example 49: Synthesis of amino lipid compound 297 [ka]

[0261] Following the general synthetic process, 830 mg of amino lipid compound 297 was prepared from M5 (1.0 g, 1.29 mmol) and 297-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 97.59% and a yield of 73%. 1 H NMR (600 MHz, CDCl3) δ 5.01-4.97 (m, 1H), 4.89-4.83 (m, 2H), 4.76-4.69 (m, 1H), 3.29-3.12 (m, 4H), 2.28-2.24 (m, 8H), 2.21 (s, 6H), 1.69 (s, 2H), 1.65-1.57 (m, 6H), 1.56-1.45 (m, 14H), 1.33-1.20 (m, 60H), 0.87 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 993.88, found 994.3.

[0262] Example 50: Synthesis of Aminolipid Compound 298 [ka]

[0263] Following the general synthetic process, 410 mg of amino lipid compound 298 was prepared from M5 (1.0 g, 1.29 mmol) and 298-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 97.33% and a yield of 36%. 1 H NMR (600 MHz, CDCl3) δ 4.90-4.80 (m, 3H), 4.76-4.70 (m, 1H), 3.30-3.12 (m, 4H), 2.28-2.25 (m, 8H), 2.21 (s, 6H), 1.67 (s, 2H), 1.65-1.57 (m, 7H), 1.57-1.44 (m, 15H), 1.35-1.21 (m, 54H), 1.19 (d, J = 6.3 Hz, 3H), 0.91-0.84 (m, 15H). LC-MS (ESI): (M + H) calculated 1007.89, found 1008.3.

[0264] Example 51: Synthesis of Aminolipid Compound 299 [ka]

[0265] Following the general synthetic process, 640 mg of amino lipid compound 299 was prepared from M5 (1.0 g, 1.29 mmol) and 299-B (0.56 g, 1.94 mmol) as a pale yellow oil with a purity of 98.14% and a yield of 57%. 1H NMR (600 MHz, CDCl3) δ 4.93-4.89 (d, J = 5.8 Hz, 1H), 4.89-4.83 (m, 2H), 4.76-4.70 (m, 1H), 3.26-3.15 (m, 4H), 2.28-2.26 (m, 8H), 2.21 (s, 6H), 1.69 (s, 2H), 1.64-1.58 (m, 7H), 1.65-1.55 (m, 15H), 1.33-1.21 (m, 56H), 1.19 (d, J = 6.2 Hz, 3H), 0.91 (t, J = 7.3 Hz, 3H), 0.87 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 1021.91, found 1022.3.

[0266] Example 52: Synthesis of Aminolipid Compound 300 [ka]

[0267] Following the general synthetic process, 550 mg of amino lipid compound 300 was prepared from M5 (1.0 g, 1.29 mmol) and 300-B (0.56 g, 1.94 mmol) as a pale yellow oil with a purity of 98.00% and a yield of 47%. 1 H NMR (600 MHz, CDCl3) δ 4.89-4.83 (m, 2H), 4.76-4.71 (m, 2H), 3.26-3.15 (m, 4H), 2.31-2.24 (m, 8H), 2.21 (s, 6H), 1.68 (s, 2H), 1.67-1.57 (m, 8H), 1.57-1.44 (m, 16H), 1.35-1.20 (m, 54H), 0.87 (t, J = 7.1 Hz, 18H). LC-MS (ESI): (M + H) calculated 1021.91, found 1022.1.

[0268] Example 53: Synthesis of Aminolipid Compound 301 [ka]

[0269] Following the general synthetic process, 750 mg of amino lipid compound 301 was prepared from M5 (1.0 g, 1.29 mmol) and 301-B (0.47 g, 1.94 mmol) as a pale yellow oil with a purity of 98.18% and a yield of 67%. 1 H NMR (600 MHz, CDCl3) δ 5.03-4.95 (m, 1H), 4.89-4.83 (m, 2H), 4.76-4.70 (m, 1H), 3.28-3.18 (m, 4H), 2.26 (t, J = 7.5 Hz, 8H), 2.21 (s, 6H), 1.69 (s, 2H), 1.60 (dt, J = 14.5, 7.4 Hz, 6H), 1.51 (t, J = 14.9 Hz, 14H), 1.32-1.21 (m, 58H), 0.87 (t, J = 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 979.86, found 980.1.

[0270] Example 54: Synthesis of Aminolipid Compound 302 [ka]

[0271] Following the general synthetic process, 680 mg of amino lipid compound 302 was prepared from M5 (1.0 g, 1.29 mmol) and 302-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 97.32% and a yield of 60%. 1H NMR (600 MHz, CDCl3) δ 4.89-4.79 (m, 3H), 4.75-4.69 (m, 1H), 3.30-3.13 (m, 4H), 2.32-2.23 (m, 8H), 2.21 (s, 6H), 1.74-1.65 (m, 2H), 1.63-1.55 (m, 8H), 1.55-1.47 (m, 14H), 1.32-1.21 (m, 52H), 1.19 (d, J = 6.2 Hz, 3H), 0.90-0.85 (m, 15H). LC-MS (ESI): (M + H) calculated 993.88, found 994.1.

[0272] Example 55: Synthesis of Aminolipid Compound 303 [ka]

[0273] Following the general synthetic process, 390 mg of amino lipid compound 303 was prepared from M5 (1.0 g, 1.29 mmol) and 303-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 95.21% and a yield of 34%. 1 H NMR (600 MHz, CDCl3) δ 4.93-4.90 (m, 1H), 4.88-4.83 (m, 2H), 4.76-4.69 (m, 1H), 3.27-3.19 (m, 4H), 2.31-2.22 (t, J = 7.5 Hz, 8H), 2.20 (s, 6H), 1.69 (s, 2H), 1.61-1.57 (m, 8H), 1.53-1.47 (m, 14H), 1.33-1.21 (m, 54H), 1.19 (d, J = 6.2 Hz, 3H), 0.91-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 1007.89, found 1008.1.

[0274] Example 56: Synthesis of Aminolipid Compound 304 [ka]

[0275] Following the general synthetic process, 660 mg of amino lipid compound 304 was prepared from M5 (1.0 g, 1.29 mmol) and 304-B ​​(0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 98.51% and a yield of 57%. 1 H NMR (600 MHz, CDCl3) δ 4.89-4.83 (m, 2H), 4.77-4.70 (m, 2H), 3.27-3.17 (m, 4H), 2.31 (s, 2H), 2.26 (t, J = 7.5 Hz, 6H), 2.21 (s, 6H), 1.69 (s, 2H), 1.63-1.44 (m, 24H), 1.35-1.18 (m, 52H), 0.87 (t, J = 7.0 Hz, 18H). LC-MS (ESI): (M + H) calculated 1007.89, found 1008.1.

[0276] Example 57: Synthesis of amino lipid compound 305 [ka]

[0277] Following the general synthetic process, 490 mg of amino lipid compound 305 was prepared from M5 (1.0 g, 1.29 mmol) and 305-B (0.56 g, 1.94 mmol) as a pale yellow oil with a purity of 97.96% and a yield of 42%. 1H NMR (600 MHz, CDCl3) δ 4.94-4.83 (m, 3H), 4.76-4.69 (m, 1H), 3.28-3.18 (m, 4H), 2.31-2.25 (m, 8H), 2.21 (s, 6H), 1.69 (s, 2H), 1.63-1.56 (m, 8H), 1.54-1.46 (m, 14H), 1.35-1.21 (m, 56H), 1.19 (d, J = 6.2 Hz, 3H), 0.90-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 1021.91, found 1022.1.

[0278] Example 58: Synthesis of Aminolipid Compound 306 [ka]

[0279] Following the general synthetic process, 410 mg of amino lipid compound 306 was prepared from M5 (1.0 g, 1.29 mmol) and 306-B (0.56 g, 1.94 mmol) as a pale yellow oil with a purity of 96.94% and a yield of 35%. 1 H NMR (600 MHz, CDCl3) δ 4.89-4.79 (m, 3H), 4.75-4.69 (m, 1H), 3.29-3.15 (m, 4H), 2.31 (s, 2H), 2.27-2.23 (m, 6H), 2.20 (s, 6H), 1.69 (s, 2H), 1.64-1.44 (m, 24H), 1.36-1.19 (m, 54H), 0.88 (dt, J = 14.0, 7.3 Hz, 18H). LC-MS (ESI): (M + H) calculated 1021.91, found 1022.1.

[0280] Example 59: Synthesis of amino lipid compound 307 [ka]

[0281] Following the general synthetic process, 160 mg of amino lipid compound 307 was prepared from M5 (1.0 g, 1.29 mmol) and 307-B (0.45 g, 1.94 mmol) as a pale yellow oil with a purity of 92.29% and a yield of 16%. 1 H NMR (600 MHz, CDCl3) δ5.02-4.98 (m, 1H), 4.88-4.84(m, 2H), 4.75-4.71(m, 1H), 3.27-3.23 (m, 4H), 2.28-2.25 (m, 8H), 2.21(m, 6H), 1.83 (m, 2H), 1.73-1.69 (m, 2H), 1.61-1.59 (m, 4H), 1.50-1.49 (m, 12H), 1.28-1.22 (m, 58H), 0.87 (t, J=6.9Hz, 12H). LC-MS (ESI): (M + H) calculated 965.85, found 966.1.

[0282] Example 60: Synthesis of Aminolipid Compound 308 [ka]

[0283] Following the general synthetic process, 660 mg of amino lipid compound 308 was prepared from M5 (1.0 g, 1.29 mmol) and 308-B (0.47 g, 1.94 mmol) as a pale yellow oil with a purity of 98.69% and a yield of 59%. 1 H NMR (600 MHz, CDCl3) δ4.88-4.82 (m, 3H), 4.75-4.71 (m, 1H), 3.26-3.22 (m, 4H), 2.27-2.25 (m, 8H), 2.20 (s, 6H), 1.84 (m, 2H), 1.69 (m, 2H), 1.61-1.49 (m, 18H), 1.29-1.25 (m, 52H), 1.19 (d, J=6.2Hz, 3H), 0.89-0.85 (m, 15H). LC-MS (ESI): (M + H) calculated 979.86, found 980.1.

[0284] Example 61: Synthesis of Aminolipid Compound 309 [ka]

[0285] Following the general synthetic process, 740 mg of amino lipid compound 309 was prepared from M5 (1.0 g, 1.29 mmol) and 309-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 97.45% and a yield of 65%. 1 H NMR (600 MHz, CDCl3) δ4.94-4.84 (m, 3H), 4.75-4.71 (m, 1H), 3.27-3.23 (m, 4H), 2.28-2.25 (m, 8H), 2.21 (s, 6H), 1.84 (m, 2H), 1.70 (m, 2H), 1.63-1.48 (m, 18H), 1.31-1.26 (m, 54H), 1.19 (d, J=6.2Hz, 3H), 0.92-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 993.88, found 994.1.

[0286] Example 62: Synthesis of Aminolipid Compound 310 [ka]

[0287] Following the general synthetic process, 650 mg of amino lipid compound 310 was prepared from M5 (1.0 g, 1.29 mmol) and 310-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 99.28% and a yield of 57%. 1H NMR (600 MHz, CDCl3) δ4.88-4.84 (m, 2H), 4.78-4.72 (m, 2H), 3.27-3.23 (m, 4H), 2.30-2.26 (m, 8H), 2.21 (s, 6H), 1.86 (m, 2H), 1.70 (m, 2H), 1.62-1.50 (m, 20H), 1.30-1.26 (m, 52H), 0.87 (t, J=7.0Hz, 18H). LC-MS (ESI): (M + H) calculated 993.88, found 994.3.

[0288] Example 63: Synthesis of Aminolipid Compound 311 [ka]

[0289] Following the general synthetic process, 420 mg of amino lipid compound 311 was prepared from M5 (1.0 g, 1.29 mmol) and 311-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 97.21% and a yield of 36%. 1 H NMR (600 MHz, CDCl3) δ4.92-4.84 (m, 3H), 4.75-4.71 (m, 1H), 3.27-3.23 (m, 4H), 2.28-2.25 (m, 8H), 2.21 (m, 6H), 1.84 (m, 2H), 1.70 (m, 2H), 1.62-1.48 (m, 18H), 1.28-1.25 (m, 56H), 1.19 (d, J=6.3Hz, 3H), 0.90-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 1007.89, found 1008.1.

[0290] Example 64: Synthesis of Aminolipid Compound 312 [ka]

[0291] Following the general synthetic process, 650 mg of amino lipid compound 312 was prepared from M5 (1.0 g, 1.29 mmol) and 312-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 97.79% and a yield of 56%. 1 H NMR (600 MHz, CDCl3) δ4.88-4.81 (m, 3H), 4.76-4.71 (m, 1H), 3.27-3.23 (m, 4H), 2.28-2.25 (m, 8H), 2.20 (s, 6H), 1.85 (m, 2H), 1.70 (m, 2H), 1.62-1.49 (m, 20H), 1.27-1.25 (m, 54H), 0.91-0.86 (m, 18H). LC-MS (ESI): (M + H) calculated 1007.89, found 1008.1.

[0292] Example 65: Synthesis of amino lipid compound 313 [ka]

[0293] Following the general synthetic process, 330 mg of amino lipid compound 313 was prepared from M5 (1.0 g, 1.29 mmol) and 313-B (0.56 g, 1.94 mmol) as a pale yellow oil with a purity of 96.97% and a yield of 28%. 1 H NMR (600 MHz, CDCl3) δ4.92-4.84 (m, 3H), 4.75-4.71 (m, 1H), 3.27-3.23 (m, 4H), 2.28-2.25 (m, 8H), 2.20 (s, 6H), 1.84 (s, 2H), 1.70 (s, 2H), 1.61-1.50 (m, 18H), 1.30-1.26 (m, 58H), 1.19 (d, J=6.3Hz, 3H), 0.89-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 1021.91, found 1022.2.

[0294] Example 66: Synthesis of Aminolipid Compound 314 [ka]

[0295] Following the general synthetic process, 122 mg of amino lipid compound 314 was prepared from M5 (1.0 g, 1.29 mmol) and 314-B (0.56 g, 1.94 mmol) as a pale yellow oil with a purity of 91.54% and a yield of 10%. 1 H NMR (600 MHz, CDCl3) δ4.88-4.84 (m, 2H), 4.83-4.79 (m, 1H), 4.76-4.71 (m, 1H), 3.27-3.23 (m, 4H), 2.29-2.25 (m, 8H), 2.21 (s, 6H), 1.85 (m, 2H), 1.70 (m, 2H), 1.61-1.49 (m, 20H), 1.28-1.25 (m, 56H), 0.89-0.86 (m, 18H). LC-MS (ESI): (M + H) calculated 1021.91, found 1022.3.

[0296] Example 67: Synthesis of amino lipid compound 315 [ka]

[0297] Following the general synthetic process, 480 mg of amino lipid compound 315 was prepared from M5 (1.0 g, 1.29 mmol) and 315-B (0.56 g, 1.94 mmol) as a pale yellow oil with a purity of 98.27% and a yield of 41%. 1H NMR (600 MHz, CDCl3) δ4.92-4.85 (m, 3H), 4.76-4.72 (m, 1H), 3.27-3.23 (m, 4H), 2.28-2.25 (m, 8H), 2.21 (s, 6H), 1.84 (m, 2H), 1.70 (m, 2H), 1.63-1.58 (m, 4H), 1.53-1.46 (m, 16H), 1.28-1.26 (m, 56H), 0.91-0.86 (m, 18H). LC-MS (ESI): (M + H) calculated 1021.91, found 1022.1.

[0298] Example 68: Synthesis of Aminolipid Compound 316 [ka]

[0299] Following the general synthetic process, 300 mg of amino lipid compound 316 was prepared from M5 (1.0 g, 1.29 mmol) and 316-B (0.42 g, 1.94 mmol) as a pale yellow oil with a purity of 94.90% and a yield of 28%. 1 H NMR (600 MHz, CDCl3) δ5.02-4.98 (m, 1H), 4.88-4.84 (m, 2H), 4.75-4.73 (m, 1H), 3.52-3.48 (m, 2H), 3.29-3.25 (m, 2H), 2.56-2.51 (m, 2H), 2.27 (t, J=7.5Hz, 6H), 2.21 (m, 6H), 1.70 (m, 2H), 1.63-1.58 (m, 4H), 1.51-1.50 (m, 12H), 1.30-1.23 (m, 58H), 0.87 (t, J=6.9Hz, 12H). LC-MS (ESI): (M + H) calculated 951.83, found 952.0.

[0300] Example 69: Synthesis of Aminolipid Compound 317 [ka]

[0301] Following the general synthetic process, 300 mg of amino lipid compound 317 was prepared from M5 (1.0 g, 1.29 mmol) and 317-B (0.45 g, 1.94 mmol) as a pale yellow oil with a purity of 92.50% and a yield of 27%. 1 H NMR (600 MHz, CDCl3) δ4.88-4.82 (m, 3H), 4.74 (m, 1H), 3.52-3.48 (m, 2H), 3.29-3.25 (m, 2H), 2.58-2.53 (m, 2H), 2.27 (t, J=7.6Hz, 6H), 2.21 (s, 6H), 1.69 (m, 2H), 1.61-1.49 (m, 18H), 1.28-1.19 (m, 55H), 0.87(t, J=7.0Hz, 15H). LC-MS (ESI): (M + H) calculated 965.85, found 966.0.

[0302] Example 70: Synthesis of amino lipid compound 318 [ka]

[0303] Following the general synthetic process, 283 mg of amino lipid compound 318 was prepared from M5 (1.0 g, 1.29 mmol) and 318-B (0.47 g, 1.94 mmol) as a pale yellow oil with a purity of 94.86% and a yield of 25%. 1H NMR (600 MHz, CDCl3) δ4.93-4.90 (m, 1H), 4.88-4.84 (m, 2H), 4.74 (m, 1H), 3.51-3.47 (m, 2H), 3.28-3.25 (m, 2H), 2.57-2.52 (m, 2H), 2.28-2.25 (m, 6H), 2.21 (m, 6H), 1.69 (m, 2H), 1.61-1.49 (m, 18H), 1.27-1.19 (m, 57H), 0.90-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 979.86, found 980.1.

[0304] Example 71: Synthesis of amino lipid compound 319 [ka]

[0305] Following the general synthetic process, 350 mg of amino lipid compound 319 was prepared from M5 (1.0 g, 1.29 mmol) and 319-B (0.47 g, 1.94 mmol) as a pale yellow oil with a purity of 97.46% and a yield of 31%. 1 H NMR (600 MHz, CDCl3) δ4.88-4.84 (m, 2H), 4.76-4.73 (m, 2H), 3.52-3.48 (m, 2H), 3.29-3.25 (m, 2H), 2.61-2.55 (m, 2H), 2.28-2.25 (m, 6H), 2.20 (s, 6H), 1.69 (m, 2H), 1.61-1.49 (m, 20H), 1.27-1.25 (m, 52H), 0.88-0.86 (m, 18H). LC-MS (ESI): (M + H) calculated 979.86, found 980.0.

[0306] Example 72: Synthesis of Aminolipid Compound 320 [ka]

[0307] Following the general synthetic process, 350 mg of amino lipid compound 320 was prepared from M5 (1.0 g, 1.29 mmol) and 320-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 96.53% and a yield of 31%. 1 H NMR (600 MHz, CDCl3) δ4.90-4.83 (m, 3H), 4.73 (m, 1H), 3.51-3.47 (m, 2H), 3.28-3.24 (m, 2H), 2.57-2.51 (m, 2H), 2.26 (t, J=7.5Hz, 6H), 2.20 (s, 6H), 1.69 (m, 2H), 1.61-1.49 (m, 18H), 1.27-1.19 (m, 59H), 0.89-0.85 (m, 15H). LC-MS (ESI): (M + H) calculated 993.88, found 994.1.

[0308] Example 73: Synthesis of Aminolipid Compound 321 [ka]

[0309] Following the general synthetic process, 450 mg of amino lipid compound 321 was prepared from M5 (1.0 g, 1.29 mmol) and 321-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 98.04% and a yield of 40%. 1 H NMR (600 MHz, CDCl3) δ4.88-4.81 (m, 3H), 4.73 (m, 1H), 3.51-3.48 (m, 2H), 3.28-3.25 (m, 2H), 2.59-2.54 (m, 2H), 2.27-2.25 (m, 6H), 2.20 (s, 6H), 1.69 (m, 2H), 1.61-1.49 (m, 20H), 1.27-1.25 (m, 54H), 0.89-0.85 (m, 18H). LC-MS (ESI): (M + H) calculated 993.88, found 994.1.

[0310] Example 74: Synthesis of Aminolipid Compound 322 [ka]

[0311] Following the general synthetic process, 350 mg of amino lipid compound 322 was prepared from M5 (1.0 g, 1.29 mmol) and 322-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 97.74% and a yield of 30%. 1 H NMR (600 MHz, CDCl3) δ4.91-4.84 (m, 3H), 4.73 (m, 1H), 3.51-3.47 (m, 2H), 3.28-3.24 (m, 2H), 2.57-2.52 (m, 2H), 2.28-2.25 (m, 6H), 2.20 (m, 6H), 1.69 (m, 2H), 1.61-1.49 (m, 18H), 1.27-1.19 (m, 61H), 0.89-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 1007.89, found 1008.1.

[0312] Example 75: Synthesis of amino lipid compound 323 [ka]

[0313] Following the general synthetic process, 520 mg of amino lipid compound 323 was prepared from M5 (1.0 g, 1.29 mmol) and 323-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 99.64% and a yield of 45%. 1H NMR (600 MHz, CDCl3) δ4.88-4.84 (m, 2H), 4.83-4.80 (m, 1H), 4.74 (m, 1H), 3.52-3.48 (m, 2H), 3.29-3.25 (m, 2H), 2.60-2.55 (m, 2H), 2.28-2.25 (m, 6H), 2.20 (s, 6H), 1.69- 1.50 (m, 22H), 1.28-1.26 (m, 56H), 0.88-0.86 (m, 18H). LC-MS (ESI): (M + H) calculated 1007.89, found 1008.0.

[0314] Example 76: Synthesis of amino lipid compound 325 [ka]

[0315] Following the general synthesis process, 620 mg of amino lipid compound 325 was prepared from M5 (1.0 g, 1.29 mmol) and 325-B (0.56 g, 1.94 mmol) as a pale yellow oil with a purity of 93.75% and a yield of 54%. 1 H NMR (600 MHz, CDCl3) δ4.91-4.84 (m, 3H), 4.74 (m, 1H), 3.51-3.47 (m, 2H), 3.28-3.23 (m, 2H), 2.57-2.52 (m, 2H), 2.26 (t, J=7.5Hz, 6H), 2.21 (m, 6H), 1.69 (m, 2H), 1.61-1.59 (m, 18H), 1.27-1.19 (m, 63H), 0.89-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 1021.91, found 1022.2.

[0316] Example 77: Synthesis of Aminolipid Compound 326 [ka]

[0317] Following the general synthetic process, 700 mg of amino lipid compound 326 was prepared from M5 (1.0 g, 1.29 mmol) and 326-B (0.56 g, 1.94 mmol) as a pale yellow oil with a purity of 98.56% and a yield of 60%. 1 H NMR (600 MHz, CDCl3) δ4.88-4.84 (m, 2H), 4.83-4.81 (m, 1H), 4.74 (m, 1H), 3.52-3.49 (m, 2H), 3.29-3.26 (m, 2H), 2.60-2.55 (m, 2H), 2.60-2.55 (m, 2H), 2.28-2.26 (m, 6H), 2.21 (s, 6H), 1.70-1.50 (m, 22H), 1.28-1.26 (m, 58H), 0.88-0.86 (m, 18H). LC-MS (ESI): (M + H) calculated 1021.91, found 1022.3.

[0318] Example 78: Synthesis of Aminolipid Compound 327 [ka]

[0319] Following the general synthetic process, 550 mg of amino lipid compound 327 was prepared from M5 (1.0 g, 1.29 mmol) and 327-B (0.56 g, 1.94 mmol) as a pale yellow oil with a purity of 98.53% and a yield of 46%. 1 H NMR (600 MHz, CDCl3) δ4.91-4.84 (m, 3H), 4.74 (m, 1H), 3.52-3.48 (m, 2H), 3.29-3.25 (m, 2H), 2.59-2.54 (m, 2H), 2.28-2.25 (m, 6H), 2.21 (s, 6H), 1.70 (m, 2H), 1.62-1.59 (m, 4H), 1.51-1.50 (m, 16H), 1.28-1.26 (m, 58H), 0.90-0.86 (m, 18H). LC-MS (ESI): (M + H) calculated 1021.91, found 1022.2.

[0320] Example 79: Synthesis of Aminolipid Compound 328 [ka]

[0321] Following the general synthetic process, 220 mg of amino lipid compound 328 was prepared from M5 (1.0 g, 1.29 mmol) and 328-B (0.39 g, 1.94 mmol) as a pale yellow oil with a purity of 97.73% and a yield of 21%. 1 H NMR (600 MHz, CDCl3) δ5.06-5.00 (m, 1H), 4.88-4.84 (m, 2H), 4.78-1.70 (m, 1H), 3.96 (m, 1H), 3.88 (s, 1H), 3.37-3.29 (m, 2H), 2.28-2.25 (m, 6H), 2.21-2.20 (m, 6H), 1.71 (m, 2H), 1.62-1.58 (m, 4H), 1.50-1.49 (m, 12H), 1.27-1.23 (m, 58H), 0.87 (m, 12H). LC-MS (ESI): (M + H) calculated 937.81, found 938.3.

[0322] Example 80: Synthesis of Aminolipid Compound 329 [ka]

[0323] Following the general synthetic process, 650 mg of amino lipid compound 329 was prepared from M5 (1.0 g, 1.29 mmol) and 329-B (0.42 g, 1.94 mmol) as a pale yellow oil with a purity of 98.08% and a yield of 60%. 1H NMR (600 MHz, CDCl3) δ4.90-4.84 (m, 3H), 4.78-4.70 (m, 1H), 3.98 (s, 1H), 3.94-3.87 (m, 1H), 3.40-3.29 (m, 2H), 2.28-2.25 (m, 6H), 2.20-2.19 (m, 6H), 1.74-1.67 (m, 2H), 1.61-1.49 (m, 18H), 1.27-1.20 (m, 55H), 0.90-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 951.83, found 952.3.

[0324] Example 81: Synthesis of amino lipid compound 330 [ka]

[0325] Following the general synthetic process, 740 mg of amino lipid compound 330 was prepared from M5 (1.0 g, 1.29 mmol) and 330-B (0.45 g, 1.94 mmol) as a pale yellow oil with a purity of 92.28% and a yield of 67%. 1 H NMR (600 MHz, CDCl3) δ4.98-4.92 (m, 1H), 4.88-4.85 (m, 2H), 4.78-4.71 (m, 1H), 3.97 (dd, J1=19.6Hz, J2=17.6Hz, 1H), 3.90 (dd, J1=25Hz, J2=18Hz, 1H), 3.40-3.30 (m, 2H), 2.27 (t, J=7.5Hz, 6H), 2.22-2.21 (m, 6H), 1.74-1.45 (m, 20H), 1.36-1.21 (m, 57H), 0.92-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 965.85, found 966.6.

[0326] Example 82: Synthesis of Aminolipid Compound 331 [ka]

[0327] Following the general synthetic process, 338 mg of amino lipid compound 331 was prepared from M5 (1.0 g, 1.29 mmol) and 331-B (0.45 g, 1.94 mmol) as a pale yellow oil with a purity of 93.50% and a yield of 31%. 1 H NMR (600 MHz, CDCl3) δ4.89-4.84 (m, 2H), 4.82-4.71 (m, 2H), 4.01 (s, 1H), 3.94 (s, 1H), 3.38-3.30 (m, 2H), 2.27 (t, J=7.5Hz, 6H), 2.21-2.20 (m, 6H), 1.74-1.46 (m, 22H), 1.28-1.26 (m, 52H), 0.89-0.86 (m, 18H). LC-MS (ESI): (M + H) calculated 965.85, found 966.6.

[0328] Example 83: Synthesis of Aminolipid Compound 332 [ka]

[0329] Following the general synthetic process, 517 mg of amino lipid compound 332 was prepared from M5 (1.0 g, 1.29 mmol) and 332-B (0.47 g, 1.94 mmol) as a pale yellow oil with a purity of 96.71% and a yield of 46%. 1 H NMR (600 MHz, CDCl3) δ4.95-4.90 (m, 1H), 4.89-4.84 (m, 2H), 4.78-4.71 (m, 1H), 3.98 (s, 1H), 3.94-3.87 (m, 1H), 3.39-3.30 (m, 2H), 2.28-2.26 (m, 6H), 2.21-2.20 (m, 6H), 1.73-1.47 (m, 20H), 1.33-1.21(m, 59H), 0.91-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 979.86, found 980.3.

[0330] Example 84: Synthesis of amino lipid compound 333 [ka]

[0331] Following the general synthetic process, 519 mg of amino lipid compound 333 was prepared from M5 (1.0 g, 1.29 mmol) and 333-B (0.47 g, 1.94 mmol) as a pale yellow oil with a purity of 96.56% and a yield of 46%. 1 H NMR (600 MHz, CDCl3) δ4.89-4.84 (m, 3H), 4.78-4.71 (m, 1H), 4.00 (s, 1H), 3.93 (s, 1H), 3.37-3.30 (m, 2H), 2.28-2.26 (m, 6H), 2.21-2.20 (m, 6H), 1.71-1.49 (m, 22H), 1.30-1.26 (m, 54H), 0.91-0.86 (m, 18H). LC-MS (ESI): (M + H) calculated 979.86, found 980.4.

[0332] Example 85: Synthesis of Aminolipid Compound 334 [ka]

[0333] Following the general synthetic process, 935 mg of amino lipid compound 334 was prepared from M5 (1.0 g, 1.29 mmol) and 334-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 93.38% and a yield of 82%. 1H NMR (600 MHz, CDCl3) δ4.95-4.90 (m, 1H), 4.89-4.84 (m, 2H), 4.78-4.71 (m, 1H), 3.98 (s, 1H), 3.93-3.86 (m, 1H), 3.39-3.30 (m, 2H), 2.28-2.26 (m, 6H), 2.22-2.20 (m, 6H), 1.74-1.69 (m, 2H), 1.63-1.49 (m, 18H), 1.29-1.21 (m, 61H), 0.88 (t, J=6.8Hz, 15H). LC-MS (ESI): (M + H) calculated 993.88, found 994.3.

[0334] Example 86: Synthesis of amino lipid compound 335 [ka]

[0335] Following the general synthetic process, 432 mg of amino lipid compound 335 was prepared from M5 (1.0 g, 1.29 mmol) and 335-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 94.97% and a yield of 46%. 1 H NMR (600 MHz, CDCl3) δ4.88-4.83 (m, 3H), 4.78-4.71 (m, 1H), 4.00 (s, 1H), 3.93 (s, 1H), 3.37-3.30 (m, 2H), 2.28-2.26 (m, 6H), 2.21-2.20 (m,6H), 1.74-1.68 (m, 2H), 1.62-1.50 (20H), 1.28-1.26 (m, 56H), 0.89-0.86 (m, 18H). LC-MS (ESI): (M + H) calculated 993.88, found 994.3.

[0336] Example 87: Synthesis of Aminolipid Compound 336 [ka]

[0337] Following the general synthetic process, 375 mg of amino lipid compound 336 was prepared from M5 (1.0 g, 1.29 mmol) and 336-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 95.91% and a yield of 33%. 1 H NMR (600 MHz, CDCl3) δ4.96-4.91 (m, 1H), 4.88-4.84 (m, 2H), 4.78-4.71 (m, 1H), 3.99 (s, 1H), 3.92 (s, 1H), 3.35-3.30 (m, 2H), 2.28-2.26 (m, 6H), 2.21-2.20 (m, 6H), 1.73-1.69 (m, 2H), 1.63-1.58 (m, 4H), 1.54-1.46 (m, 16H), 1.28-1.26 (m, 56H), 0.92-0.86 (m, 18H). LC-MS (ESI): (M + H) calculated 993.88, found 994.3.

[0338] Example 88: Synthesis of Aminolipid Compound 338 [ka]

[0339] Following the general synthetic process, 460 mg of amino lipid compound 338 was prepared from M5 (1.0 g, 1.29 mmol) and 338-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 96.25% and a yield of 40%. 1H NMR (600 MHz, CDCl3) δ4.88-4.83 (m, 3H), 4.77-4.72 (m, 1H), 4.00 (s, 1H), 3.92 (s, 1H), 3.36-3.31 (m, 2H), 2.28-2.25 (m, 6H), 2.21-2.20 (m, 6H), 1.82-1.46 (m, 22H), 1.28-1.26 (m, 58H), 0.87 (t, J=6.7Hz, 18H). LC-MS (ESI): (M + H) calculated 1007.90, found 1008.4.

[0340] Example 89: Synthesis of Aminolipid Compound 339 [ka]

[0341] Following the general synthetic process, 207 mg of amino lipid compound 339 was prepared from M5 (1.0 g, 1.29 mmol) and 339-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 92.98% and a yield of 18%. 1 H NMR (600 MHz, CDCl3) δ4.93-4.90 (m, 1H), 4.88-4.84 (m, 2H), 4.78-4.71 (m, 1H), 3.99 (s, 1H), 3.91 (s, 1H), 3.37-3.29 (m, 2H), 2.28-2.21 (m, 12H), 1.78-1.50 (m, 22H), 1.28-1.26 (m, 58H), 0.90-0.86 (m, 18H). LC-MS (ESI): (M + H) calculated 1007.89, found 1008.3.

[0342] Example 90: Synthesis of amino lipid compound 365 [ka]

[0343] Following the general synthetic process, 750 mg of amino lipid compound 365 was prepared from M5 (1.0 g, 1.29 mmol) and 365-B (0.52 g, 1.94 mmol) as a pale yellow oil with a purity of 96.10% and a yield of 65%. 1 H NMR (600 MHz, CDCl3) δ4.88-4.84 (m, 2H), 4.75-4.71 (m, 1H), 4.14-4.10 (m, 2H), 3.28-3.18 (m, 4H), 2.49-2.44 (m, 6H), 2.30-2.26 (m, 6H), 1.79-1.50 (m, 26H), 1.30-1.24 (m, 57H), 0.87 (t, J=6.8Hz, 12H). LC-MS (ESI): (M + H) calculated 1005.88, found 1006.4.

[0344] Example 91: Synthesis of Aminolipid Compound 366 [ka]

[0345] Following the general synthetic process, 750 mg of amino lipid compound 366 was prepared from M5 (1.0 g, 1.29 mmol) and 366-B (0.55 g, 1.94 mmol) as a pale yellow oil with a purity of 93.80% and a yield of 64%. 1 H NMR (600 MHz, CDCl3) δ4.88-4.84 (m, 2H), 4.73-4.69 (m, 1H), 4.13-4.10 (m, 2H), 3.21-3.17 (m, 4H), 2.48 (m, 6H), 2.29-2.25 (m, 6H), 1.77 (m, 4H), 1.62-1.59 (m, 6H), 1.50-1.49 (m, 18H), 1.27-1.22 (m, 57H), 0.87 (t, J=6.8Hz, 12H). LC-MS (ESI): (M + H) calculated 1019.89, found 1020.4.

[0346] Example 92: Synthesis of Aminolipid Compound 367 [ka]

[0347] Following the general synthetic process, 650 mg of amino lipid compound 367 was prepared from M5 (1.0 g, 1.29 mmol) and 367-B (0.42 g, 1.94 mmol) as a pale yellow oil with a purity of 96.49% and a yield of 60%. 1 H NMR (600 MHz, CDCl3) δ4.88-4.84 (m, 2H), 4.78-4.70 (m, 1H), 4.19-4.14 (m, 2H), 3.99 (s, 1H), 3.92 (s, 1H), 3.40-3.32 (m, 2H), 2.51-2.44 (m, 6H), 2.26 (t, J=7.5Hz, 4H), 1.77 (m, 6H), 1.61-1.59 (m, 4H),1.50-1.48 (m, 12H), 1.30-1.22 (m, 55H), 0.87 (t, J=7.0Hz, 12H). LC-MS (ESI): (M + H) calculated 949.81, found 950.2.

[0348] Example 93: Synthesis of Aminolipid Compound 368 [ka]

[0349] Following the general synthetic process, 550 mg of amino lipid compound 368 was prepared from M5 (1.0 g, 1.29 mmol) and 368-B (0.44 g, 1.94 mmol) as a pale yellow oil with a purity of 93.24% and a yield of 50%. 1H NMR (600 MHz, CDCl3) δ4.88-4.84 (m, 2H), 4.77-4.70 (m, 1H), 4.19-4.14 (m, 2H), 3.97 (s, 1H), 3.89 (s, 1H), 3.35-3.27 (m, 2H), 2.49-2.44 (m, 6H), 2.27 (t, J=7.5Hz, 4H), 1.78 (m, 4H), 1.61-1.47 (m, 20H), 1.30-1.22 (m, 55H), 0.87 (t, J=7.0Hz, 12H). LC-MS (ESI): (M + H) calculated 963.83, found 964.3.

[0350] Example 94: Synthesis of Aminolipid Compound 369 [ka]

[0351] Following the general synthetic process, 206 mg of amino lipid compound 369 was prepared from M6 (1.0 g, 1.34 mmol) and 369-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 92.16% and a yield of 15%. 1 H NMR (600 MHz, CDCl3) δ4.75-4.71 (m, 1H), 4.06 (t, J= 6.3 Hz, 2H), 3.96 (d, J= 5.9Hz, 4H), 3.20 (m, 4H), 2.29-2.27 (m, 8H), 2.23-2.22 (m, 6H), 1.69-1.27 (m, 74H), 0.93 (t, J= 7.5Hz, 3H) 0.89-0.86 (m, 12H). LC-MS (ESI): (M + H) calculated 979.86, found 980.3.

[0352] Example 95: Synthesis of amino lipid compound 370 [ka]

[0353] Following the general synthetic process, 155 mg of amino lipid compound 370 was prepared from M6 (1.0 g, 1.34 mmol) and 370-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 92.69% and a yield of 11%. 1 H NMR (600 MHz, CDCl3) δ4.75-4.71 (m, 1H), 4.06 (t, J= 6.7 Hz, 2H), 3.97 (d, J= 5.6Hz, 4H), 3.25-3.21 (m, 4H), 2.32-2.26 (m, 8H), 2.21 0.93 (t, J= 7.5Hz, 3H) 0.90-0.87 (m, 12H). LC-MS (ESI): (M + H) calculated 965.85, found 966.3.

[0354] Example 96: Synthesis of Aminolipid Compound 371 [ka]

[0355] Following the general synthetic process, 300 mg of amino lipid compound 371 was prepared from M6 (1.0 g, 1.34 mmol) and 371-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 93.23% and a yield of 22%. 1H NMR (600 MHz, CDCl3) δ4.75-4.71 (m, 1H), 4.05 (t, J=6.8Hz, 2H), 3.95 (d, J= 5.9Hz, 4H), 3.26-3.23 (m, 4H), 2.28 (t, J=7.5Hz, 8H), 2.21 (s, 6H), 1.84 (m, 2H), 1.70 (m, 2H), 1.62-1.58 (m, 8H), 1.50 (m, 4H), 1.32-1.26 (m, 56H), 0.91-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 965.85, found 966.2.

[0356] Example 97: Synthesis of amino lipid compound 372 [ka]

[0357] Following the general synthetic process, 150 mg of amino lipid compound 372 was prepared from M6 (1.0 g, 1.34 mmol) and 372-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 94.31% and a yield of 10%. 1 H NMR (600 MHz, CDCl3) δ4.74 (m, 1H), 4.06 (m, 2H), 3.97 (d, J=5.6Hz, 4H), 3.52-3.49 (m, 2H), 3.29-3.25 (m, 2H), 2.60-2.55 (m, 2H), 2.30-2.26 (m, 6H), 2.21 (m, 6H), 1.73-1.67 (m, 2H), 1.62-1.58 (m, 8H), 1.51 (m, 4H), 1.28-1.23 (m, 58H), 0.90-0.87 (m, 15H). LC-MS (ESI): (M + H) calculated 965.85, found 966.3.

[0358] Example 98: Synthesis of amino lipid compound 373 [ka]

[0359] Following the general synthetic process, 296 mg of amino lipid compound 373 was prepared from M6 (1.0 g, 1.34 mmol) and 373-B (0.53 g, 1.94 mmol) as a pale yellow oil with a purity of 91.57% and a yield of 22%. 1 H NMR (600 MHz, CDCl3) δ4.74 (m, 1H), 4.06 (m, 2H), 3.96 (d, J=5.6Hz, 4H), 3.52-3.49 (m, 2H), 3.28-3.24 (m, 2H), 2.59-2.54 (m, 2H), 2.30-2.25 (m, 6H), 2.21 (s, 6H), 1.69 (m, 2H), 1.61-1.59 (m, 8H), 1.51 (m, 4H), 1.28 (m, 58H), 0.89-0.86 (m, 15H). LC-MS (ESI): (M + H) calculated 979.86, found 980.3.

[0360] Example 99: Synthesis of amino lipid compound 374 [ka]

[0361] Following the general synthetic process, 405 mg of amino lipid compound 374 was prepared from M6 (1.0 g, 1.34 mmol) and 374-B (0.37 g, 1.94 mmol) as a pale yellow oil with a purity of 93.54% and a yield of 31%. 1H NMR (600 MHz, CDCl3) δ 4.78-4.70 (m, 1H), 4.19-4.14 (m, 2H), 4.00-3.92 (m, 6H), 3.38-3.25 (m, 2H), 2.29 (t, J-7.5 Hz, 6H), 2.22 (d, J = 16.7 Hz, 6H), 1.75-1.70 (m, 2H),1.61-1.26 (m, 65H), 0.90-0.87 (m, 12H). LC-MS (ESI): (M + H) calculated 895.77, found 896.2.

[0362] Example 100: Synthesis of amino lipid compound 375 [ka]

[0363] Following the general synthetic process, 977 mg of amino lipid compound 375 was prepared from M6 (1.0 g, 1.34 mmol) and 375-B (0.50 g, 1.94 mmol) as a pale yellow oil with a purity of 92.77% and a yield of 70%. 1 H NMR (600 MHz, CDCl3) δ 4.79-4.70 (m, 1H), 4.12-4.08 (m, 2H), 4.00-3.92 (m, 6H), 3.37-3.25 (m, 2H), 2.29 (t, J-7.5 Hz, 6H), 2.22 (d, J = 10.2 Hz, 6H), 1.74-1.27 (m, 74H), 0.90-0.87 (m, 15H). LC-MS (ESI): (M + H) calculated 965.85, found 966.6.

[0364] Example 101: Synthesis of Aminolipid Compound 376 [ka]

[0365] Following the general synthetic process, 380 mg of amino lipid compound 376 was prepared from M5 (1.0 g, 1.29 mmol) and 376-B (0.45 g, 1.94 mmol) as a pale yellow oil with a purity of 93.62% and a yield of 27%. 1 H NMR (600 MHz, CDCl3) δ4.85-4.82 (m, 2H), 4.72-4.71 (m, 1H), 3.65 (s, 3H), 3.20(m, 4H), 2.30-2.24 (m, 8H), 2.21 (bs, 6H), 1.69 (m, 2H), 1.62-1.59 (m, 6H), 1.49 (m, 14H), 1.26-1.25 (m, 54H), 0.87-0.84 (m, 12H). LC-MS (ESI): (M + H) calculated 965.85, found 966.3.

[0366] Example 102: Synthesis of amino lipid compound 377 [ka]

[0367] Following the general synthetic process, 468 mg of amino lipid compound 377 was prepared from M5 (1.0 g, 1.29 mmol) and 377-B (0.34 g, 1.94 mmol) as a pale yellow oil with a purity of 91.13% and a yield of 41%. 1H NMR (600 MHz, CDCl3) δ 4.88-4.84 (m, 2H), 4.78-4.70 (m, 1H), 4.02, (s, 1H), 3.94 (s, 1H), 3.71 (d, J= 7.6 Hz, 3H), 3.36 (t, J= 7.1 Hz, 1H), 3.32 (t, J= 7.1 Hz, 1H), 2.33-2.25 (m, 6H), 2.22 (d, J= 14.2 Hz, 6H), 1.75-1.67 (m, 2H), 1.62-1.58 (m, 4H), 1.53-1.46 (m, 12H), 1.33-1.25 (m, 52H), 0.87 (t, J= 7.0 Hz, 12H). LC-MS (ESI): (M + H) calculated 909.78, found 910.2.

[0368] Example 103: Synthesis of amino lipid compound 181 (1) Synthesis of compound 181-B [ka]

[0369] 1) Synthesis of (2-bromoethoxy)-tert-butyldimethylsilane (181-A) 2-Bromoethanol (5 g, 40 mmol), DCM (100 ml) and imidazole (4.08 g, 60 mmol) were added to a 250 mL round bottom flask, cooled to 0° C. with stirring, TBSCl (7.23 g, 48 mmol) was added, heated to room temperature and reacted at room temperature for 14 hours. The reaction mixture was quenched by adding saturated aqueous sodium bicarbonate solution and extracted twice with DCM (200 ml). The organic phases were combined, washed with 50 ml of saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to give 10.78 g of crude product, which was used directly in the next reaction.

[0370] 2) Synthesis of N1-(2-(tert-butyldimethylsilyloxy)ethyl)-N3,N3-dimethylpropane-1,3-diamine (181-B) Potassium carbonate (3.87 g, 28 mmol), acetonitrile (80 ml) and 3-dimethylaminopropylamine (7.55 ml, 60 mmol) were added to a 250 mL round bottom flask and stirred at room temperature for 1 h, then cooled to -10°C. 181-A (9.57 g, 40 mmol, dissolved in 20 mL acetonitrile) was added slowly through a dropping funnel. The mixture was reacted overnight at -10°C and filtered. The filtrate was concentrated and extracted with water (60 ml) and ethyl acetate (60 ml). The organic phase was separated, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to give 7.8 g of crude 181-B, which was purified by silica gel column chromatography eluted with EA:MeOH=5:1 to give 3.98 g of 181-B as a pale yellow oil.

[0371] (2) Synthesis of amino lipid compound 181 [ka]

[0372] 1) Synthesis of compound 181-C 181-B (0.50 g, 1.92 mmol) and THF (20 ml) were added to a 25 ml one-neck flask and stirred at 0°C for 10 min, then potassium carbonate (0.19 g, 1.41 mmol) was added and M5 (0.99 g, 1.28 mmol, dissolved in 5 ml THF) was added dropwise. After the dropwise addition was completed, the mixture was reacted for 30 min. The reaction solution was extracted by adding water (50 ml) and DCM (50 ml). The organic phase was separated and the aqueous phase was extracted twice with DCM (100 ml). The organic phases were combined, washed with saturated aqueous sodium chloride solution (50 ml), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated, and the concentrate was purified by silica gel column chromatography and eluted with EA to obtain 597 mg of 181-C as a pale yellow oil in a yield of 47%.

[0373] 2) Synthesis of compound 181 181-C (0.60 g, 0.6 mmol) and THF (5 ml) were added to a 50 ml one-neck flask, and TEA-3HF (863 mg, 5.36 mmol) was added dropwise with stirring at room temperature, and the mixture was allowed to react with stirring for 1 h. The reaction was quenched by adding 10 ml of saturated aqueous ammonium chloride solution, and extracted with ethyl acetate (50 ml) and water (50 ml). The organic phase was separated, and the aqueous phase was extracted twice with ethyl acetate (100 ml). The organic phases were combined, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated, and the concentrate was purified by silica gel column chromatography eluting with DCM:MeOH=20:1 to obtain 236 mg of amino lipid compound 181 as a pale yellow oil with a purity of 93.86% and a yield of 44.67%. 1 H NMR (600 MHz, CDCl3) δ 5.34 (s, 1H), 4.94-4.85 (m, 2H), 4.78 (m, 1H), 3.75 (t, J = 4.8 Hz, 2H), 3.56-3.33 (m, 4H), 2.78 (s, 1H), 2.55 (s, 4H), 2.42 (s, 3H), 2.31 (t, J = 7.5 Hz, 4H), 2.08 (m, 1H), 1.91 (m, 1H), 1.68-1.59 (m, 4H), 1.52 (m, 12H), 1.38-1.20 (m, 52H), 0.91 (t, J = 6.5 Hz, 12H). LC-MS (ESI): (M + H) calculated 881.42, found 882.2.

[0374] Example 104: Synthesis of amino lipid compound 187 [ka]

[0375] Following the general synthetic process, amino lipid compound 187-C was prepared from compound M5 (1.0 g, 1.29 mmol) and 187-B (0.53 g, 1.9 mmol), and 382 mg of compound 187 was prepared as a colorless oil with a yield of 33.1% and a purity of 93.90% from 187-C. 1 H NMR (600 MHz, CDCl3) δ 5.34 (s, 1H), 4.95-4.86 (m, 2H), 4.81 (m, 1H), 3.62-3.55 (t, J = 4.8 Hz, 2H), 3.53-3.41 (t, J = 6.6 Hz, 2H), 3.31-3.19 (t, J = 6.9 Hz, 2H), 2.42-2.25 (m, 12H), 1.76 (m, 4H), 1.69-1.61 (m, 4H), 1.53 (m, 12H), 1.38-1.21 (m, 52H), 0.96-0.85 (m, 12H). LC-MS (ESI): (M + H) calculated 895.45, found 896.2.

[0376] Biological testing The amino lipid compounds 118, SM102, and ALC-0315 used in the biological tests of this disclosure have the following structures: [ka]

[0377] Amino lipid compound 118 can be prepared according to the synthesis method of compound 10 (Example 9) described in Chinese patent application CN107922364A; SM102 and ALC-0315 are commercially available or can be prepared according to techniques well known in the art.

[0378] Experimental Example 1: Preparation of lipid nanoparticles encapsulating luciferase mRNA (Fluc mRNA) (1) Mixture: Predetermined amounts of Fluc mRNA stock solution, 0.2 M sodium acetate buffer, and DEPC water were added to a container and mixed well to obtain an aqueous phase; The amino lipid compound of the present disclosure, the helper lipid, the structural lipid, and the PEG lipid were dissolved separately in absolute ethanol to prepare solutions with concentrations of 20 mg / mL, 10 mg / mL, 20 mg / mL, and 25 mg / mL, respectively. The above four solutions were pipetted in a lipid:DSPC:CHO-HP:M-DMG-2000 molar ratio of 48:10:40.5:1.5 and mixed well to prepare an alcohol phase.

[0379] (2) Encapsulation: Using a microfluidic preparation device (MPE-L2), the above aqueous phase and the above alcohol phase were injected into a microfluidic chip at a flow rate of aqueous phase:alcohol phase = 12 mL / min:4 mL / min, and encapsulation was performed at a flow rate of aqueous phase:alcohol phase = 9 mL / min:3 mL / min to obtain mRNA-encapsulated lipid nanoparticles (mRNA-LNPs).

[0380] (3) Dialysis: The product of step (2) was placed in a dialysis bag and then placed in a Tris Buffer-8% sucrose solution and exchanged to remove components such as residual ethanol, unformulated lipids, etc. Dialysis was performed for 2 hours at room temperature with magnetic stirring in the dark (the dialysis solution was exchanged every hour).

[0381] (4) The product of step (3) was sterilized by passing it through a 0.22 μm microporous membrane and packaged. A lipid nanoparticle formulation encapsulating Fluc mRNA was prepared with a Fluc mRNA concentration of 0.2 μg / μL, a Fluc mRNA to lipid mass ratio of 1:10, a particle diameter of 80-130 nm, and an encapsulation efficiency of 85% or more.

[0382] Experimental Example 2: Evaluation of in vivo delivery performance of lipid nanoparticles Animal preparation: 6-8 week-old female BALB / c mice were selected and bred in an SPF grade breeding room. Animal experiments were carried out in strict compliance with the guidelines of the national medical institution and the requirements of animal ethics.

[0383] In vivo delivery: Before injecting the test LNP formulation, the LNP formulation was gently inverted repeatedly to thoroughly mix the compounded sample. The corresponding amount of formulation sample was aspirated into a 1 ml insulin syringe, and the LNP formulation was injected via tail vein injection (IV) into three mice in duplicate per formulation. Each mouse was injected with 75 μL of the luciferase mRNA (Fluc mRNA)-encapsulated lipid nanoparticle formulation prepared in Experimental Example 1.

[0384] Six hours after injection of the LNP formulation, the mice were injected with 200 μL of D-Luciferin luciferase expression substrate (catalog no. 122799; manufacturer: Perkin Elmer). After substrate injection, the mice were anesthetized with isoflurane inhalation, and the injection time of the luciferase expression substrate was recorded. Ten minutes after substrate injection, the animals were placed in a supine position, and the signal distribution and expression intensity of luciferase in the animal's body and in various organs were observed using an In Vivo Imaging System (IVIS).

[0385] Table 4 shows the encapsulation efficiency of representative amino lipid compounds in lipid nanoparticles encapsulating luciferase mRNA (Fluc mRNA) and the fluorescence expression intensity induced thereby, using amino lipid compound 118 as a control.

[0386] [Table 4]

[0387] Table 5 shows the splenic delivery / total delivery fold of the fluorescence expression intensity induced by lipid nanoparticles in which luciferase mRNA (Fluc mRNA) was encapsulated with amino lipid compound 118.

[0388] [Table 5]

[0389] As can be seen from Table 5, all amino lipid compounds except amino lipid compounds 108 and 109 showed a stronger preference for delivery to the spleen compared to amino lipid compound 118.

[0390] Experimental Example 3: Evaluation of delivery performance of lipid nanoparticles to lymph nodes Animal preparation: 6-8 week-old female BALB / c mice were selected and bred in an SPF grade breeding room. Animal experiments were carried out in strict compliance with the guidelines of the national medical institution and the requirements of animal ethics.

[0391] In vivo delivery: Before injecting the test LNP formulation, the LNP formulation was gently inverted repeatedly to thoroughly mix the prepared sample. A corresponding amount of the formulation sample was drawn into a 1 ml insulin syringe, and the LNP formulation was injected into three mice per formulation via tail vein injection (IV). Each mouse was injected with 75 μL of the luciferase mRNA (Fluc mRNA)-encapsulated lipid nanoparticle formulation prepared in Experimental Example 1.

[0392] Six hours after injection of the LNP formulation, the mice were injected with 200 μL of D-Luciferin luciferase expression substrate (catalog no. 122799; manufacturer: Perkin Elmer). After substrate injection, the mice were anesthetized with isoflurane inhalation, and the injection time of the luciferase expression substrate was recorded. Ten minutes after substrate injection, the animals were placed in a supine position, and the signal distribution and expression intensity of luciferase in the lymph nodes were observed using an In Vivo Imaging System (IVIS).

[0393] Table 6 shows the fluorescence expression intensity induced by lipid nanoparticles in which luciferase mRNA (Fluc mRNA) was encapsulated with a representative amino lipid compound, using SM-102 as a control.

[0394] [Table 6]

[0395] As can be seen from Table 6, amino lipid compounds 250 and 268 are more effective at delivering to lymph nodes than SM102, indicating a stronger preference for lymph nodes.

[0396] Experimental Example 4: In vivo safety evaluation of lipid nanoparticles Following the method described in Experimental Example 1, luciferase mRNA (Fluc mRNA) was replaced with human erythropoietin mRNA (hEPO mRNA), and a human erythropoietin mRNA (hEPO mRNA)-encapsulated lipid nanoparticle formulation was prepared with an hEPO mRNA concentration of 0.2 μg / μL, a hEPO mRNA to lipid mass ratio of 1:10, a particle diameter of 90 to 130 nm, and an encapsulation efficiency of 90% or more.

[0397] Animal preparation: 6-8 week-old female BALB / c mice were selected and bred in an SPF grade breeding room. Animal experiments were carried out in strict compliance with the guidelines of the national medical institution and the requirements of animal ethics.

[0398] In vivo delivery: Prior to injection of the test LNP formulations, the LNP formulations were gently inverted repeatedly to thoroughly mix the compounded samples. A corresponding amount of formulation sample was drawn into a 1 ml insulin syringe and the LNP formulations were injected IV into five mice per formulation via the tail vein. Each mouse was injected with 300 μL of the human erythropoietin mRNA (hEPO mRNA)-encapsulated lipid nanoparticle formulation.

[0399] Collection of serum: 12 hours after injection, blood was collected from the mice and placed in a tube without anticoagulant. Blood was allowed to clot naturally at room temperature for 30-60 minutes, and then centrifuged at 3500 rpm for 10 minutes to obtain the supernatant, which was used as serum.

[0400] Alanine transaminase was detected according to the kit (Nanjing Jiancheng Bioengineering Institute, Catalog No. C009-2-1) instructions, and a standard curve was constructed using the standard provided with the kit. D-PBS was used in the experiment, which was purchased from Sangon Biotech (Shanghai) Co., Ltd. (Cat. No. E607009-0600).

[0401] The method for detecting the enzyme activity of alanine aminotransferase (ALT) in mouse serum is as follows:

[0402] Preparation of ALT standard curve: (1) Enzyme reaction: 0, 2, 4, 6, 8, and 10 μL of 2 μmol / mL sodium pyruvate standard solution were added to 5 μL of 0.1 mol / L phosphate buffer solution in sequence, and the matrix solution was added to make up to 25 μL. The mixture was then mixed well by repeatedly aspirating and dispensing with a pipette;

[0403] (2) Addition reaction: 20 μL of 2,4-dinitrophenylhydrazine solution was added to all reaction wells in (1), mixed by aspirating and dispensing, and then placed in a 37° C. incubator for reaction for 20 minutes;

[0404] (3) Development: 200 μL of 0.4 mol / L NaOH solution was added to all reaction wells in (2) to stop the reaction, mixed by aspirating and dispensing, and incubated at room temperature for 15 minutes. The OD value of each well was measured at 510 nm with a microplate reader; and

[0405] (4) Data processing of the standard curve: The absolute OD value of each well was calculated by subtracting the OD value of the 0 μL well from the OD measurement value of each well, and the corresponding ALT Karmen units were set to 0, 28, 57, 97, 150, and 200 U / L, respectively. The standard curve was obtained by plotting the absolute OD value on the horizontal axis and the corresponding Carmen unit on the vertical axis.

[0406] Detection of ALT enzyme activity in serum samples: (1) Preparation of reagents: The ALT matrix solution was placed in a 37°C incubator and preheated; (2) Enzyme reaction: 5 μL of diluted serum was aspirated and added to a 96-well plate, and 20 μL of matrix solution was added to the corresponding sample wells, mixed by repeatedly aspirating and dispensing to avoid air bubbles, and then placed in a 37°C incubator for 30 minutes; (3) Addition reaction: 20 μL of 2,4-dinitrophenylhydrazine was added to all reaction wells in (2), mixed by aspirating and dispensing, and then reacted in a 37° C. incubator for 20 minutes; (4) Development: 200 μL of 0.4 mol / L NaOH solution was added to all reaction wells in (3) to stop the reaction, and the wells were mixed by aspirating and dispensing, and then incubated at room temperature for 15 minutes. The OD value of each well was measured at a wavelength of 510 nm using a microplate reader; and (5) Calculation of ALT enzyme activity in serum: The absolute OD value of the corresponding sample well was calculated by subtracting the OD value of the control well from the OD value of the sample well obtained, and the ALT enzyme activity (Carmen units) of the corresponding serum sample was calculated by substituting it into the standard curve equation.

[0407] Figure 1 shows the results of in vivo safety evaluation of lipid nanoparticles encapsulating human erythropoietin (hEPO) mRNA using representative amino lipid compounds, with ALC00315 and SM-102 as controls.

[0408] As can be seen from Figure 1, compared with commercially available ALC0315 and SM-102, the representative amino lipid compounds showed equivalent or lower ALT enzyme activity (Carmen units), and 252, 255, 259, 260, 263, 264, 266, 267, 270, 272, and 273 had significantly lower ALT enzyme activity (Carmen units) and were therefore safer.

[0409] Experimental Example 5: IFN-γ Elispot Cellular Immunoassay Following the method described in Experimental Example 1, luciferase mRNA (Fluc mRNA) was replaced with IN002.5.1 mRNA, and an IN002.5.1 mRNA-encapsulated lipid nanoparticle formulation was prepared with an IN002.5.1 mRNA concentration of 0.2 μg / μL, an IN002.5.1 mRNA to lipid mass ratio of 1:10, a particle diameter of 80 to 130 nm, and an encapsulation efficiency of 90% or more.

[0410] The sequence of IN002.5.1 mRNA is that in SEQ ID NO. 1, all uracils (u) are replaced by N1-methylpseudouridine. It is noteworthy that the t (thymine) in the RNA sequence of the sequence listing, SEQ ID NO. 1, is actually u (uracil) according to the WIPO Standard ST.26 for Nucleotide or Amino Acid Sequence Listings.

[0411] Animal preparation: 6-8 week-old female BALB / c mice were selected and bred in an SPF grade breeding room. Animal experiments were carried out in strict compliance with the guidelines of the national medical institution and the requirements of animal ethics.

[0412] Immunization of mice: Prior to injection of the test LNP formulation, the LNP formulation was gently inverted repeatedly to thoroughly mix the compounded sample. A corresponding amount of the formulation sample was drawn into a 1 ml insulin syringe and the LNP formulation was injected intramuscularly (IM) in the tail into 8 mice per formulation. Each mouse was injected with 50 μL of the IN002.5.1 mRNA-loaded lipid nanoparticle formulation.

[0413] Spleen collection: On day 7 after immunization, 3 to 4 mice were selected from each LNP immunization group, euthanized, and the spleens were collected in a super clean bench.

[0414] Collection of serum: On the 14th day after immunization, 150 μL of orbital blood was collected from five mice in each LNP-immunized group, placed in a tube without anticoagulant, allowed to clot naturally at room temperature for 30 to 60 minutes, and then centrifuged at 3,500 rpm for 10 minutes, and the supernatant was used as serum.

[0415] ELISpot test for mouse lymphocytes: Isolation of lymphocytes: Mouse spleens were removed on a super clean bench. 7 mL of mouse lymphocyte separation solution was added to a 6-well cell culture plate. Mouse spleen cells were crushed with a syringe piston, and the spleen cell suspension was filtered through a cell screen and immediately transferred to a 15 mL centrifuge tube. 1000 μL of RPMI1640 medium was slowly added while distinguishing the liquid interface. After centrifugation at 800 g for 30 minutes at room temperature in a horizontal rotor, obvious stratification was observed. The lymphocyte layer was aspirated, 10 mL of RPMI1640 medium was added, and the tube was inverted to wash. The cells were collected by centrifugation at 250 g for 10 minutes at room temperature, and red blood cells were lysed. After red blood cell lysis was completed, the supernatant was poured off, the cells were suspended in the medium, and counted.

[0416] Addition of stimuli and culture of lymphocytes: Precoated plates were activated with RPMI-1640 medium containing 10% fetal bovine serum. After at least 30 min at room temperature, the medium was removed and the cell suspension with adjusted concentration was added (100 μL / well). The medium used to resuspend the cells was used as background negative control. 10 μL of positive stimuli working solution (PMA+ionomycin (dissolved in DPBS)) was added to positive control wells; 10 μL of medium used to resuspend the cells was added to negative control wells; and 10 μL / well of peptide library diluted in RPMI1640 was added to experimental wells. After addition of all samples and stimuli, the plates were covered and incubated in an incubator at 37°C with 5% CO2 for 16-24 h.

[0417] ELISPOT detection after incubation: The cells and culture medium in the wells were discarded, and the wells were lysed with 200 μL / well of ice-cold deionized water at 4°C for 10 minutes. The liquid in the wells was shaken and washed five times with PBS buffer. The wells were incubated with 100 μL / well of 1 μg / ml detection antibody at room temperature for 2 hours; after washing the plate five times with PBS, the wells were incubated with 100 μL / well of 1000-fold diluted enzyme-labeled avidin (Streptavidin-HRP) at room temperature for 1 hour. After washing the plate five times with PBS, it was incubated with TMB substrate developer equilibrated to room temperature in the dark for 15 minutes at room temperature. The developer was discarded, and the front, back and base of the plate were washed 3 to 5 times with deionized water to stop the development.

[0418] Spot counting of ELISPOT plates: The plates were placed in a cool and dark place at room temperature, and the bottom was closed after air drying. The plates were read by an enzyme-linked immunospot analyzer, and various parameters of the spots were recorded.

[0419] The results of IFN-γ ELISPOT cellular immunoassay using lipid nanoparticles in which IN002.5.1 mRNA was encapsulated with a representative amino lipid compound are shown in Figures 2, 3 and 4, with ALC0315 as a control.

[0420] The test results are shown in Figures 2, 3 and 4. From Figure 2, it can be seen that amino lipid compounds 270, 271, 272, 273 and 274 show better cellular immune effects than commercially available ALC0315. From Figure 3, it can be seen that amino lipid compounds 263, 264, 265, 267 and 268 show better cellular immune effects than commercially available ALC0315. From Figure 4, it can be seen that amino lipid compounds 302 and 307 show better cellular immune effects than commercially available ALC0315.

[0421] Experimental Example 6: Humoral immunity test for total binding IgG antibody Using the serum obtained in Experimental Example 5, a humoral immune test of antigen-specific IgG against the expression of IN002.5.1 mRNA was carried out.

[0422] Preparation of test reagents: Washing solution: Take an appropriate amount of coating solution, add Tween 20 to a final concentration of 0.05%, and mix thoroughly for later use. Blocking solution: Accurately weigh out BSA and add to the washing solution to make 3% w / v, mix thoroughly for later use (prepare and use immediately). Sample Diluent: Accurately weigh BSA and add to Wash Solution at 1% w / v and mix thoroughly for later use (prepare and use immediately).

[0423] Operating Procedure Coating: Coat protein with SARS-COV-2 antigen protein (Acro, # SPN-C52He) diluted with PBS buffer to the required concentration for testing and mixed thoroughly for later use; 100μL / well, seal with sealing film, place at 2-8℃ overnight (16-20 hours) or incubate at 37℃ for 2 hours. Plate washing: After incubation, mechanically wash three times with 300 μl / well of washing solution and pat dry with clean paper. Blocking: Add 250 μl / well of blocking solution to the ELISA plate, seal the plate with sealing film, and incubate at 37 °C for 40 to 60 min. Plate washing: After blocking, wash with 300 μl / well of washing solution three times using a washer and pat dry on clean paper. Sample preparation: Vortex the previously separated serum and measure the IgG titer (if storing in a -80°C refrigerator, dissolve at 4°C beforehand). Dilution of serum samples: Collect the separated serum, determine the initial dilution factor (generally 300-3000 times) according to the difference in immunization time, and use this dilution factor as the initial dilution factor to perform a total of 8 gradient dilutions; add 100 μl / well of the diluted serum to the ELISA plate and incubate at 37 °C (incubation time varies depending on the test requirements). Plate washing: After incubation, mechanically wash the plate 3 times with 300 μl / well washing solution and pat dry with clean paper. Enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody with sample dilution solution to a certain ratio, add 100 μl / well, and incubate at 37°C (incubation time varies depending on the test requirements). Plate washing: After incubation, mechanically wash the plate 3 times with 300 μl / well of washing solution and pat dry with clean paper. Development: Equilibrate TMB one-component developer to room temperature in advance, add 100 μl / well to the plate, and incubate at room temperature in the dark. Stop: After development, add stop solution at 50 μl / well. Reading: Select a detection wavelength of 450 nm and a reference wavelength of 630 nm, and read and analyze with a microplate reader.

[0424] Calculation result Data analysis was performed using the SoftMax Pro microplate reader. 450 The cut-off value was calculated based on the following formula: Cut-off value = mean OD of negative serum solutions 450 ×2.1 NOTE: OD of the negative serum solution 450 If the average value of OD is less than 0.05, it is calculated as 0.05. 450 If the average value of is greater than 0.05, the actual OD 450 The cutoff value is calculated to three decimal places. The antibody titer was calculated by comparing the OD of the control serum and the test serum. 450The maximum dilution rate corresponding to the mean value > the cutoff value was defined as the maximum dilution rate. The results of a total anti-IgG binding humoral immunity test using lipid nanoparticles in which IN002.5.1 mRNA was encapsulated with a representative amino lipid compound are shown in Figures 5 and 6, with ALC00315 as a control. The test results are shown in Figures 5 and 6. Figure 5 shows that amino lipid compounds 269, 270, 271, 273 and 274 have better humoral immune effects than commercially available ALC0315. Figure 6 shows that amino lipid compounds 263, 264, 265 and 266 have better humoral immune effects than commercially available ALC0315.

[0425] IN002.5.1 DNA sequence corresponding to the mRNA sequence (SEQ ID NO.1):

[0426] Various modifications of the present disclosure in addition to those described herein will be apparent to those skilled in the art from the foregoing description, and such modifications are intended to fall within the scope of the appended claims.

Claims

1. An amino lipid compound having the structure of formula (I) or a pharma- ceutically acceptable salt or stereoisomer thereof: 【Chemistry 1】 During the ceremony: Z １ , Z ２ , Z ３ , Z ４ , Z ５ , and Z ６ Each independently represents -CH(OR ７ )-, -C=C-, -C≡C-, -O-, -C(=O)O-, -OC(=O)-, -N(R ６ )C(=O)-, -C(=O)N(R ６ ) -, -N(R ６ )C(=O)N(R ６ )-, -OC(=O)N(R ６ ) -, -N(R ６ )C(=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -S-S- or a bond; A １ , A ２ , A ３ , A ４ , A ５ , A ６ , and A ７ are each independently １ -C １２ Hydrocarbylene, C ３ -C ７ cyclohydrocarbyl, phenyl, benzyl, a 4- to 7-membered heterocycle, or a bond; R １ and R ２ are each independently H or C １ -C １８ Hydrocarbyl, or C ３ -C ７ cyclohydrocarbyl, phenyl, benzyl, or a 4- to 7-membered heterocycle; or R １ and R ２ together with the nitrogen atom to which they are attached form a 4-7 membered heterocycle having said nitrogen atom and 0, 1, 2 or 3 additional heteroatoms independently selected from N, O and S in the ring, said heterocycle being １ -C ８ Alkyl, C １ -C ８ Haloalkyl, —O—C １ -C ８ Alkyl, —O—C １ -C ８ Haloalkyl, halogen, OH, CN, nitro, NH ２ , -NH(C １ -C ６ alkyl), and -N(C １ -C ６ Alkyl) ２ and optionally substituted with one, two, three or more substituents independently selected from: R ３ is H or C １ -C １８ Hydrocarbyl, or C ３ -C ７ cyclohydrocarbyl, phenyl, benzyl, 4- to 7-membered heterocycle; R 4 and R 5 are each independently １ -C １８ Hydrocarbyl, or C ３ -C ７ cyclohydrocarbyl, phenyl, benzyl, or a 4- to 7-membered heterocycle; R ６ is H or C １ -C １８ Hydrocarbyl or -OH, -O-optionally substituted C ２ -C １８ Hydrocarbyl, or -C=C-, or -C≡C- optionally substituted C ４ -C １８ Hydrocarbyl, or C １ -C １８ is heterohydrocarbyl; and R ７ is H or C １ -C １２ It is a hydrocarbyl.

2. A １ , A ２ , A ３ , A ４ , A ５ , A ６ , and A ７ are each independently １ -C １２ Alkylene, C having 1, 2, 3, 4 or more double bonds ２ -C １２ Alkenylene, C ３ -C ６ Cycloalkyl, phenyl, benzyl, or a 5- to 6-membered heterocycle or a bond, preferably C １ -C １２ Alkylene, C having 1, 2, 3, 4 or more double bonds ２ -C １２ alkenylene or a bond; Preferably, A １ and A ２ are each independently １ -C ６ alkylene or a bond, more preferably C １ , C ２ , C ３ , C ４ , or C ５ alkylene or a bond, more preferably C １ Or C ２ is an alkylene or a bond; Preferably, A ３ is C １ -C ６ alkylene or a bond, more preferably C ２ , C ３ , C ４ , or C ５ alkylene, more preferably C ２ , C ３ , or C ４ is alkylene; Preferably, A ４ is C １ -C ６ alkylene or a bond, more preferably C １ , C ２ , C ３ , C ４ , or C ５ alkylene or a bond, more preferably C １ , C ２ , C ３ , or C ４ is an alkylene or a bond; Preferably, A ５ is C １ -C ８ alkylene or a bond, more preferably C １ , C ２ , C ３ , C ４ , or C ５ alkylene or a bond, more preferably C １ , C ２ , C ３ , or C ４ is an alkylene or a bond; Preferably, A ６ and A ７ are each independently ５ -C １２ alkylene, more preferably C ６ -C １１ alkylene, more preferably C ７ , C ８ , C ９ , or C １０ is alkylene, 2. The amino lipid compound of claim 1.

3. R １ and R ２ are each independently H or C １ -C １８ Alkyl, C with 1, 2, 3, 4 or more double bonds ２ -C １８ Alkenyl or C ３ -C ６ Cycloalkyl, phenyl, benzyl, or a 5- to 6-membered heterocycle, preferably C １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ alkenyl; or R １ and R ２ together with the nitrogen atom to which they are attached form a 4- to 7-membered heterocycle having said nitrogen atom and 0, 1, 2 or 3 additional heteroatoms independently selected from N, O and S in the ring, said heterocycle being １ -C ８ Alkyl, C １ -C ８ Haloalkyl, —O—C １ -C ８ Alkyl, —O—C １ -C ８ Haloalkyl, halogen, OH, CN, nitro, NH ２ , -NH(C １ -C ６ alkyl), and -N(C １ -C ６ Alkyl) ２ and optionally substituted with one, two, three or more substituents independently selected from: Preferably, R １ and R ２ are each independently C １ -C ６ Alkyl or a bond, preferably C １ , C ２ , C ３ , C ４ , or C ５ alkyl or a bond, more preferably methyl, ethyl, n-propyl, or isopropyl; or Preferably, R １ and R ２ form, together with the nitrogen atom to which they are bonded, a 4- to 7-membered heterocycle, preferably a 5- or 6-membered heterocycle, having the nitrogen atom in the ring; 3. The amino lipid compound according to claim 1 or 2.

4. R ３ is H, C １ -C １８ Alkyl, C with 1, 2, 3, 4 or more double bonds ２ -C １８ Alkenyl, or C ３ -C ６ Cycloalkyl, phenyl, benzyl, or 5-6 membered heterocycle, preferably H, C １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ alkenyl; R ３ is preferably C １ -C １６ Alkyl, more preferably C １ -C １４ Alkyl, more preferably C １ , C ２ , C ３ , C ４ , C ５ , C ６ , C ７ , C ８ , C ９ Or C １０ is alkyl, The amino lipid compound according to any one of claims 1 to 3.

5. R ４ and R ５ are each independently １ -C １８ Alkyl, C with 1, 2, 3, 4 or more double bonds ２ -C １８ Alkenyl or C ３ -C ６ Cycloalkyl, phenyl, benzyl, or a 5- to 6-membered heterocycle, preferably C １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ alkenyl, Preferably, R ４ and R ５ are each independently a branched or linear C １ -C １８ Alkyl, more preferably branched or linear C ８ , C ９ , C １０ , C １１ , C １２ , C １３ Or C １４ Alkyl, more preferably branched C １２ Or C １３ is alkyl, The amino lipid compound according to any one of claims 1 to 4.

6. R ６ is H, C １ -C １８ Alkyl, C with 1, 2, 3, 4 or more double bonds ２ -C １８ alkenyl, —OH, —O-optionally substituted C ２ -C １８ Alkyl, -O-C having 1, 2, 3, 4 or more double bonds, optionally substituted ２ -C １８ Alkenyl, or -C=C-, -C≡C- optionally substituted C ４ -C １８ Alkyl, or -C≡C- C with 1, 2, 3, 4 or more double bonds, optionally substituted ４ -C １８ Alkyl, or C containing O, N or S １ -C １８ Heteroalkyl, or C containing O, N or S ２ -C １８ Heteroalkenyl, or C containing O, N or S ２ -C １８ is heteroalkynyl; or R ６ is H, C １ -C ８ Alkyl, C having 1, 2 or 3 double bonds ２ -C １２ alkenyl, —OH, —O-optionally substituted C ２ -C １２ Alkyl, -O-C optionally substituted with 1, 2, or 3 double bonds ２ -C １２ Alkenyl, -C=C-, or -C≡C- optionally substituted C ４ -C １２ Alkyl, or -C≡C- optionally substituted C with 1, 2 or 3 double bonds ４ -C １２ Alkyl, or C containing O, N or S １ -C ８ Heteroalkyl, or C containing O, N or S ２ -C １２ Heteroalkenyl, or C containing O, N or S ２ -C １２ Heteroalkynyl, The amino lipid compound according to any one of claims 1 to 5.

7. R ７ is H, C １ -C １２ Alkyl or C having 1, 2 or 3 double bonds ２ -C １２ Alkenyl, preferably H, or C １ -C １２ is alkyl, The amino lipid compound according to any one of claims 1 to 6.

8. Z ５ and Z ６ The amino lipid compound according to any one of claims 1 to 7, wherein each of the groups is independently -OC(=O)-, -C(=O)O-, or a bond.

9. Z １ and Z ２ At least one of Z is a bond, and preferably １ and Z ２ are bonds, The amino lipid compound according to any one of claims 1 to 8.

10. A １ and A ２ At least one of A is a bond, preferably １ and A ２ are bonds; and / or A ５ is a bond; and / or A ６ and A ７ At least one of A is a bond, preferably ６ and A ７ are bonds; and / or Z ４ is a bond, The amino lipid compound according to any one of claims 1 to 9.

11. Z ３ is -CH(OR ７ )-, -(C=O)O-, -O(C=O)-, or a bond; The amino lipid compound according to any one of claims 1 to 10.

12. Z １ , Z ２ , Z ４ are each independently a bond; Z ３ is -CH(OR ７ )-, -C(=O)O-, -OC(=O)-, or a bond; Z ５ and Z ６ are each independently -C(=O)O- or -OC(=O)-; A １ , A ２ , and A ５ teeth 、 each independently is a bond; A ３ , A ６ , and A ７ are each independently １ -C １２ Alkylene, C having 1, 2, 3, 4 or more double bonds ２ -C １２ alkenylene; A ４ is C １ -C １２ Alkylene, C having 1, 2, 3, 4 or more double bonds ２ -C １２ alkenylene or a bond; R １ and R ２ are each independently １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ alkenyl; or R １ and R ２ together with the nitrogen atom to which they are attached form a 4-7 membered heterocycle having said nitrogen atom and 0, 1, 2 or 3 additional heteroatoms independently selected from N, O and S in the ring, said heterocycle being １ -C ８ Alkyl, C １ -C ８ Haloalkyl, —O—C １ -C ８ Alkyl, —O—C １ -C ８ Haloalkyl, halogen, OH, CN, nitro, NH ２ , -NH(C １ -C ６ alkyl), and -N(C １ -C ６ Alkyl) ２ and optionally substituted with one, two, three or more substituents independently selected from: R ３ is H, C １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ alkenyl; R ４ and R ５ are each independently １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ alkenyl; and R ７ is H, C １ -C １２ Alkyl or C having 1, 2 or 3 double bonds ２ -C １２ Alkenyl, preferably H, or C １ -C １２ is alkyl, The amino lipid compound according to any one of claims 1 to 11.

13. Z ３ is a bond; A ４ is a bond; R １ and R ２ are each independently １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ alkenyl; or R １ and R ２ together with the nitrogen atom to which they are attached form a 4-7 membered heterocycle having said nitrogen atom and 0, 1, or 2 additional heteroatoms independently selected from N, O, and S in the ring, said heterocycle being １ -C ８ Alkyl, C １ -C ８ Haloalkyl, —O—C １ -C ８ Alkyl, —O—C １ -C ８ Haloalkyl, halogen, OH, CN, nitro, NH ２ , -NH(C １ -C ６ alkyl), and -N(C １ -C ６ Alkyl) ２ and R ３ is C １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ Alkenyl, preferably C １ -C １８ is alkyl, The amino lipid compound according to any one of claims 1 to 12.

14. Z ３ is -CH(OR ７ )—and R １ and R ２ are each independently １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ Alkenyl, preferably C １ -C １８ is alkyl, The amino lipid compound according to any one of claims 1 to 12.

15. R ７ The amino lipid compound of claim 14, wherein is H.

16. A 4 The amino lipid compound of claim 15, wherein is a bond.

17. A ４ is C １ -C １２ Alkylene, C having 1, 2, 3, 4 or more double bonds ２ -C １２ Alkenylene, preferably C １ -C １０ Alkenylene, more preferably C １ -C ８ Alkylene, more preferably -(CH ２ ) ｎ４ -, where n4=1, 2, 3, 4, 5, 6, 7 or 8; 16. The amino lipid compound of claim 15.

18. R ３ is C １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ Alkenyl, preferably C １ -C １８ is alkyl, The amino lipid compound according to any one of claims 15 to 17.

19. R ７ is C １ -C １２ Alkyl or C having 1, 2 or 3 double bonds ２ -C １２ Alkenyl, preferably C １ -C １２ is alkyl, 11. The amino lipid compound of claim 10.

20. R ３ The amino lipid compound of claim 19, wherein is H.

21. R ３ is C １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ Alkenyl, preferably C １ -C １８ is alkyl, 20. The amino lipid compound of claim 19.

22. A ４ 22. The amino lipid compound of claim 20 or 21, wherein is a bond.

23. A ４ is C １ -C １２ Alkylene or C having 1, 2, 3, 4 or more double bonds ２ -C １２ Alkenylene, preferably C １ -C １０ Alkylene, more preferably C １ -C ８ Alkylene, more preferably -(CH ２ ) ｎ４ -, where n4=1, 2, 3, 4, 5, 6, 7 or 8; 22. The amino lipid compound of claim 20 or 21.

24. Z ３ is -C(=O)O- or -OC(=O)-; The amino lipid compound according to any one of claims 1 to 8.

25. Z ３ is -C(=O)O-, where C(=O) is A ４ is bound to; and R ３ is C １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ alkenyl, 25. The amino lipid compound of claim 24.

26. A ４ The amino lipid compound of claim 24 or 25, wherein is a bond.

27. A ４ is C １ -C １２ Alkylene or C having 1, 2, 3, 4 or more double bonds ２ -C １２ Alkenylene, preferably C １ -C １０ Alkylene, more preferably C １ -C ８ Alkylene, more preferably -(CH ２ ) ｎ４ -, where n4=1, 2, 3, 4, 5, 6, 7 or 8; 26. An amino lipid compound according to claim 24 or 25.

28. A １ , A ２ , A ３ , A ４ , A ５ , A ６ , or A ７ Each of the hydrocarbylene or alkylene groups defined above is independently １ -C １０ is alkylene, 28. An amino lipid compound according to any one of claims 1 to 27.

29. A １ , A ２ , A ３ , A ４ , A ５ , A ６ , or A ７ Each of the hydrocarbylenes or alkylenes defined above is independently a C having 1, 2, 3, 4 or more double bonds. ２ -C １０ is alkenylene; 28. An amino lipid compound according to any one of claims 1 to 27.

30. A ３ is C １ -C ８ Alkylene or C having 1 or 2 double bonds ２ -C ８ Alkenylene, preferably C １ -C ６ Alkylene or C having one double bond ２ -C ６ Alkenylene, more preferably C ２ -C ４ Alkylene or C having one double bond ２ -C ４ Alkenylene, and more preferably -(CH ２ ) ２ -, -(CH ２ ) ３ - or - (CH ２ ) ４ - is, 30. The amino lipid compound according to any one of claims 1 to 29.

31. A ６ and A ７ are each independently １ -C １０ Alkylene, C having 1, 2, 3, 4 or more double bonds ２ -C １０ Alkenylene, preferably A ６ and A ７ are each independently ５ -C １０ Alkylene, C having 1, 2, 3, 4 or more double bonds ５ -C １０ Alkenylene, more preferably C ６ -C ９ Alkylene, C having 1, 2, 3, 4 or more double bonds ６ -C ９ Alkenylene, more preferably C ７ -C ８ Alkylene, C having one, two or more double bonds ７ -C ８ is alkenylene; 31. The amino lipid compound according to any one of claims 1 to 30.

32. R １ Or R ２ Hydrocarbyl or alkyl as defined for C １ -C ８ Alkyl, preferably C １ -C ６ Alkyl, more preferably C １ -C ４ Alkyl, more preferably C １ -C ２ is alkyl, 32. The amino lipid compound according to any one of claims 1 to 31.

33. R １ and R ２ are 5-7 membered heterocycles which they form together with the nitrogen atom to which they are attached, and which have in the ring the nitrogen atom and 0, 1, or 2 additional heteroatoms independently selected from N, O, and S, and which are selected from C １ -C ６ Alkyl, C １ -C ６ Haloalkyl, —O—C １ -C ６ Alkyl, —O—C １ -C ６ Haloalkyl, halogen, OH, CN, nitro, NH ２ , -NH(C １ -C ６ alkyl), and -N(C １ -C ６ Alkyl) ２ optionally substituted with 1, 2, or 3 substituents independently selected from Preferably, the heterocycle is a 5- to 6-membered heterocycle having the nitrogen atom and no additional heteroatoms in the ring, and the heterocycle is C １ -C ４ Alkyl, preferably C １ -C ３ Optionally substituted with 1, 2, or 3 substituents independently selected from alkyl, preferably 【Chemistry 2】 The amino lipid compound according to any one of claims 1 to 31,

34. R ３ The hydrocarbyl or alkyl defined for １ -C １２ Alkyl, preferably C １ -C １０ Alkyl, more preferably -(CH ２ ) ｎ３１ -CH ３ , or -CH((CH ２ ) ｎ３２ -CH ３ )-(CH ２ ) ｎ３３ -CH ３ wherein n31 is 0, 1, 2, 3, 4, 5, 6, 7, or 8; n32 is 0, 1, 2, 3, 4, 5, or 6, preferably 0, 1, 2, or 3; and n33 is 0, 1, 2, 3, 4, 5, 6, 7, or 8, preferably 0, 1, 2, 3, 4, 5, or 6.

34. An amino lipid compound according to any one of claims 1 to 33.

35. R ３ C, wherein the hydrocarbyl or alkenyl as defined above has 1, 2 or 3 double bonds. ２ -C １２ Alkenyl, preferably C having one double bond ２ -C １０ alkenyl, 34. An amino lipid compound according to any one of claims 1 to 33.

36. R ７ Hydrocarbyl or alkyl as defined for C １ -C １０ Alkyl, preferably C １ -C ８ Alkyl, more preferably -(CH ２ ) ｎ７ -CH ３ where n7 is 0, 1, 2, 3, 4, 5, 6, or 7; 36. An amino lipid compound according to any one of claims 1 to 35.

37. R ７ The hydrocarbyl or alkenyl defined for ２ -C １０ Alkenyl, preferably C having 1 or 2 double bonds ２ -C ８ alkenyl, 36. An amino lipid compound according to any one of claims 1 to 35.

38. Z ５ and Z ６ At least one of is -C(=O)O-, and preferably Z ５ and Z ６ are both -C(=O)O-, more preferably the C(=O) moiety is A ６ Or A ７ is bound to 38. An amino lipid compound according to any one of claims 1 to 37.

39. R ４ Or R ５ The hydrocarbyl or alkyl defined for is branched, preferably branched C ３ -C １８ Alkyl, more preferably branched C ８ -C １８ Alkyl, e.g., branched C １１ -C １８ Alkyl or branched C １３ -C １５ Alkyl, preferably 【Chemistry 3】 That is, 39. An amino lipid compound according to any one of claims 1 to 38.

40. R ４ Or R ５ The hydrocarbyl or alkenyl defined for is branched, preferably branched C ３ -C １８ Alkenyl, more preferably branched C ８ -C １８ Alkenyl, e.g., branched C １１ -C １８ Alkenyl or branched C １３ -C １５ alkenyl, said alkenyl having 1, 2 or 3 double bonds; 40. An amino lipid compound according to any one of claims 1 to 39.

41. 2. The amino lipid compound of claim 1, wherein the amino lipid compound has a structure represented by formula (II): 【Chemistry 4】 During the ceremony, Z ３ , Z ５ and Z ６ are each independently -C(=O)O- or -OC(=O)-; A ３ is C ２ -C ５ is alkylene; A ４ is C １ -C ５ is an alkylene or a bond; R １ and R ２ are each independently H or C １ -C ３ alkyl; or R １ and R ２ form, together with the nitrogen atom to which they are attached, a 4- to 7-membered heterocycle having the nitrogen atom in the ring, and the heterocycle is １ -C ８ Alkyl, C １ -C ８ Haloalkyl, —O—C １ -C ８ Alkyl, —O—C １ -C ８ Haloalkyl, halogen, OH, CN, nitro, NH ２ , -NH(C １ -C ６ alkyl), and -N(C １ -C ６ Alkyl) ２ and optionally substituted with one, two, three or more substituents independently selected from: R ３ is C １ -C １０ is alkyl; and R ４ and R ５ are each independently a branched C １ -C １８ It is an alkyl.

42. Z ３ The amino lipid compound of claim 41, wherein is -C(=O)O-.

43. Z ５ The amino lipid compound of any one of claims 41 or 32, wherein is -C(=O)O-.

44. Z ６ The amino lipid compound according to any one of claims 41 to 43, wherein is -C(=O)O-.

45. A ３ is C ３ -C ４ 45. The amino lipid compound according to any one of claims 41 to 44, which is an alkylene.

46. A ４ is C １ -C ２ Alkylene, C ３ -C ４ 46. ​​The amino lipid compound of any one of claims 41 to 45, which is an alkylene or a bond.

47. R １ 47. The amino lipid compound of any one of claims 41 to 46, wherein is methyl, ethyl, n-propyl, or isopropyl.

48. R ２ 48. The amino lipid compound of any one of claims 41 to 47, wherein is methyl, ethyl, n-propyl, or isopropyl.

49. R １ and R ２ form, together with the nitrogen atom to which they are attached, a 5- to 6-membered heterocycle having the nitrogen atom in the ring, and the heterocycle is １ -C ８ Alkyl, C １ -C ８ Haloalkyl, —O—C １ -C ８ Alkyl, —O—C １ -C ８ Haloalkyl, halogen, OH, CN, nitro, NH ２ , -NH(C １ -C ６ alkyl), and -N(C １ -C ６ Alkyl) ２ Optionally substituted with one, two, three or more substituents independently selected from, for example, methyl; 49. The amino lipid compound according to any one of claims 41 to 48.

50. The heterocycle 【Chemistry 5】 50. The amino lipid compound of claim 49,

51. R ３ C １ , C ２ , C ３ , C ４ , C ５ , C ６ , C ７ Or C ８ alkyl, said alkyl being linear or branched; 51. The amino lipid compound according to any one of claims 41 to 50.

52. R ４ Branch C １３ -C １５ 52. The amino lipid compound of any one of claims 41 to 51, which is alkyl.

53. R ４ but 【Chemistry 6】 53. The amino lipid compound of claim 52, wherein

54. R ５ Branch C １３ -C １５ 54. The amino lipid compound of any one of claims 41 to 53, which is alkyl.

55. R ５ but 【Chemistry 7】 55. The amino lipid compound of claim 54,

56. 56. The amino lipid compound of any one of claims 1 to 55, having one of the following structures:

57. A lipid nanoparticle comprising an amino lipid compound according to any one of claims 1 to 56.

58. The lipid nanoparticle further comprises one or more of a helper lipid, a structural lipid, and a PEG-lipid; Preferably, the lipid nanoparticle further comprises a helper lipid, a structural lipid, and a PEG lipid.

59. 58. The lipid nanoparticle of claim 58, comprising an amino lipid compound according to any one of claims 1 to 56 in an amount (mol percent) of about 25.0% to 75.0%, for example, about 25.0%-28.0%, 28.0%-32.0%, 32.0%-35.0%, 35.0%-40.0%, 40.0%-42.0%, 42.0%-45.0%, 45.0%-46.3%, 46.3%-48.0%, 48.0%-49.5%, 49.5%-50.0%, 50.0%-55.0%, 55.0%-60.0%, 60.0%-65.0%, or 65.0%-75.0%, based on the total amount of the amino lipid compound according to any one of claims 1 to 56, the helper lipid, the structural lipid, and the PEG-lipid.

60. The lipid nanoparticle of claim 58 or 59, comprising about 5.0% to 45.0% of the helper lipid, for example about 5.0%-9.0%, 9.0%-9.4%, 9.4%-10.0%, 10.0%-10.5%, 10.5%-11.0%, 11.0%-15.0%, 15.0%-16.0%, 16.0%-18.0%, 18.0%-20.0%, 20.0%-25.0%, 25.0%-33.5%, 33.5%-37.0%, 37.0%-40.0%, 40.0%-42.0%, or 42.0%-45.0% (mol percent) based on the total amount of the amino lipid compound, helper lipid, structural lipid, and PEG-lipid of any one of claims 1 to 56.

61. The lipid nanoparticles may be prepared by mixing the amino lipid compound according to any one of claims 1 to 56, the helper lipid, the structural lipid, and the PEG-lipid in an amount of about 0.0% to 50.0%, for example, about 0.0%-10.0%, 10.0%-15.5%, 15.5%-18.5%, 18.5%-22.5%, 22.5%-23.5%, 23.5%-28.5%, 28.5%-33.5%, 33.5%-35.0%, 35.0%-36.5%, 36.5%-38.0%, 61. The lipid nanoparticle of any one of claims 58 to 60, comprising the structural lipid in an amount (mol percent) of 38.0%-38.5%, 38.5%-39.0%, 39.0%-39.5%, 39.5%-40.5%, 40.5%-41.5%, 41.5%-42.5%, 42.5%-42.7%, 42.7%-43.0%, 43.0%-43.5%, 43.5%-45.0%, 45.0%-46.5%, 46.5%-48.5%, or 46.5%-50.0%.

62. The lipid nanoparticle of any one of claims 58 to 61, comprising the PEG-lipid in an amount (mol percent) of about 0.5% to 5.0%, for example, about 0.5%-1.0%, 1.0%-1.5%, 1.5%-1.6%, 1.6%-2.0%, 2.0%-2.5%, 2.5%-3.0%, 3.0%-3.5%, 3.5%-4.0%, 4.0%-4.5%, or 4.5%-5.0%, based on the total amount of the amino lipid compound, helper lipid, structural lipid, and PEG-lipid of any one of claims 1 to 56. The lipid nanoparticle of any one of claims 58 to 61, comprising the PEG-lipid in an amount (mol percent) of about 0.5% to 5.0%.

63. The helper lipid is selected from the group consisting of DSPC, DOPE, DOPC, DOPS, DSPG, DPPG, DPPC, DGTS, and lysophospholipids, preferably one or more selected from the group consisting of DSPC, DOPE, DOPC, and DOPS, more preferably DSPC and / or DOPE. The lipid nanoparticle according to any one of claims 58 to 62.

64. The structured lipid is at least one selected from the group consisting of cholesterol, cholesterol esters, sterol hormones, sterol vitamins, bile acids, cholesterin, ergosterol, β-sitosterol, and oxidized cholesterol derivatives, preferably at least one selected from the group consisting of cholesterol, cholesteryl esters, sterol hormones, sterol vitamins, and bile acids, more preferably cholesterol, and even more preferably high-purity cholesterol, particularly high-purity cholesterol of injection grade such as CHO-HP. The lipid nanoparticles according to any one of claims 58 to 63.

65. The PEG-lipid is selected from the group consisting of PEG-DMG and PEG-DSPE, preferably PEG-DMG; Preferably, the PEG-DMG is a polyethylene glycol (PEG) derivative of 1,2-dimyristoyl-sn-glycerol; Preferably, the PEG has an average molecular weight of about 2,000 to 5,000, preferably about 2,000.

66. The molar ratio of amino lipid compound:helper lipid:structural lipid:PEG-lipid according to any one of claims 1 to 56 is about 45:10:42.5:2.5, or 45:11:41.5:2.5, or 42.0:10.5:45.0:2.5, or 42.0:16.0:39.5:2.5, or 40.0:16.0:41.5:2.5, or 40.0:18.0:39.5:2.5, or 35.0: 16.0:46.5:2.5, or 35.0:25.0:36.5:3.5, or 28.0:33.5:35.0:3.5, or 32.0:37.0:40.5:0.5, or 35.0:40.0:22.5:2.5, or 40.0:42.0:15.5:2.5, or 40.0:20.0:38.5:1.5, or 45.0:15.0:38.5:1.5, or 55.0:5.0:3 8.5:1.5, or 60.0:5.0:33.5:1.5, or 45.0:20.0:33.5:1.5, or 50.0:20.0:28.5:1.5, or 55.0:20.0:23.5:1.5, or 60.0:20.0:18.5:1.5, or 40.0:15.0:43.5:1.5, or 50.0:15.0:33.5:1.5, or 55.0:15.0:28.5:

1. 5, or 60.0:15.0:23.5:1.5, or 40.0:10.0:48.5:1.5, or 45.0:10.0:43.5:1.5, or 55.0:10.0:33.5:1.5, or 40.0:5.0:53.5:1.5, or 45.0:5.0:48.5:1.5, or 50.0:5.0:43.5:1.

5.

67. The lipid nanoparticle of claim 66, wherein the helper lipid is DOPE and the structured lipid is CHO-HP.

68. The molar ratio of amino lipid compound according to any one of claims 1 to 56 to helper lipid:structural lipid:PEG-lipid is about 50.0:10.0:38.5:1.5, or 50.0:9.0:38.0:3.0, or 49.5:10.0:39.0:1.5, or 48.0:10.0:40.5:1.5, or 46.3:9.4:42.7:1.6, or 45.0:9.0:43.0:3.0, or 4 5.0:11.0:41.5:2.5, or 42.0:10.5:45.0:2.5, or 42.0:16.0:39.5:2.5, or 40.0:16.0:41.5:2.5, or 40.0:18.0:39.5:2.5, or 35.0:40.0:22.5:2.5, or 40.0:20.0:38.5:1.5, or 45.0:15.0:38.5:1.5, or 55.0:5.0 :38.5:1.5, or 60.0:5.0:33.5:1.5, or 45.0:20.0:33.5:1.5, or 50.0:20.0:28.5:1.5, or 55.0:20.0:23.5:1.5, or 60.0:20.0:18.5:1.5, or 40.0:15.0:43.5:1.5, or 50.0:15.0:33.5:1.5, or 55.0:15.0:28.5:1 60.0:15.0:23.5:1.5, or 40.0:10.0:48.5:1.5, or 45.0:10.0:43.5:1.5, or 55.0:10.0:33.5:1.5, or 40.0:5.0:53.5:1.5, or 45.0:5.0:48.5:1.5, or 50.0:5.0:43.5:1.

5.

69. The lipid nanoparticle of claim 68, wherein the helper lipid is DSPC and the structured lipid is CHO-HP.

70. Further contains biologically active ingredients, Preferably, the biologically active component is a nucleic acid. A lipid nanoparticle according to any one of claims 57 to 69.

71. The lipid nanoparticle of claim 70, wherein the mass ratio of the amino lipid compound of any one of claims 1 to 56 to the nucleic acid is about (5-30):1, such as about (5-10):1, (10-15):1, (15-20):1, (20-25):1, or (25-30):1, preferably about 10:

1.

72. said nucleic acid is selected from the group consisting of RNA, antisense oligonucleotides, and DNA; Preferably, the RNA is selected from the group consisting of messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), short interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single guide RNA (sgRNA), Cas9 mRNA, or a mixture thereof; Preferably, the DNA is a plasmid.

72. The lipid nanoparticle of claim 70 or 71.

73. 73. A pharmaceutical composition comprising an amino lipid compound according to any one of claims 1 to 56 or a lipid nanoparticle according to any one of claims 57 to 72, together with a pharma- ceutically acceptable carrier, diluent or excipient.

74. further comprising a buffer; Preferably, the buffer is selected from phosphate buffer and Tris buffer, preferably phosphate buffer; and / or Preferably, the buffer has a concentration of about 5 mmol / L to about 30 mmol / L, preferably about 10 mmol / L; and / or Preferably, the buffer has a pH of about 6 to 8, preferably about 7 to 8, more preferably about 7 to 7.

5.

74. The pharmaceutical composition of claim 73.

75. Further comprising a cryoprotectant, Preferably, the cryoprotectant is selected from sucrose and trehalose, preferably sucrose; Preferably, the cryoprotectant has a concentration of about 50 mg / ml to 100 mg / ml.

75. A pharmaceutical composition according to any one of claims 73 or 74.

76. 57. Use of an amino lipid compound according to any one of claims 1 to 56 in the manufacture of a vehicle for delivering an active ingredient.

77. The delivery vehicle is a lipid nanoparticle according to any one of claims 57 to 72; and / or the active ingredient is a pharma- ceutical active ingredient, preferably a nucleic acid; and / or said nucleic acid is selected from the group consisting of RNA, antisense oligonucleotides, and DNA; Preferably, the RNA is selected from the group consisting of messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), short interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single guide RNA (sgRNA), Cas9 mRNA, or a mixture thereof; Preferably, the DNA is a plasmid.

77. The use according to claim 76.

78. 20. Use of an amino lipid compound according to any one of claims 1 to 56, a lipid nanoparticle according to any one of claims 57 to 72, or a pharmaceutical composition according to any one of claims 73 to 75 in the manufacture of a medicament.

79. 79. The use according to claim 78, wherein the medicament is for the treatment and / or prevention of a disease, preferably the medicament is for use in gene therapy, protein replacement therapy, antisense therapy, therapy with interfering RNA or genetic vaccination.

80. 80. The use of claim 79, wherein the disease is selected from the group consisting of a rare disease, an infectious disease, a cancer, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, and a metabolic disease.

81. 81. The use of claim 80, wherein the cancer is selected from one or more of lung cancer, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer, or prostate cancer; and the genetic disease is selected from one or more of hemophilia, thalassemia, and Gaucher disease.

82. 80. The use according to claim 79, wherein said genetic vaccination is for use in the treatment and / or prevention of cancer, allergy, toxicity and pathogen infection.

83. 83. The use of claim 82, wherein the pathogen is one or more selected from the group consisting of a virus, a bacterium, or a fungus.

84. Use of an amino lipid compound according to any one of claims 1 to 56, a lipid nanoparticle according to any one of claims 57 to 72, or a pharmaceutical composition according to any one of claims 73 to 75 in the manufacture of a pharmaceutical for nucleic acid introduction.

85. said nucleic acid is selected from the group consisting of RNA, antisense oligonucleotides, and DNA; Preferably, the RNA is selected from the group consisting of messenger RNA (mRNA), ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), short interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA), single guide RNA (sgRNA), Cas9 mRNA, or a mixture thereof; 85. The use according to claim 84, wherein preferably the DNA is a plasmid.

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