Aminolipid compounds, methods for preparing the same, compositions thereof, and their uses

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] This disclosure relates to aminolipid compounds that can be used in the preparation of lipid nanoparticles for delivering active ingredients, and to methods for preparing the same. This disclosure also relates to compositions comprising the above aminolipid compounds, in particular lipid nanoparticles, and the use thereof. [Background technology]

[0002] Gene therapies involve introducing genes containing specific genetic information into target cells through artificial means. The expressed target proteins exert regulatory, therapeutic, or even curative effects on diseases caused by congenital or acquired gene deficiencies, or the gene series interferes with or modulates the expression of related genes to exert clinical therapeutic effects. However, gene therapies still face several challenges, including the difficulty of directly introducing nucleic acids into cells and their high susceptibility to degradation by nucleases in the cytoplasm. Therefore, to facilitate the achievement of the therapeutic and / or preventive objectives of gene therapies, there is a need to develop more aminolipid compounds that can be used to deliver nucleic acids like active ingredients, as well as related preparation methods and applications. [Overview of the project]

[0003] Summary of the Invention In one aspect of this disclosure, an aminolipid compound represented by formula (I) is provided. [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 defined as described below. Another aspect of this disclosure provides a method for preparing the above-mentioned aminolipid compounds. Another aspect of this disclosure provides the use of the above-mentioned aminolipid compound in the manufacture of a vehicle for active ingredients. Another aspect of this disclosure provides lipid nanoparticles containing the above-mentioned aminolipid compound. Another aspect of the present disclosure provides a composition comprising the above-mentioned aminolipid compound. Another aspect of this disclosure provides the use of the above-mentioned aminolipid compounds, lipid nanoparticles, or compositions in the manufacture of pharmaceuticals. Another aspect of this disclosure provides the use of the above-mentioned aminolipid compounds, lipid nanoparticles, or compositions in the manufacture of a nucleic acid delivery pharmaceutical. [Brief explanation of the drawing]

[0005] [Figure 1] The results of the ALT enzyme activity test 12 hours after in vivo delivery of lipid nanoparticles in Experimental Example 4 are shown. [Figure 2] The results of an erythropoietic cell immunoassay against mouse splenic lymphocytes using representative aminolipid compounds in Experiment Example 5 are shown. [Figure 3] The results of an erythropoietic cell immunoassay against mouse splenic lymphocytes using representative aminolipid compounds in Experiment Example 5 are shown. [Figure 4] The results of the erythropoietic cell immunization test against splenic lymphocytes of mice treated with representative aminolipid compounds in Experimental Example 5 are shown. [Figure 5] The IgG titers in the serum of mice immunized for 14 days with LNPs containing IN002.5.1 mRNA encapsulated in representative aminolipid compounds, as in Experimental Example 6, are shown. [Figure 6] The IgG titers in the serum of mice immunized for 14 days with LNPs containing N002.5.1 mRNA encapsulated in a representative aminolipid compound, as in Experimental Example 6, are shown. [Modes for carrying out the invention]

[0006] Detailed description of the invention definition Unless otherwise defined herein, all technical and scientific terms used in this specification are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques herein are intended to mean techniques commonly understood in the art including variations apparent to one of ordinary skill in the art or substitutions of equivalent techniques. The following terms are considered well understood by one of ordinary skill in the art, but the following definitions are provided to better explain the present 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-recited elements or method steps.

[0008] As used herein, the term "hydrocarbyl" refers to the residue remaining after removal of one hydrogen atom from an aliphatic hydrocarbon and includes straight-chain or branched, saturated or unsaturated hydrocarbyls. Hydrocarbyl groups include, but are not limited to, alkyl, alkenyl, and alkynyl. Preferably, the hydrocarbyl group has from 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 hydrocarbyl). 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 10Examples of hydrocarbyls include, but are not limited to, hydrocarbyls, C1-C8 hydrocarbyls, C1-C7 hydrocarbyls, C1-C6 hydrocarbyls, C1-C4 hydrocarbyls, C1-C3 hydrocarbyls, C1-C2 hydrocarbyls, C2-C8 hydrocarbyls, C2-C4 hydrocarbyls, C4-C8 hydrocarbyls, C4-C9 hydrocarbyls, C5-C8 hydrocarbyls, C1-C4 hydrocarbyls, C2-C8 hydrocarbyls, C3 hydrocarbyls, C4 hydrocarbyls, C5 hydrocarbyls, C6 hydrocarbyls, C7 hydrocarbyls, and C8 hydrocarbyls. Unless otherwise expressly stated herein, hydrocarbyls are optionally substituted, and the definition of "optionally substituted" below applies to such substituents. In certain embodiments, hydrocarbyls are either unbranched (i.e., linear) or have one branch, two branches, or more branches.

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

[0010] In this specification, the term "alkyl" means a linear or branched, saturated monovalent hydrocarbyl. Preferably, the alkyl group has 1 to 24 carbon atoms (C1-C24). 24 Alkyl atoms, 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 It has an alkyl group. An example of an alkyl group is 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 groups 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 tridecanoyl. Unless otherwise expressly stated herein, alkyl groups are optionally substituted.

[0011] In this specification, the term "alkylene" refers to the remaining divalent group after the removal of one more hydrogen atom from the alkyl group as defined above. Unless otherwise explicitly stated herein, alkylenes are also optionally substituted.

[0012] In this specification, the term "alkenyl" refers to a monovalent hydrocarbyl, either linear or branched, containing one or more double bonds (C=C). Preferably, the alkenyl group has 2 to 24 carbon atoms (C2-C). 24 Alkenyls), 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 It has an alkenyl group and has 1, 2, 3, 4, or more double bonds. An example of an alkenyl group is a C2-C group with 1, 2, 3, 4, or more double bonds. 24 Alkenyl, C2-C 20 Alkenyl, C2-C 18 Alkenyl, C2-C 16 Alkenyl, C2-C 12 Alkenyl, C2-C 10Examples include, but are not limited to, alkenyls, C2-C8 alkenyls, C2-C7 alkenyls, C2-C6 alkenyls, C2-C4 alkenyls, C2-C3 alkenyls, C4-C8 alkenyls, C4-C9 alkenyls, and C5-C8 alkenyls. Some more specific examples include, but are not limited to, ethenyls, propenyls, buto-1-enyls, buto-2-enyls, pento-1-enyls, pento-2-enyls, hexy-1-enyls, hexy-2-enyls, hexy-3-enyls, hept-1-enyls, hept-2-enyls, hept-3-enyls, octo-1-enyls, octo-2-enyls, octo-3-enyls, non-1-enyls, non-2-enyls, and non-3-enyls. In some preferred embodiments, the alkenyl group has one double bond. Unless otherwise explicitly stated herein, alkenils are optionally substituted.

[0013] In this specification, the term "alkenylene" refers to the remaining divalent group after an additional hydrogen atom has been removed from the alkenyl as defined above. Unless otherwise expressly stated in the specification, alkenylenes are optionally substituted.

[0014] In this specification, the term "alkynyl" refers to a linear or branched monovalent alkynyl group containing one or more triple bonds (C≡C). Preferably, the alkynyl group contains 2 to 24 carbon atoms (C2-C). 24 Alkynnyl) 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 It has an alkynyl group and has 1, 2, 3, 4, or more triple bonds. The alkynyl group has 1, 2, 3, 4, or more triple bonds, C2-C 24 Alkinyl, C2-C 20 Alkinyl, C2-C 18 Alkinyl, C2-C 16 Alkinyl, C2-C 12 Alkinyl, 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 groups. Some more specific examples include, but are not limited to, ethynyl, propynyl, buto-1-inyl, buto-2-inyl, pento-1-inyl, pento-2-inyl, hexy-1-inyl, hexy-2-inyl, hexy-3-inyl, hepto-1-inyl, hepto-2-inyl, hepto-3-inyl, octo-1-inyl, octo-2-inyl, octo-3-inyl, non-1-inyl, non-2-alkynyl, and non-3-alkynyl groups. In some preferred embodiments, the alkynyl group has one triple bond. Unless otherwise explicitly stated herein, alkynyl is optionally substituted.

[0015] In this specification, the term "alkynylene" refers to the remaining divalent group after one more hydrogen atom has been removed from the alkynyl as defined above. Unless otherwise explicitly stated in the specification, alkynylenes are optionally substituted.

[0016] In this specification, the terms “cyclohydrocarbyl,” “cyclohydrocarbylene,” and “hydrocarbon ring” refer to monocyclic or polycyclic hydrocarbon rings having ring carbon atoms that are 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). In certain embodiments, “cyclohydrocarbyl,” “cyclohydrocarbylene,” and “hydrocarbon ring” have, for example, 3 to 10, preferably 3 to 8, more preferably 3 to 6, for example 5 to 6, or 5 to 7 ring carbon atoms. Examples of "cyclohydrocarbyl," "cyclohydrocarbylene," and "hydrocarbon ring" include, but are not limited to, cyclopropyl(len)(ring), cyclobutyl(len)(ring), cyclopentyl(len)(ring), cyclohexyl(len)(ring), cycloheptyl(len)(ring), cyclooctyl(len)(ring), cyclononyl(len)(ring), and cyclohexenyl(len)(ring). Unless otherwise expressly stated herein, cyclohydrocarbyl, cyclohydrocarbylene, and hydrocarbon rings are optionally substituted.

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

[0018] In this specification, the term “heterohydrocarbyl” or its sub-concepts (e.g., heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, etc.) refers to a stable linear, branched, or cyclic hydrocarbon radical or combination thereof, comprising a specific number of carbon atoms and at least one heteroatom. A heteroatom is an atom other than carbon and hydrogen. In certain embodiments, a heterohydrocarbyl contains one, two, three or more heteroatoms. In certain embodiments, a heterohydrocarbyl contains one or more (e.g., two or three) identical heteroatoms, or a plurality (e.g., two or three) different heteroatoms. Preferably, the heteroatoms are selected from O, N, and S. Examples of heterohydrocarbyls 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 otherwise explicitly stated herein, heterohydrocarbyls or their sub-concepts (e.g., heteroalkyls, heteroalkenyls, heteroalkynyls, heteroaryls, etc.) are optionally substituted.

[0019] In this specification, the term "heterohydrocarbilene" or its sub-concepts (heteroalkylene, heteroalkenylene, heteroalkylynylene, heteroarylene, etc.) refers to the remaining divalent group after one more hydrogen atom has been lost from the heterohydrocarbyl as defined above. Unless otherwise explicitly stated herein, "heterohydrocarbilene" or its sub-concepts (heteroalkylene, heteroalkenylene, heteroalkylynylene, heteroarylene, etc.) are substituted as appropriate.

[0020] In this specification, the terms “heterocycle,” “heterocyclyl,” or “heterocyclylene” mean a cyclic group having a cyclic structure and containing one or more heteroatoms in the ring-forming atoms. In certain embodiments, the ring-forming atoms contain one or more identical or different heteroatoms. 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 are 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- to 10-membered heterocycle, e.g., a 4- to 7-membered heterocycle, a 5- to 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, and morpholine. A heterocycle may be optionally substituted with one or more substituents, the definition of "optionally substituted" below shall apply to substituents. Unless otherwise expressly stated herein, heterocycles, heterocyclyls, or heterocyclylenes are optionally substituted.

[0021] In this specification, the terms “aryl” and “aromatic ring” refer to a monocyclic or polycyclic aromatic group with a conjugated π-electron system. For example, as used herein, “C 6-10 "Aryl (en)" and "C 6-10 The term "aromatic ring" refers to an aromatic group containing 6 to 10 carbon atoms, such as phenyl (benzene ring) or naphthyl (naphthalene ring). Unless otherwise explicitly stated herein, aryl and aromatic rings are optionally substituted.

[0022] In this specification, the terms “heteroaryl” or “heteroaryl ring” mean a monocyclic, bicyclic, or tricyclic aromatic ring system having, for example, 5, 6, 8, 9, 10, 11, 12, 13, or 14 ring atoms, and particularly including 1, 2, 3, 4, 5, 6, 9, or 10 carbon atoms, and containing at least one heteroatom selected from N, O, and S, which may in each case be further benzo-condensed. For example, heteroaryl or heteroaryl rings 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), etc., and their benzo derivatives; or from pyridyl(ring), pyridazinyl(ring), pyrimidinyl(ring), pyrazinyl(ring), triazinyl(ring), etc., and their benzo derivatives. Unless expressly stated otherwise herein, heteroaryl or heteroaryl rings are optionally substituted.

[0023] In this specification, the term "optionally substituted" means that one or more hydrogen atoms bonded to an atom or group are independently either unsubstituted or substituted with one or more substituents (e.g., 1, 2, 3, or 4). The substituents can be 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-membered 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-C6alkyl-O-C1-C6alkyl, OC1-C6 haloalkyl, NH2, C1-C6alkyl-NH2 (preferably C1-C3alkyl-NH2), -N(C1-C6alkyl)2 (preferably -N(C1-C3alkyl)2), -NH(C1-C6alkyl) (preferably -NH(C1-C3alkyl)), -N(C1-C6alkyl)(C1-C6alkylphenyl), -NH(C1-C6alkylphenyl), nitro, -C(O)-OH, -C(O)OC1-C6alkyl (preferably -C(O)OC1-C3alkyl), -CONRiRii (wherein Ri and Rii are H, D, and C1-C6alkyl, preferably C1-C3alkyl).), -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-membered 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 the formula contains alkyl, cycloalkyl, phenyl, aryl, heterocyclyl, and heteroaryl. Each of these further includes 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 The substituents are selected from, but are not limited to, those optionally substituted with one or more substituents selected from, -NH(C1-C6 alkyl)C(O)(C1-C6 alkyl), -SO2(C1-C6 alkyl), -SO2(phenyl), -SO2(C1-C6 haloalkyl), -SO2NH2, -SO2NH(C1-C6 alkyl), -SO2NH(phenyl), -NHSO2(C1-C6 alkyl), -NHSO2(phenyl), and -NHSO2(C1-C6 haloalkyl). If an atom or group is substituted with multiple substituents, the multiple substituents may be the same or different.

[0024] In certain embodiments, substituents can independently be halogens (e.g., chlorine, bromine, fluorine, iodine), carboxylic acids (e.g., -C(=O)-OH), oxygen (e.g., =O), sulfur (e.g., =S), hydroxyl (e.g., -OH), ester groups (e.g., -C(=O)ORiii or -OC(=O)Riii), aldehyde groups (e.g., -C(=O)H), carbonyls (e.g., -C(=O)Riii or represented by C=O), acyl halides (e.g., -C(=O)X, where X is bromine, fluorine, chlorine, or iodine), carboxylic acid ester groups (e.g., -OC(=O)ORiii), alkoxy groups (e.g., -ORiii), acetals (e.g., -C(ORiii)2Riii, where each ORiii is the same or different alkoxy group), phosphates (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), amide (e.g., -C(=O)N(Riii)2 or -N(Riii)C(=O)Riii), azide (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)NH2), sulfonamides (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., heterocyclo containing one or more heteroatoms selected from S, N and O) Roalkyl (or heterocyclyl containing one or more heteroatoms selected from S, N, and O), aryl (e.g., phenyl or fused ring group), heteroaryl (e.g., 8- to 10-membered bicyclic heteroaryl containing 1 to 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,This is not limited to these. 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 as defined herein. 12 Alkyl, or C1-C 12 Alkenyl, or C1-C 12 Alkinyl, or C1-C 12 Heteroalkyl, or C1-C 12 Heteroalkenyl, or C1-C 12 It is a heteroalkynyl.

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

[0026] In this specification, the term “pharmaceutically acceptable salt” means a basic salt of an organic or inorganic acid, including, but not limited to, hydrochlorides, hydrobroms, hydroiodides, sulfates, phosphates, acetates, trifluoroacetates, thiocyans, maleates, hydroxymaleates, glutarates, methanesulfons, ethanesulfons, benzenesulfons, p-toluenesulfons, benzoates, salicylates, phenylacetates, cinnamates, lactates, malons, pivaphosphates, succinates, fumarates, malates, mandelates, tartrates, gallates, glucons, laurates, palmitates, pectins, piclates, citrates, or combinations thereof.

[0027] In this specification, the term "halo" or "halogen group" is defined to include F, Cl, Br, or I.

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

[0029] In this specification, the term "isomer" means different compounds having the same molecular formula. A "stereoisomer" is an isomer that differs only in the arrangement of its atoms in space. An "atropisomer" is a stereoisomer arising from a hindered rotation with respect to a single bond. An "enantiomer" is a pair of stereoisomers that are mirror images of each other but do not overlap. A mixture of any ratio of a pair of enantiomers is sometimes called a "racemic" mixture. A "diastereoisomer" is a stereoisomer that has at least two asymmetric atoms and is not a mirror image of each other. A "tautomer" is an isomer of a compound that is in equilibrium with each other. The concentration of an isomer depends on the environment in which the compound exists, for example, whether the compound is a solid, in an organic solution, or in an aqueous solution.

[0030] In certain embodiments, “stereoisomers” also include E isomers, Z isomers, or mixtures thereof, cis isomers, trans isomers, or mixtures thereof.

[0031] Nucleic acids and / or polynucleotides useful in this disclosure include a coding region encoding the polypeptide of interest, a 5'-UTR at the 5'-terminus of the coding region, and a 3'-UTR at the 3'-terminus of the coding region. In some embodiments, the nucleic acid or polynucleotide further includes at least one polyadenylated 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 substituted nucleosides, e.g., 5-substituted uridine (e.g., 5-methoxyuridine), 1-substituted pseudouridine (e.g., 1-methyl-pseudridine or 1-ethyl-pseudridine), and / or 5-substituted cytidine (e.g., 5-methyl-cytidine).

[0032] The term “5'-UTR” or “5'-untranslated region” can refer to an RNA sequence in mRNA located upstream of the coding sequence and not translated into protein. The 5'-UTR of a gene typically begins at the transcription start site and ends at a nucleotide upstream of the translation start codon in the coding sequence. The 5'-UTR may contain elements that regulate gene expression, such as ribosome binding sites, 5'-terminal oligopyrimidine tracts, and translation start signals such as Kozak sequences. mRNA may be post-transcriptionally modified by the addition of a 5' cap. Therefore, the 5'-UTR in mature mRNA can also refer to the RNA sequence between the 5' cap and the start codon. In this specification, the term “3'-untranslated region” or “3'-UTR” can refer to an RNA sequence in mRNA located upstream of the coding sequence and not translated into protein. The 3'-UTR in mRNA is located between the stop codon and the poly(A) sequence in the coding 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 coding region sequence. The 3'-UTR may consist of 3'-UTRs derived from at least one of the following genes: albumin, α-globin, β-globin, tyrosine hydroxylase, lipoxygenase, and collagen α.

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

[0034] In this specification, the term “5' cap structure” refers to a 5' cap structure typically located at the 5' end of mature mRNA. In some embodiments, the 5' cap structure is linked to the 5' end of mRNA by a 5'-5'-triphosphate bond. The 5' cap structure is typically formed from a modified (e.g., methylated) ribonucleotide (particularly from guanine nucleotide derivatives). For example, m7GpppN (cap 0, or “cap 0”, is a cap structure formed by the interaction of the 5'-phosphate group of hnRNA 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 may include, but is not limited to, cap 0, cap 1 (a cap structure formed by further methylating the 2'-OH of the ribose on the first nucleotide of hnRNA based on cap 0, or "cap 1"), cap 2 (a cap structure formed by further methylating the 2'-OH of the ribose on the second nucleotide of hnRNA based on cap 1, or "cap 2"), cap 4, cap 0 analogue, cap 1 analogue, cap 2 analogue, or cap 4 analogue.

[0035] Aminolipid compounds In one embodiment, the present disclosure provides an aminolipid compound represented by the following formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof. [ka]

[0036] During the ceremony, Z1, Z2, Z3, Z4, Z5, and Z6 are, independently of each other, -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 Hydrocarbylene, cyclohydrocarbyl, phenyl, benzyl, heterocyclic, or bonded; R1 and R2 are independently H, or C1-C 18 It is hydrocarbyl, or cyclohydrocarbyl, phenyl, benzyl, or heterocyclic; or R1 and R2, together with the nitrogen atom to which they are bonded, form a 5- to 7-membered heterocyclic ring; R3 is H or C1-C 18 Hydrocarbyl, or cyclohydrocarbyl, phenyl, benzyl, heterocyclic, R4 and R5 are independently C1-C 18 Hydrocarbyl, or cyclohydrocarbyl, phenyl, benzyl, or heterocyclic; R6 is H or C1-C 18 Hydrocarbyl or C2-C with any -OH, -O- substitution 18 Hydrocarbyl, or -C=C-, or -C≡C-, or any other substituted C4-C 18 It is hydrocarbyl; Preferably, Z5 and Z6 are independently -OC(=O)-, -C(=O)O-, or a bond.

[0037] In some embodiments, the disclosure provides an aminolipid compound of formula (I) above, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein either Z5 and Z6 are -(C=O)O-, or either of them are -(C=O)O-. In some embodiments, the disclosure provides an aminolipid compound of formula (I) above, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein either Z5 and Z6 are -O(C=O)-, or either either are -O(C=O)-. In some embodiments, the disclosure provides an aminolipid compound of formula (I) above, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein either Z1 or Z2 is a bond; or both Z1 and Z2 are bonds. In some embodiments, the disclosure provides an aminolipid compound of formula (I) above, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein either A1 or A2 is bonded; or both are bonded. In some embodiments, the present disclosure is that Z3 is -(C=O)O- or -O(C=O)-; furthermore, R4 and R5 are the C3-C of the branch. 18 The present invention provides an aminolipid compound of formula (I) described above, which is hydrocarbyl, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0038] In another aspect, the present disclosure provides an aminolipid compound represented by the following formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof: [ka]

[0039] During the ceremony: Z1, Z2, Z3, Z4, Z5, and Z6 are 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 combination; A1, A2, A3, A4, A5, A6, and A7 are each independently C1-C 12Hydrocarbylene, C3-C7 cyclohydrocarbyl, phenyl, benzyl, 4-7 membered heterocycle, or bond; R1 and R2 are independently H, or C1-C 18 It is a 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 bonded, form a 4- to 7-membered heterocycle having in the ring an additional heteroatom of 0, 1, 2, or 3 independently selected from the nitrogen atom and N, O, and S, wherein the heterocycle is 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 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 C2-C with any -OH, -O- substitution 18 Hydrocarbyl, or -C=C-, or -C≡C-, or any other substituted C4-C 18 Hydrocarbyl, or C1-C 18 It is heterohydrocarbyl; and R7 is H or C1-C 12 It is hydrocarbil.

[0040] In some embodiments, R6 is H or C1-C 18 Hydrocarbyl or C2-C with any -OH, -O- substitution 18 Hydrocarbyl, or -C=C-, or -C≡C-, or any other substituted C4-C 18It is hydrocarbil.

[0041] In some embodiments, this disclosure is, 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 5- to 6-membered heterocycle or 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 It is an alkenylene or bond. Preferably, A1 and A2 are each independently a C1-C6 alkylene or bond, more preferably a C1, C2, C3, C4, or C5 alkylene or bond, and even more preferably a C1 or C2 alkylene or bond. Preferably, A3 is a C1-C6 alkylene or bond, more preferably a C2, C3, C4, or C5 alkylene, and even more preferably a C2, C3, or C4 alkylene. Preferably, A4 is a C1-C6 alkylene or bond, more preferably a C1, C2, C3, C4, or C5 alkylene or bond, and even more preferably a C1, C2, C3, or C4 alkylene or bond. Preferably, A5 is a C1-C8 alkylene or bond, more preferably a C1, C2, C3, C4, or C5 alkylene or bond, and even more preferably a C1, C2, C3, or C4 alkylene or bond. Preferably, A6 and A7 are independently C5-C 12 Alkylene, more preferably C6-C 11 Alkylene, more preferably C7, C8, C9, or C 10 The present invention provides an aminolipid compound of formula (I) described above, which is alkylene, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0042] In some of the embodiments described above, R1 and R2 are independently H or C1-C 18alkyl, C2-C having 1, 2, 3, 4 or more double bonds 18 alkenyl or C3-C6 cycloalkyl, phenyl, benzyl, or a 5- to 6-membered heterocycle, preferably C1-C 18 alkyl, or C2-C having 1, 2, 3, 4 or more double bonds 18 is alkenyl; or R1 and R2, together with the nitrogen atom to which they are attached, form a 4- to 7-membered heterocycle having 0, 1, 2 or 3 additional heteroatoms independently selected from N, O and S with respect to said nitrogen atom in the ring, and said heterocycle is 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. In some preferred embodiments, R1 and R2 are each independently C1-C 18 alkyl or a bond, preferably C1, C2, C3, C4, or C5 alkyl or a bond, more preferably methyl, ethyl, n-propyl, or isopropyl. In other preferred embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 4- to 7-membered heterocycle, preferably a 5- to 6-membered heterocycle, having said nitrogen atom in the ring.

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

[0044] In some of the above-described embodiments, R4 and R5 are each independently C1-C 18 alkyl, C2-C having 1, 2, 3, 4 or more double bonds 18 alkenyl or C3-C6 cycloalkyl, phenyl, benzyl, or a 5- to 6-membered heterocycle. Preferably, R4 and R5 are each independently C1-C 18 alkyl, or C2-C having 1, 2, 3, 4 or more double bonds 18 alkenyl. Preferably, R4 and R5 are each independently branched or straight-chain C1-C 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 is alkyl.

[0045] In some of the above-described embodiments, R6 is H, C1-C 18 alkyl, C2-C having 1, 2, 3, 4 or more double bonds 18 alkenyl, -OH, -O-C2-C optionally substituted 18 alkyl, -O-C2-C having 1, 2, 3, 4 or more double bonds optionally substituted 18 alkenyl, or -C=C-, -C≡C-C4-C optionally substituted 18 alkyl, or -C≡C-C4-C having 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 18It is a heteroalkynyl. Preferably, R6 is H, C1-C8 alkyl, C2-C having a double bond of 1, 2, or 3. 12 C2-C with alkenyl, -OH, or -O- substitutions as desired. 12 C2-C with alkyl, -O-optionally substituted 1, 2, or 3 double bonds 12 C4-C is arbitrarily substituted with alkenyl, -C=C-, and -C≡C-. 12 C4-C with 1, 2, or 3 double bonds that are alkyl or -C≡C- optionally substituted. 12 Alkyl, or C1-C8 heteroalkyl containing O, N, or S, or C2-C containing O, N, or S 12 Heteroalkenyl, or C2-C2 containing O, N, or S 12 It is a heteroalkynyl

[0046] In some of the embodiments described above, R6 is H, C1-C 18 Alkyl, C2-C with 1, 2, 3, 4 or more double bonds 18 C2-C with alkenyl, -OH, or -O- substitutions as desired. 18 C2-C with 1, 2, 3, 4 or more double bonds, optionally substituted with alkyl or -O- 18 C4-C is an alkenyl, or a C4-C that is arbitrarily substituted with -C=C- or -C≡C-. 18 C4-C having 1, 2, 3, 4 or more double bonds that are alkyl or -C≡C- optionally substituted. 18 Preferably, R6 is an alkyl group, and R6 is H, C1-C8 alkyl, C2-C having a double bond of 1, 2, or 3. 12 C2-C with alkenyl, -OH, or -O- substitutions as desired. 12 C2-C with alkyl, -O-optionally substituted 1, 2, or 3 double bonds 12 C4-C is arbitrarily substituted with alkenyl, -C=C-, and -C≡C-. 12 C4-C with 1, 2, or 3 double bonds that are alkyl or -C≡C- optionally substituted. 12 It is alkyl.

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

[0048] In some preferred embodiments, this disclosure is, Z1, Z2, and Z4 are each independent connections; Z3 is -CH(OR7)-, -C(=O)O-, -OC(=O)-, or a bond; Z5 and Z6 are independently -C(=O)O- or -OC(=O)-; A1, A2, and A5 are 、 Each is independent, yet they are a combination; A3, A6, and A7 are each independently C1-C 12 Alkylene, C2-C with 1, 2, 3, 4 or more double bonds 12 It is an alkenylene; A4 is C1-C 12Alkylene, C2-C with 1, 2, 3, 4 or more double bonds 12 It is an alkenylene or bond; R1 and R2 are independently C1-C 18 C2-C with alkyl or 1, 2, 3, 4 or more double bonds 18 It is an alkenyl; or R1 and R2, together with the nitrogen atom to which they are bonded, form a 4- to 7-membered heterocycle having in the ring an additional heteroatom of 0, 1, 2, or 3 independently selected from the nitrogen atom and N, O, and S, wherein the heterocycle is 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 having 1, 2, 3, 4 or more double bonds 18 It is an alkenil; R4 and R5 are independently C1-C 18 Alkyl, or C2-C having 1, 2, 3, 4 or more double bonds 18 It is Alkenil; and R7 is H, C1-C 12 Alkyl, or C2-C having 1, 2, or 3 double bonds 12 Alkenyl, preferably H, or C1-C 12 It is alkyl. The present invention provides an aminolipid compound of formula (I) described above, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0049] In some of such embodiments, this disclosure refers to, Z3 is a coupling; A4 is a combined sheet; R1 and R2 are independently C1-C 18 C2-C with alkyl or 1, 2, 3, 4 or more double bonds 18They are alkenyls; or R1 and R2 form a 4- to 7-membered heterocycle having, together with the nitrogen atom to which they are bonded, an additional heteroatom of 0, 1, or 2 independently selected from N, O, and S, wherein the heterocycle is 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 having 1, 2, 3, 4 or more double bonds 18 Alkenyl, preferably C1-C 18 It is alkyl. The present invention provides an aminolipid compound of formula (I) described above, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0050] In other such embodiments, this disclosure is, Z3 is -CH(OR7)-; R1 and R2 are independently C1-C 18 C2-C with alkyl or 1, 2, 3, 4 or more double bonds 18 Alkenyl, preferably C1-C 18 It is alkyl. The present invention provides an aminolipid compound of formula (I) described above, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0051] In some of the embodiments described above, R7 is H. In some of those embodiments, A4 is bonded. In other embodiments, A4 is 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) n4-. In some of the embodiments described above, R3 is C1-C 18 Alkyl, or C2-C having 1, 2, 3, 4 or more double bonds 18 Alkenyl, preferably C1-C 18 It is alkyl.

[0052] In the other embodiments described above, R7 is H, C1-C 12 Alkyl, or C2-C having 1, 2, or 3 double bonds 12 It is an alkenyl. Preferably, R7 is C1-C 12 It is alkyl. In some such embodiments, R3 is H. In other embodiments, R3 is C1-C 18 Alkyl, or C2-C having 1, 2, 3, 4 or more double bonds 18 It is an alkenyl. Preferably, R3 is C1-C 18 It is alkyl. In some embodiments described above, A4 is a bond. In other embodiments, A4 is C1-C 12 Alkylene, C2-C with 1, 2, 3, 4 or more double bonds 12 It is an 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 - is

[0053] In other such embodiments, this disclosure is, The present invention provides an aminolipid compound of formula (I) above, wherein Z3 is -C(=O)O- or -OC(=O)-, or a pharmaceutically acceptable salt or stereoisomer thereof.

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

[0055] In some such embodiments, A4 is a bond. In other embodiments, A4 is C1-C 12 Alkylene, C2-C with 1, 2, 3, 4 or more double bonds 12 It is an 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 - is

[0056] In some of the embodiments described above, the hydrocarbylene or alkylene defined for A1, A2, A3, A4, A5, A6, or A7 is independently C1-C 10 It is an alkenylene. In other embodiments, the hydrocarbylene or alkylene defined for A1, A2, A3, A4, A5, A6, or A7 is each independently a C2-C with 1, 2, 3, 4 or more double bonds. 10 It is alkenylene.

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

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

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

[0060] In the other embodiments described above, the heterocycle formed with R1 and R2 and the nitrogen atom to which they are bonded is a 5- to 7-membered heterocycle having the nitrogen atom and an additional heteroatom of 0, 1, or 2 independently selected from N, O, and S, wherein the heterocycle is optionally substituted with substituents of 1, 2, or 3 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- to 6-membered heterocycle having the nitrogen atom and no additional heteroatoms, wherein the heterocycle is optionally substituted with substituents of 1, 2, or 3 independently selected from C1-C4 alkyl, preferably C1-C3 alkyl. More preferably, the heterocycle [ka] That is the case.

[0061] In some of the embodiments described above, the hydrocarbyl or alkyl defined for R3 is C1-C 12 Alkyl, preferably C1-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 of the embodiments described above, the hydrocarbyl or alkenyl defined for R3 is a C2-C bond having 1, 2, or 3 double bonds. 12 Alkenyl, preferably C2-C having one double bond 10 It is Alkenil.

[0063] In some of the embodiments described above, the hydrocarbyl or alkyl defined for R7 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 of the embodiments described above, the hydrocarbyl or alkenyl defined for R7 is a C2-C bond having 1, 2, or 3 double bonds. 10 The alkenyl is preferably a C2-C8 alkenyl having one or two double bonds.

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

[0066] In some of the embodiments described above, the hydrocarbyl or alkyl defined for R4 or R5 is branched, preferably branched C3-C 18 Alkyl, more preferably branched C8-C 18 Alkyl, for example, branched C 11 -C 18 Alkyl or branched C 13 -C 15It is alkyl, and preferably, [ka] That is the case.

[0067] In some of the embodiments described above, the hydrocarbyl or alkenyl defined for R4 or R5 is branched, preferably branched C3-C 18 Alkenyl, more preferably branched C8-C 18 Alkenil, for example, branch C 11 -C 18 Alkenyl or branched C 13 -C 15 It is an alkenyl group, where the alkenyl group has 1, 2, or 3 double bonds.

[0068] In some embodiments, an aminolipid compound represented by formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof has a structure represented by 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 bond; R1 and R2 are each independently H or C1-C3 alkyl; or R1 and R2, together with the nitrogen atom to which they are bonded, form a 4- to 7-membered heterocycle having the nitrogen atom in the ring, wherein the heterocycle is 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 C1-C10 It is alkyl; and R4 and R5 are independently branched C1-C 18 It is alkyl.

[0070] In some embodiments, this disclosure provides an aminolipid compound represented by formula (II) as described herein, or a pharmaceutically acceptable salt or stereoisomer thereof. These include, where applicable, one or more of the following features: 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 a C3-C4 alkylene. In some embodiments, A4 is a C1-C2 alkylene, a 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 bonded, form a 5- to 6-membered heterocycle having the nitrogen atom in the ring, the 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, for example, substituted with methyl.

[0072] In some embodiments, the heteroring is [ka] That is the case.

[0073] In some embodiments, R3 is a C1, C2, C3, C4, C5, C6, C7, or C8 alkyl group, and the alkyl group can be linear or branched.

[0074] In some embodiments, R4 is branch C 13 -C 15 It is alkyl. In some embodiments, R4 is [ka] That is the case.

[0075] In some embodiments, R5 is branch C 13 -C 15 It is alkyl. In some embodiments, R5 is [ka] That is the case.

[0076] In various embodiments, the present disclosure provides an aminolipid compound of formula (I) or (II) described above, or a pharmaceutically acceptable salt or stereoisomer thereof, the aminolipid compound having one of the structures shown in Table 1 below.

[0077] [Table 1] JPEG0007886640000011.jpg239170JPEG0007886640000012.jpg240170JPEG0007886640000013.jpg243170 JPEG0007886640000014.jpg235170JPEG0007886640000015.jpg232170JPEG0007886640000016.jpg237170 JPEG0007886640000017.jpg232170JPEG0007886640000018.jpg231170JPEG0007886640000019.jpg234170 JPEG0007886640000020.jpg222170JPEG0007886640000021.jpg215170JPEG0007886640000022.jpg234170 JPEG0007886640000023.jpg226170JPEG0007886640000024.jpg232170JPEG0007886640000025.jpg238170 JPEG0007886640000026.jpg232170JPEG0007886640000027.jpg226170JPEG0007886640000028.jpg221170 JPEG0007886640000029.jpg221170JPEG0007886640000030.jpg231170JPEG0007886640000031.jpg219170 JPEG0007886640000032.jpg233170JPEG0007886640000033.jpg226170JPEG0007886640000034.jpg172170

[0078] All of the aminolipid compounds disclosed herein possess hydrophobic properties due to the presence of long nonpolar residues, and simultaneously possess hydrophilic properties due to the amino group. Due to these amphiphilic properties, the aminolipid compounds disclosed herein can be used to form lipid nanoparticles such as lipid bilayers, micelles, and liposomes.

[0079] In the context of this disclosure, the term “lipid nanoparticles” means nanometer-sized materials produced by introducing aminolipid compounds into an aqueous solution. The particles are, in particular, lipid nanoparticles, lipid bilayer vesicles (liposomes), multilayer vesicles, or micelles.

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

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

[0082] In the context of this disclosure, the term "lipid bilayer" means a thin film formed by two layers of lipid molecules. The term "micelle" means an aggregate of surfactant molecules dispersed in a liquid colloid. A typical micelle in an aqueous solution forms an aggregate with a hydrophilic head region upon contact with water, chelating the hydrophobic single tail region at the center of the micelle.

[0083] In one embodiment, the present disclosure provides the use of the aminolipid compounds of the present disclosure for the production of a vehicle for an active ingredient. In some embodiments, the vehicle is in the form of lipid nanoparticles such as lipid bilayers, micelles, or liposomes.

[0084] Lipid nanoparticles (LNPs) In another embodiment, the Disclosure provides lipid nanoparticles comprising the aminolipid compounds of the Disclosure and pharmaceutically acceptable carriers, diluents, or excipients.

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

[0086] In some further embodiments, the lipid nanoparticles further comprise helper lipids, structural lipids, and PEG lipids.

[0087] In some embodiments, the lipid nanoparticles contain aminolipid compounds in amounts ranging from 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 aminolipid compounds, helper lipids, structural lipids, and PEG-lipids.

[0088] In some embodiments, the lipid nanoparticles contain helper lipids in amounts ranging from about 5.0% to 45.0%, based on the total amount of aminolipid compounds, helper lipids, structural lipids, and PEG-lipids, 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%, respectively.

[0089] In some embodiments, the lipid nanoparticles are present in concentrations ranging from approximately 0.0% to 50.0%, based on the total amount of aminolipid compounds, helper lipids, structural lipids, and PEG-lipids, for example, approximately 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%, and 36. Contains structural lipids in amounts (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 contain PEG-lipids in amounts (mol percent) ranging from 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 aminolipid compounds, helper lipids, structural lipids, and PEG-lipids.

[0091] In the context of this disclosure, helper lipids are phospholipids. Phospholipids are generally semi-synthetic and may be naturally derived or chemically modified. Examples of phospholipids 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), and lysophospholipids. 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 this disclosure, structural lipids are sterols including, but not limited to, cholesterol, cholesterol esters, steroid hormones, steroid vitamins, bile acids, cholesteryl, ergosterol, β-sitosterol, and oxidized cholesterol derivatives. Preferably, the structural lipid is at least one selected from cholesterol, cholesteryl esters, steroid hormones, steroid vitamins, and bile acids. In some embodiments, the structural lipid is cholesterol, preferably high-purity cholesterol, particularly injection-grade high-purity cholesterol such as CHO-HP.

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

[0094] In some embodiments described above, the molar ratio of the aminolipid compound:helper lipid:structural lipid:PEG-lipid in the lipid nanoparticles is approximately 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. The ratios are 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 the other embodiments described above, in lipid nanoparticles, the molar ratio of the aminolipid compound:helper lipid:structural lipid:PEG-lipid of the Disclosure is approximately 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 0.5:1.5, 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 The ratios are 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 contain the aminolipid compounds, helper lipids, structural lipids, and PEG-lipids of this disclosure in mole percent (%) based on the total amount of aminolipid compounds, helper lipids, structural lipids, and PEG-lipids, as shown in Nos. 1 to 24 of Table 2 below:

[0097] [Table 2]

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

[0099] [Table 3]

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

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

[0102] The term “therapeutic” or “preventive” refers to any drug that, when administered to a subject, has a therapeutic, diagnostic, and / or preventive effect and / or induces a desired biological and / or pharmacological effect.

[0103] “Effective dose” or “therapeutic dose” means an amount of the aminolipid compound of the Disclosure or lipid nanoparticles containing the aminolipid compound of the Disclosure that, when administered to a mammal (preferably human), is sufficient to effectively provide treatment in the mammal (preferably human). The amount of lipid nanoparticles of the Disclosure constituting a “therapeutic dose” depends on the aminolipid compound, the symptoms and their severity, the mode of administration, and the age of the mammal being treated, but can be routinely determined by a person skilled in the art in light of their knowledge and the Disclosure.

[0104] Preferably, the pharmaceutically active ingredient is a biologically active ingredient, which is a substance that, when introduced into a cell or host, has a biological effect, for example, by stimulating an immune or inflammatory response, by exerting enzymatic activity, by complementing mutations, 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 component is nucleic acid.

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

[0107] Lipid nanoparticles, when they contain active ingredients within their internal aqueous space, are sometimes called "lipid nanoparticle pharmaceuticals."

[0108] In the context of this disclosure, the term “cell” is a general term and includes individual cells, tissues, organs, insect cells, avian cells, fish cells, amphibian cells, mammalian cells, primary cells, serial cell lines, stem cells, and / or cultures of genetically modified cells (e.g., recombinant cells expressing heterologous polypeptides or proteins). Recombinant cells include, for example, cells expressing heterologous polypeptides or proteins (such as growth factors or blood factors).

[0109] In some of the embodiments described above, the lipid nanoparticles of this disclosure further comprise nucleic acids.

[0110] In some embodiments, the mass ratio of the aminolipid compound of this disclosure to nucleic acids in lipid nanoparticles is about (5-30):1, for example, 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), nuclear small RNA (snRNA), small hairpin RNA (shRNA), single guide RNA (sgRNA), Cas9 mRNA, or mixtures thereof.

[0113] In some embodiments, messenger RNA (mRNA) encodes the polypeptide and / or protein of interest. This includes any polypeptide that is naturally occurring, non-natural, or otherwise modified. In some embodiments, the polypeptide and / or protein encoded by mRNA may have therapeutic and / or preventive effects when expressed in cells.

[0114] In some embodiments, the RNA is an siRNA that can selectively reduce or downregulate the expression of a gene of interest. For example, the siRNA may be selected so that a gene associated with a particular disease, disorder, or symptom is silenced when lipid nanoparticles containing the siRNA are administered to a target that needs it. The siRNA may contain a sequence complementary to the mRNA sequence encoding the gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

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

[0116] In some embodiments, the RNA is shRNA or a vector or plasmid encoding it. The shRNA may be produced in the target cell after a suitable construct has been delivered into the nucleus. Constructs and mechanisms related to shRNA are well known in the relevant art.

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

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

[0119] Pharmaceutical composition In another embodiment, the disclosure provides a pharmaceutical composition comprising lipid nanoparticles as described above and a pharmaceutically 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, and is preferably sucrose. In some embodiments, the cryoprotectant has a concentration of about 50 mg / ml to 100 mg / ml.

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

[0123] Purpose The lipid nanoparticles of this disclosure possess excellent properties for encapsulating biologically active ingredients. Lipid nanoparticles containing biologically active ingredients can be used to deliver any of a variety of therapeutic agents into cells. This disclosure includes the use of such lipid nanoparticles for delivering biologically active ingredients into cells. This disclosure also provides a method for delivering biologically active ingredients to cells, tissues, or organs, which includes contacting the lipid nanoparticles of this disclosure containing the biologically active ingredient with the cells, tissues, or organs. This provides new therapeutic possibilities to the target.

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

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

[0126] This disclosure provides a method for producing a target polypeptide and / or protein in mammalian cells, comprising contacting the cells with lipid nanoparticles containing mRNA encoding the target polypeptide and / or protein, wherein contact with the cells with the lipid nanoparticles allows the mRNA to be taken up into the cells and translated to produce the target polypeptide and / or protein.

[0127] In yet another embodiment, the Disclosure provides the use of the aminolipid compounds, lipid nanoparticles, or pharmaceutical compositions of the Disclosure in the manufacture of pharmaceuticals. Preferably, the pharmaceutical compositions are used for the treatment and / or prevention of diseases.

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

[0129] Pharmaceuticals are used in various applications, such as gene therapy, protein replacement therapy, antisense therapy, interfering RNA therapy, and gene vaccination.

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

[0131] In some embodiments, gene vaccination is preferably used to treat and / or prevent cancer, allergies, toxic diseases, and pathogen infections. In some embodiments, the pathogen is selected from one or more viruses, bacteria, or fungi.

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

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

[0134] In yet another aspect, the present disclosure provides the use of the amino lipid compounds, lipid nanoparticles, or pharmaceutical compositions of the present disclosure in the manufacture of a medicament for nucleic acid delivery. In some embodiments, the nucleic acid is selected from the group consisting of RNA, antisense oligonucleotides, 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 mixtures 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: [Chemical formula]

[0136] Wherein, 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 above method includes the following: (1) Subjecting M1 and M_{2}-a to a reductive amination reaction to obtain M3, or subjecting M1 and M_{2}-b to an alkylation reaction to obtain M3; (2) Reacting M4 with an acylating agent (or triphosgene, chloroformate, DIC, DSC) to obtain M5; and (3) Reacting M5 with M3 in the presence of a base (such as triethylamine or pyridine, DMAP) to obtain the amino lipid compound of formula (I).

[0137] In yet another embodiment, the lipid nanoparticles or compositions of the present disclosure may be prepared according to methods known in the art. For example, the method may include the following steps: (1) Formulation: Formulate a suitable aqueous phase; and formulate an organic phase comprising the aminolipid compounds of the Disclosure, and optionally helper lipids, structural lipids, and / or PEG lipids; (2) Encapsulation: Mix an appropriate amount of the above aqueous phase with the above organic phase; (3) Dialysis: Optionally, dialyze the mixture from step (2); and (4) Sterilization: Optionally, sterilize the product from step (3) using a sterilization filter, such as a 0.22 μm microporous membrane.

[0138] In some embodiments, lipid nanoparticles or compositions of the present disclosure, particularly those containing nucleic acids and mRNA, may be prepared by a method comprising the following steps: (1) Formulation: Formulate an aqueous phase containing nucleic acids; and formulate an organic phase (e.g., an ethanol phase) containing the aminolipid compounds of the Disclosure, and optionally helper lipids, structural lipids, and / or PEG lipids; (2) Encapsulation: Mix an appropriate amount of the above aqueous phase with the above organic phase; (3) Dialysis: Optionally, dialyze the mixture from step (2); (4) Sterilization: Optionally, sterilize the product from step (3) using a sterilization filter, such as a 0.22 μm microporous membrane.

[0139] This disclosure also includes the following embodiments: Embodiment 1. An aminolipid compound having the structure of formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof: [ka]

[0140] During the ceremony, Z1, Z2, Z3, Z4, Z5, and Z6 are 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 Hydrocarbylene, cyclohydrocarbyl, phenyl, benzyl, heterocyclic, or bonded; R1 and R2 are independently H, or C1-C 18 It is hydrocarbyl, or cyclohydrocarbyl, phenyl, benzyl, or heterocyclic; or R1 and R2, together with the nitrogen atom to which they are bonded, form a 5- to 7-membered heterocyclic ring; R3 is H or C1-C 18 Hydrocarbyl or cyclohydrocarbyl, phenyl, benzyl, heterocyclic; R4 and R5 are independently C1-C 18 Hydrocarbyl, or cyclohydrocarbyl, phenyl, benzyl, heterocyclic; R6 is H or C1-C 18 Hydrocarbyl or C2-C with any -OH, -O- substitution 18 Hydrocarbyl, or C4-C with any substitution of -C=C- or -C≡C-. 18 It is hydrocarbil.

[0141] Embodiment 2. The aminolipid compound of Embodiment 1, wherein Z5 and Z6 are independently -OC(=O)-, -C(=O)O-, or a bond. Embodiment 3. An aminolipid compound according to any one of Embodiments 1 to 2, wherein one of Z5 and Z6 is -C(=O)O-. Embodiment 4. The aminolipid compounds of Embodiments 1-2, wherein both Z5 and Z6 are -C(=O)O-. Embodiment 5. An aminolipid compound from any one of Embodiments 1 to 2, wherein one of Z5 and Z6 is -OC(=O)-. Embodiment 6. The aminolipid compounds of Embodiments 1-2, wherein both Z5 and Z6 are -OC(=O)-. Embodiment 7. An aminolipid compound from any one of Embodiments 1-2, wherein one of Z1 and Z2 is a bond. Embodiment 8. An aminolipid compound from any one of Embodiments 1-2, wherein both Z1 and Z2 are bonds. Embodiment 9. An aminolipid compound from any one of Embodiments 1-2, wherein one of A1 and A2 is a bond. Embodiment 10. An aminolipid compound from any one of Embodiments 1-2, wherein both A1 and A2 are conjugates. Embodiment 11.Z3 is an aminolipid compound from any one of Embodiments 1 to 2, wherein Z3 is -C(=O)O- or -OC(=O)-. Embodiment 12. R4 and R5 are branches C3-C 18 The aminolipid compound of Embodiment 11, which is a hydrocarbyl group. Embodiment 13. Lipid nanoparticles comprising one aminolipid compound from Embodiments 1 to 12 and a pharmaceutically acceptable carrier, diluent, or excipient. Embodiment 14. A pharmaceutical composition comprising one aminolipid compound from any one of Embodiments 1 to 12 and a pharmaceutically acceptable carrier, diluent, or excipient.

[0142] Use in the manufacture of a medicament for gene therapy, gene vaccination, antisense therapy, or therapy with interfering RNA of the amino lipid compound according to any one of Embodiments 1 to 12, the lipid nanoparticles according to Embodiment 13, and the pharmaceutical composition according to Embodiment 14. 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, gastric cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain tumor, lymphatic cancer, blood cancer, or prostate cancer; and the genetic disease is selected from one or more of hemophilia, thalassemia, and Gaucher's disease. Embodiment 18. The use according to Embodiment 17, wherein the gene vaccination is useful for the treatment of cancer, allergy, toxicity, and pathogen infection. Embodiment 19. The use according to Embodiment 18, wherein the pathogen is selected from one or more of viruses, bacteria, or fungi. Embodiment 20. Use in the manufacture of a medicament for nucleic acid delivery of the lipid nanoparticles according to Embodiment 13 or the amino lipid compound according to any one of Embodiments 1 to 12, 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 compound of the present disclosure can form vehicles such as lipid nanoparticles having excellent properties for encapsulating biologically active components, and can be used to deliver biologically active components, particularly water-insoluble drugs or active components (such as nucleic acids) that are easily degraded or decomposed, and can improve their bioavailability and effectiveness, or transfection efficiency (in the case of nucleic acids), or safety, or preference for specific organs or tissues.

Example

[0144] To further clarify the purpose, technical solutions, and benefits of this disclosure, the disclosure is described below with reference to specific embodiments. The following embodiments are illustrative and not intended to limit the disclosure.

[0145] example The following examples are provided for illustrative purposes only and are not limiting. In the examples, experimental methods for which specific conditions are not specified are usually conventional conditions or conditions recommended by the raw material or product manufacturer, and reagents of unspecified origin are usually commercially available conventional reagents. The meanings of the abbreviations used in the example are as follows: DSC N,N'-disucine imidyl carbonate; TLC (Thin-Layer Chromatography); EA ethyl acetate DCM Dichloromethane TEA (triethylamine); PMA (Phosphomolybdate) 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-Glycerol; h time min

[0146] Example 1: Synthesis of aminolipid 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, purged with nitrogen three times, then filled with hydrogen to 2.0 MPa, reduced pressure 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 hours.

[0148] TLC using EA:n-hexane=1:5 as the developing solvent was used as a monitor, and the starting material heptanal with Rf=0.8 was detected. After development with methanol:DCM=1:1 and fumigation with iodine, the product (108-M3) with Rf=0.2 and the starting material N,N-dimethylaminopropylamine (108-M1) with Rf=0.1 were detected. After the reaction was complete, the reaction mixture was filtered by suction, the palladium on the 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 52% yield.

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

[0150] 1) Synthesis of compound M4-1 Dimethyl sebacate (M4-0) (170g, 738.2 mmol) was added to a 2L round-bottom flask, 850mL of DMF was added, and the mixture was cooled to -10°C. 46g 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 1000mL of water, extracted with 500mL of EA, and the solvent was evaporated to obtain the crude product. This crude product was purified by silica gel column chromatography using EA:n-hexane = 1:5 elution to obtain 80.0g of monomethyl sebacate (M4-1) in 50% yield.

[0151] 2) Synthesis of compound M4-2 M4-1 (120g, 555 mmol), dimethylamine hydrochloride (59g), EDCl (149g), and DMAP (6.8g) were sequentially added to a 2L round-bottom flask, dissolved in 1.2L DCM, and stirred well. Finally, pyridine (123g) was added, and the mixture was stirred overnight at room temperature. The mixture was diluted with 1L of water, and the pH was adjusted to 3 with dilute hydrochloric acid. The organic phase was separated, and the aqueous phase was extracted with 500mL of DCM. The organic phases were combined, and the solvent was removed by evaporation to obtain 126g 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-necked flask and 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 mixture was reacted at 0°C for 2 hours. 600 mL of water was slowly added, and the mixture was 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 obtain 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] 133g of M4-2-2 was placed in a 1L single-neck flask, hydrobromic acid (500ml, 48% aqueous solution) was added, and the mixture was refluxed at 120°C overnight. The mixture was cooled to room temperature while stirring, stirred at -10°C for 20 minutes, and then filtered by suction. The filtered cake was washed with approximately 100mL of water to obtain 120g of crude 10-oxononadecanedioic acid (M4-2).

[0154] Crude M4-2 (120g) and acetonitrile (1L) were added to a 2L necked flask and heated to 85°C under reflux for 0.5 hours. After cooling to room temperature, the mixture was stirred at -10°C for 20 minutes and filtered by suction. The filtered cake was washed with approximately 100mL of cold acetonitrile, transferred to a flask, and the residual solvent was evaporated to obtain 45g 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 value 341.23, Measured value 341.4.

[0155] 3) Synthesis of compound M4-3 EDCl (16.8g), DCM (100ml), triethylamine (8.86g), DMAP (1.78g), 7-tridecanol (10.5g), and M4-2 (10.0g, 29.2mol) were sequentially added to a 500mL round-bottom flask and stirred overnight at room temperature. TLC monitoring confirmed that the conversion of 7-tridecanol was almost complete (developed with EA:n-hexane=1:25, stained with PMA, Rf=0.4 for the starting material 7-tridecanol, Rf=0.5 for the product DTN-T), and the reaction was terminated. 200mL of DCM and 300mL of water were added for extraction, the liquid was separated, the organic phase was recovered, and the solvent was removed by evaporation to obtain 30g of a yellow oily crude product, which was purified by silica gel column chromatography and eluted with EA:n-hexane=1:25 to obtain 15.72g 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 30.0 g of M4-3 (42.4 mmol) was added to a 1 L necked flask, dissolved in methanol (300 ml), and cooled to 0°C. 1.7 g of sodium borohydride was slowly added with stirring, and the mixture was reacted at 0°C for 1 hour. The pH was then adjusted to 6 with dilute hydrochloric acid. The solvent was removed by evaporation. 300 mL of DCM and 300 mL of water were added for extraction, and the organic phase was recovered. 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 (24g, 33.8 mmol), toluene (240 ml), and TEA (5.1 g) were sequentially added to a 500 ml neck flask. Stirring was started at room temperature, and triphosgene (15 g) was slowly added. After 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 the starting material, Rf=0.8 for the product) until the reaction was complete.

[0158] The reaction mixture was cooled to room temperature and filtered by suction. The filtered 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, and eluted with EA:n-hexane = 1:5-25 to obtain 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 aminolipid compound 108 [ka]

[0160] 108-M3 (266 mg, 1.55 mmol), THF (10 ml), and potassium carbonate (178.3 mg) were sequentially added to a 50 ml 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 added dropwise to the neck flask over approximately 10 minutes. After the addition was complete, the mixture was stirred at 0°C for 5 minutes and then allowed to react at room temperature. After 1.5 hours, the reaction was monitored by TLC (EA:n-hexane=30:1, developed with PMA, product and starting material 108-M3 were at the origin, and compound 108-M3 had an Rf of approximately 0.5) until the reaction was complete. 100 ml of water and 100 ml of EA were added to the reaction mixture for extraction, the organic phase was recovered, 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 obtain 1.1 g of crude product. This was purified by silica gel column chromatography using EA:n-hexane = 1:5 to obtain 630 mg of aminolipid compound 108 as a colorless oil in a yield of 53.8% and a purity of 97.41%. 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 value 907.8, Measured value 908.2.

[0161] Example 2: Synthesis of aminolipid compound 109 [ka]

[0162] Following a general synthesis process, 190 mg of aminolipid compound 109 was prepared from compound M5 (1.0 g, 1.29 mmol) and 109-M3 (1.55 mmol) as a colorless oil with a yield of 21% and a purity of 94.94%. 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 value 921.9, Measured value 922.2.

[0163] Example 3: Synthesis of aminolipid compound 110 [ka]

[0164] Following a general synthesis process, 640 mg of aminolipid compound 110 was prepared from compound M5 (1.0 g, 1.29 mmol) and 110-M3 (1.55 mmol) as a colorless oil with a yield of 60% and a purity of 97.71%. 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 value 935.9, Measured value 936.3.

[0165] Example 4: Synthesis of aminolipid compound 111 [ka]

[0166] Following a general synthesis process, 510 mg of compound 111 was prepared from compound M5 (1.0 g, 1.29 mmol) and 111-M3 (1.55 mmol) as a colorless oil with a yield of 47% and a purity of 97.18%. 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 value 949.9, Measured value 950.3.

[0167] Example 5: Synthesis of aminolipid compound 112 [ka]

[0168] Following a general synthesis process, 760 mg of aminolipid compound 112 was prepared from compound M5 (1.0 g, 1.29 mmol) and 112-M3 (1.55 mmol) as a colorless oil with a yield of 69% and a purity of 94.69%. 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 value 963.9, Measured value 964.3.

[0169] Example 6: Synthesis of aminolipid compound 113 [ka]

[0170] Following a general synthesis process, 640 mg of aminolipid compound 113 was prepared from compound M5 (1.0 g, 1.29 mmol) and 113-M3 (1.55 mmol) as a colorless oil with a yield of 58% and a purity of 98.04%. 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 value 977.9, Measured value 978.4.

[0171] Example 7: Synthesis of aminolipid compound 114 [ka]

[0172] Following a general synthesis process, 270 mg of aminolipid compound 114 was prepared from compound M5 (1.0 g, 1.29 mmol) and 114-M3 (1.55 mmol) as a colorless oil with a yield of 22% and a purity of 75.59%. 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 value 933.86, Measured value 934.3.

[0173] Example 8: Synthesis of aminolipid compound 115 [ka]

[0174] Following a general synthesis process, 468 mg of aminolipid compound 115 was prepared from compound M5 (1.0 g, 1.29 mmol) and 115-M3 (1.55 mmol) as a colorless oil with a yield of 38% and a purity of 77.72%. 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 value 947.87, Measured value 948.3.

[0175] Example 9: Synthesis of aminolipid compound 117 [ka]

[0176] Following a general synthesis process, 140 mg of aminolipid compound 117 was prepared from compound M5 (1.0 g, 1.29 mmol) and 117-M3 (1.55 mmol) as a colorless oil with a yield of 13% and a purity of 97.21%. 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 value 975.9, Measured value 976.4.

[0177] Example 10: Synthesis of aminolipid compound 137 [ka]

[0178] Following a general synthesis process, 200 mg of aminolipid compound 137 was prepared as a colorless oil with a yield of 20% and a purity of 97.46% 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 value 893.82, Measured value 894.3.

[0179] Example 11: Synthesis of aminolipid compound 138 [ka]

[0180] Following a general synthesis process, 490 mg of aminolipid compound 138 was prepared from compound M5 (1.0 g, 1.29 mmol) and 138-M3 (1.55 mmol) as a colorless oil with a yield of 42% and a purity of 77.73%. 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 value 907.84, Measured value 908.3.

[0181] Example 12: Synthesis of aminolipid compound 139 [ka]

[0182] Following a general synthesis process, 470 mg of aminolipid compound 139 was prepared from compound M5 (1.0 g, 1.29 mmol) and 139-M3 (1.55 mmol) as a colorless oil with a yield of 45% and a purity of 95.87%. 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 value 921.86, Measured value 922.3.

[0183] Example 13: Synthesis of aminolipid compound 140 [ka]

[0184] Following a general synthesis process, 470 mg of aminolipid compound 140 was prepared from compound M5 (1.0 g, 1.29 mmol) and 140-M3 (1.55 mmol) as a colorless oil with a yield of 44% and a purity of 96.31%. 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 value 935.87, Measured value 936.3.

[0185] Example 14: Synthesis of aminolipid compound 141 [ka]

[0186] Following a general synthesis process, 330 mg of aminolipid compound 141 was prepared from compound M5 (1.0 g, 1.29 mmol) and 141-M3 (1.55 mmol) as a colorless oil with a yield of 31% and a purity of 95.98%. 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 value 949.89, Measured value 950.3.

[0187] Example 15: Synthesis of aminolipid compound 142 [ka]

[0188] Following a general synthesis process, 580 mg of aminolipid compound 142 was prepared from compound M5 (1.0 g, 1.29 mmol) and 142-M3 (1.55 mmol) as a colorless oil with a yield of 53% and a purity of 98.23%. 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 value 963.9, Measured value 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 neck flask and cooled to 0°C with stirring. 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 the acylchloride 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 acylchloride was added dropwise at 0°C. After the addition was complete, the reaction mixture was warmed to room temperature and reacted for 5 hours. Then, 200 mL of water was added, and the mixture was extracted twice with 200 mL of DCM. The organic phases were combined, washed with 100 mL of saline solution, dried on anhydrous sodium sulfate, and the solvent was evaporated to obtain 28 g of crude 255-A.

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

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

[0194] (2) Synthesis of aminolipid 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 with 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 addition was complete, 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 and sequentially washed with water (50 ml) and saturated sodium chloride aqueous solution (50 ml), dried on 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 aminolipid 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 value 993.88, Measured value 994.5.

[0196] Example 17: Synthesis of aminolipid compound 250 [ka]

[0197] Following a general synthesis process, 600 mg of aminolipid 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 value 979.86, Measured value 980.4.

[0198] Example 18: Synthesis of aminolipid compound 251 [ka]

[0199] Following a general synthesis process, 1000 mg of aminolipid 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 value 993.88, Measured value 994.5.

[0200] Example 19: Synthesis of aminolipid compound 252 [ka]

[0201] Following a general synthesis process, 650 mg of aminolipid 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 value 1007.89, Measured value 1008.5.

[0202] Example 20: Synthesis of aminolipid compound 253 [ka]

[0203] Following a general synthesis process, 300 mg of aminolipid compound 253 was prepared as a pale yellow oil with a purity of 96.09% and a yield of 27% from M5 (1.0 g, 1.29 mmol) and 253-B (0.45 g, 1.94 mmol). 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 value 965.85, Measured value 966.5.

[0204] Example 21: Synthesis of aminolipid compound 254 [ka]

[0205] Following a general synthesis process, 100 mg of aminolipid compound 254 was prepared as a pale yellow oil with a purity of 91.94% and a yield of 8.9% from M5 (1.0 g, 1.29 mmol) and 254-B (0.47 g, 1.94 mmol). 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 value 979.86, Measured value 980.5.

[0206] Example 22: Synthesis of aminolipid compound 256 [ka]

[0207] Following a general synthesis process, 480 mg of aminolipid compound 256 was prepared as a pale yellow oil with a purity of 92.94% and a yield of 42% from M5 (1.0 g, 1.29 mmol) and 256-B (0.53 g, 1.94 mmol). 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 value 1007.89, Measured value 1008.3

[0208] Example 23: Synthesis of aminolipid compound 257 [ka]

[0209] Following a general synthesis process, 290 mg of aminolipid compound 257 was prepared as a pale yellow oil with a purity of 95.16% and a yield of 27% from M5 (1.0 g, 1.29 mmol) and 257-B (0.42 g, 1.94 mmol). 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 value 951.83, Measured value 952.4.

[0210] Example 24: Synthesis of aminolipid compound 258 [ka]

[0211] Following a general synthesis process, 340 mg of aminolipid compound 258 was prepared as a pale yellow oil with a purity of 92.94% and a yield of 31% from M5 (1.0 g, 1.29 mmol) and 258-B (0.45 g, 1.94 mmol). 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 value 965.85, Measured value 966.2.

[0212] Example 25: Synthesis of aminolipid compound 259 [ka]

[0213] Following a general synthesis process, 320 mg of aminolipid 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 value 979.86, Measured value 980.3.

[0214] Example 26: Synthesis of aminolipid compound 260 [ka]

[0215] Following a general synthesis process, 440 mg of aminolipid 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 value 993.88, Measured value 994.2.

[0216] Example 27: Synthesis of aminolipid compound 261 [ka]

[0217] Following a general synthesis process, 140 mg of aminolipid 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 value 1007.89, Measured value 1008.3.

[0218] Example 28: Synthesis of aminolipid compound 262 [ka]

[0219] Following a general synthesis process, 500 mg of aminolipid compound 262 was prepared as a pale yellow oil with a purity of 88.60% and a yield of 47% from M5 (1.0 g, 1.29 mmol) and 262-B (0.39 g, 1.94 mmol). 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 value 937.81, Measured value 938.2.

[0220] Example 29: Synthesis of aminolipid compound 263 [ka]

[0221] Following a general synthesis process, 580 mg of aminolipid 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 value 951.83, Measured value 952.2.

[0222] Example 30: Synthesis of aminolipid compound 264 [ka]

[0223] Following a general synthesis process, 220 mg of aminolipid compound 264 was prepared as a pale yellow oil with a purity of 98.65% and a yield of 20% from M5 (1.0 g, 1.29 mmol) and 264-B (0.45 g, 1.94 mmol). 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 value 965.85, Measured value 966.2.

[0224] Example 31: Synthesis of aminolipid compound 265 [ka]

[0225] Following a general synthesis process, 450 mg of aminolipid compound 265 was prepared as a pale yellow oil with a purity of 98.47% and a yield of 40% from M5 (1.0 g, 1.29 mmol) and 265-B (0.47 g, 1.94 mmol). 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 value 979.86, Measured value 980.2.

[0226] Example 32: Synthesis of aminolipid compound 266 [ka]

[0227] Following a general synthesis process, 487 mg of aminolipid 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 value 993.88, Measured value 994.3.

[0228] Example 33: Synthesis of aminolipid compound 267 [ka]

[0229] Following a general synthesis process, 360 mg of aminolipid compound 267 was prepared as a pale yellow oil with a purity of 98.16% and a yield of 31% from M5 (1.0 g, 1.29 mmol) and 267-B (0.53 g, 1.94 mmol). 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 value 1007.89, Measured value 1008.2.

[0230] Example 34: Synthesis of aminolipid compound 268 [ka]

[0231] Following a general synthesis process, 500 mg of aminolipid 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 value 923.80, Measured value 924.1.

[0232] Example 35: Synthesis of aminolipid compound 269 [ka]

[0233] Following a general synthesis process, 510 mg of aminolipid compound 269 was prepared as a pale yellow oil with a purity of 96.02% and a yield of 48% from M5 (1.0 g, 1.29 mmol) and 269-B (0.39 g, 1.94 mmol). 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 value 937.81, Measured value 938.1.

[0234] Example 36: Synthesis of aminolipid compound 270 [ka]

[0235] Following a general synthesis process, 550 mg of aminolipid compound 270 was prepared as a pale yellow oil with a purity of 97.66% and a yield of 51% from M5 (1.0 g, 1.29 mmol) and 270-B (0.42 g, 1.94 mmol). 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 value 951.83, Measured value 952.2.

[0236] Example 37: Synthesis of aminolipid compound 271 [ka]

[0237] Following a general synthesis process, 230 mg of aminolipid compound 271 was prepared as a pale yellow oil with a purity of 94.22% and a yield of 21% from M5 (1.0 g, 1.29 mmol) and 271-B (0.45 g, 1.94 mmol). 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 value 965.85, Measured value 966.2.

[0238] Example 38: Synthesis of aminolipid compound 272 [ka]

[0239] Following a general synthesis process, 450 mg of aminolipid compound 272 was prepared as a pale yellow oil with a purity of 98.47% and a yield of 40% from M5 (1.0 g, 1.29 mmol) and 272-B (0.47 g, 1.94 mmol). 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 value 979.86, Measured value 980.2.

[0240] Example 39: Synthesis of aminolipid compound 273 [ka]

[0241] Following a general synthesis process, 460 mg of aminolipid compound 273 was prepared as a pale yellow oil with a purity of 96.41% and a yield of 40% from M5 (1.0 g, 1.29 mmol) and 273-B (0.50 g, 1.94 mmol). 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 value 993.88, Measured value 994.2.

[0242] Example 40: Synthesis of aminolipid compound 274 [ka]

[0243] Following a general synthesis process, 420 mg of aminolipid 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 value 1007.89, Measured value 1008.3.

[0244] Example 41: Synthesis of aminolipid compound 275 [ka]

[0245] Following a general synthesis process, 280 mg of aminolipid 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 value 991.86, Measured value 992.1.

[0246] Example 42: Synthesis of aminolipid compound 276 [ka]

[0247] Following a general synthesis process, 110 mg of aminolipid 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 value 1005.88, Measured value 1006.3.

[0248] Example 43: Synthesis of aminolipid compound 277 [ka]

[0249] Following a general synthesis process, 350 mg of aminolipid compound 277 was prepared as a pale yellow oil with a purity of 96.47% and a yield of 33% from M5 (1.0 g, 1.29 mmol) and 277-B (0.52 g, 1.94 mmol). 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 value 1005.88, Measured value 1006.3.

[0250] Example 44: Synthesis of aminolipid compound 279 [ka]

[0251] Following a general synthesis process, 250 mg of aminolipid 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 value 1033.91, Measured value 1034.4.

[0252] Example 45: Synthesis of aminolipid compound 280 [ka]

[0253] Following a general synthesis process, 190 mg of aminolipid 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 value 1047.92, Measured value 1048.4

[0254] Example 46: Synthesis of aminolipid compound 281 [ka]

[0255] Following a general synthesis process, 480 mg of aminolipid compound 281 was prepared as a pale yellow oil with a purity of 96.03% and a yield of 41% from M5 (1.0 g, 1.29 mmol) and 281-B (0.55 g, 1.94 mmol). 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 value 1019.89, Measured value 1020.3.

[0256] Example 47: Synthesis of aminolipid compound 282 [ka]

[0257] Following a general synthesis process, 300 mg of aminolipid compound 282 was prepared as a pale yellow oil with a purity of 94.87% and a yield of 27% from M5 (1.0 g, 1.29 mmol) and 282-B (0.44 g, 1.94 mmol). 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 value 963.83, Measured value 964.2.

[0258] Example 48: Synthesis of aminolipid compound 284 [ka]

[0259] Following a general synthesis process, 530 mg of aminolipid compound 284 was prepared as a pale yellow oil with a purity of 94.29% and a yield of 45% from M5 (1.0 g, 1.29 mmol) and 284-B (0.55 g, 1.94 mmol). 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 value 1019.89, Measured value 1020.2.

[0260] Example 49: Synthesis of aminolipid compound 297 [ka]

[0261] Following a general synthesis process, 830 mg of aminolipid 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 value 993.88, Measured value 994.3.

[0262] Example 50: Synthesis of aminolipid compound 298 [ka]

[0263] Following a general synthesis process, 410 mg of aminolipid 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 value 1007.89, Measured value 1008.3.

[0264] Example 51: Synthesis of aminolipid compound 299 [ka]

[0265] Following a general synthesis process, 640 mg of aminolipid 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 value 1021.91, Measured value 1022.3.

[0266] Example 52: Synthesis of aminolipid compound 300 [ka]

[0267] Following a general synthesis process, 550 mg of aminolipid 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 value 1021.91, Measured value 1022.1.

[0268] Example 53: Synthesis of aminolipid compound 301 [ka]

[0269] Following a general synthesis process, 750 mg of aminolipid 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 value 979.86, Measured value 980.1.

[0270] Example 54: Synthesis of aminolipid compound 302 [ka]

[0271] Following a general synthesis process, 680 mg of aminolipid 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 value 993.88, Measured value 994.1.

[0272] Example 55: Synthesis of aminolipid compound 303 [ka]

[0273] Following a general synthesis process, 390 mg of aminolipid compound 303 was prepared as a pale yellow oil with a purity of 95.21% and a yield of 34% from M5 (1.0 g, 1.29 mmol) and 303-B (0.53 g, 1.94 mmol). 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 value 1007.89, Measured value 1008.1.

[0274] Example 56: Synthesis of aminolipid compound 304 [ka]

[0275] Following a general synthesis process, 660 mg of aminolipid 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 value 1007.89, Measured value 1008.1.

[0276] Example 57: Synthesis of aminolipid compound 305 [ka]

[0277] Following a general synthesis process, 490 mg of aminolipid 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 value 1021.91, Measured value 1022.1.

[0278] Example 58: Synthesis of aminolipid compound 306 [ka]

[0279] Following a general synthesis process, 410 mg of aminolipid 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 value 1021.91, Measured value 1022.1.

[0280] Example 59: Synthesis of aminolipid compound 307 [ka]

[0281] Following a general synthesis process, 160 mg of aminolipid 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 value 965.85, Measured value 966.1.

[0282] Example 60: Synthesis of aminolipid compound 308 [ka]

[0283] Following a general synthesis process, 660 mg of aminolipid 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 value 979.86, Measured value 980.1.

[0284] Example 61: Synthesis of aminolipid compound 309 [ka]

[0285] Following a general synthesis process, 740 mg of aminolipid 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 value 993.88, Measured value 994.1.

[0286] Example 62: Synthesis of aminolipid compound 310 [ka]

[0287] Following a general synthesis process, 650 mg of aminolipid 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 value 993.88, Measured value 994.3.

[0288] Example 63: Synthesis of aminolipid compound 311 [ka]

[0289] Following a general synthesis process, 420 mg of aminolipid compound 311 was prepared as a pale yellow oil with a purity of 97.21% and a yield of 36% from M5 (1.0 g, 1.29 mmol) and 311-B (0.53 g, 1.94 mmol). 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 value 1007.89, Measured value 1008.1.

[0290] Example 64: Synthesis of aminolipid compound 312 [ka]

[0291] Following a general synthesis process, 650 mg of aminolipid 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 value 1007.89, Measured value 1008.1.

[0292] Example 65: Synthesis of aminolipid compound 313 [ka]

[0293] Following a general synthesis process, 330 mg of aminolipid 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 value 1021.91, Measured value 1022.2.

[0294] Example 66: Synthesis of aminolipid compound 314 [ka]

[0295] Following a general synthesis process, 122 mg of aminolipid compound 314 was prepared as a pale yellow oil with a purity of 91.54% and a yield of 10% from M5 (1.0 g, 1.29 mmol) and 314-B (0.56 g, 1.94 mmol). 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 value 1021.91, Measured value 1022.3.

[0296] Example 67: Synthesis of aminolipid compound 315 [ka]

[0297] Following a general synthesis process, 480 mg of aminolipid 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 value 1021.91, Measured value 1022.1.

[0298] Example 68: Synthesis of aminolipid compound 316 [ka]

[0299] Following a general synthesis process, 300 mg of aminolipid 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 value 951.83, Measured value 952.0.

[0300] Example 69: Synthesis of aminolipid compound 317 [ka]

[0301] Following a general synthesis process, 300 mg of aminolipid 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 value 965.85, Measured value 966.0.

[0302] Example 70: Synthesis of aminolipid compound 318 [ka]

[0303] Following a general synthesis process, 283 mg of aminolipid 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 value 979.86, Measured value 980.1.

[0304] Example 71: Synthesis of aminolipid compound 319 [ka]

[0305] Following a general synthesis process, 350 mg of aminolipid 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 value 979.86, Measured value 980.0.

[0306] Example 72: Synthesis of aminolipid compound 320 [ka]

[0307] Following a general synthesis process, 350 mg of aminolipid 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 value 993.88, Measured value 994.1.

[0308] Example 73: Synthesis of aminolipid compound 321 [ka]

[0309] Following a general synthesis process, 450 mg of aminolipid 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 value 993.88, Measured value 994.1.

[0310] Example 74: Synthesis of aminolipid compound 322 [ka]

[0311] Following a general synthesis process, 350 mg of aminolipid 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 value 1007.89, Measured value 1008.1.

[0312] Example 75: Synthesis of aminolipid compound 323 [ka]

[0313] Following a general synthesis process, 520 mg of aminolipid 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 value 1007.89, Measured value 1008.0.

[0314] Example 76: Synthesis of aminolipid compound 325 [ka]

[0315] Following a general synthesis process, 620 mg of aminolipid 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 value 1021.91, Measured value 1022.2.

[0316] Example 77: Synthesis of aminolipid compound 326 [ka]

[0317] Following a general synthesis process, 700 mg of aminolipid compound 326 was prepared as a pale yellow oil with a purity of 98.56% and a yield of 60% from M5 (1.0 g, 1.29 mmol) and 326-B (0.56 g, 1.94 mmol). 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 value 1021.91, Measured value 1022.3.

[0318] Example 78: Synthesis of aminolipid compound 327 [ka]

[0319] Following a general synthesis process, 550 mg of aminolipid compound 327 was prepared as a pale yellow oil with a purity of 98.53% and a yield of 46% from M5 (1.0 g, 1.29 mmol) and 327-B (0.56 g, 1.94 mmol). 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 value 1021.91, Measured value 1022.2.

[0320] Example 79: Synthesis of aminolipid compound 328 [ka]

[0321] Following a general synthesis process, 220 mg of aminolipid 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 value 937.81, Measured value 938.3.

[0322] Example 80: Synthesis of aminolipid compound 329 [ka]

[0323] Following a general synthesis process, 650 mg of aminolipid 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 value 951.83, Measured value 952.3.

[0324] Example 81: Synthesis of aminolipid compound 330 [ka]

[0325] Following a general synthesis process, 740 mg of aminolipid compound 330 was prepared as a pale yellow oil with a purity of 92.28% and a yield of 67% from M5 (1.0 g, 1.29 mmol) and 330-B (0.45 g, 1.94 mmol). 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 value 965.85, Measured value 966.6.

[0326] Example 82: Synthesis of aminolipid compound 331 [ka]

[0327] Following a general synthesis process, 338 mg of aminolipid 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 value 965.85, Measured value 966.6.

[0328] Example 83: Synthesis of aminolipid compound 332 [ka]

[0329] Following a general synthesis process, 517 mg of aminolipid 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 value 979.86, Measured value 980.3.

[0330] Example 84: Synthesis of aminolipid compound 333 [ka]

[0331] Following a general synthesis process, 519 mg of aminolipid 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 value 979.86, Measured value 980.4.

[0332] Example 85: Synthesis of aminolipid compound 334 [ka]

[0333] Following a general synthesis process, 935 mg of aminolipid 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 value 993.88, Measured value 994.3.

[0334] Example 86: Synthesis of aminolipid compound 335 [ka]

[0335] Following a general synthesis process, 432 mg of aminolipid 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 value 993.88, Measured value 994.3.

[0336] Example 87: Synthesis of aminolipid compound 336 [ka]

[0337] Following a general synthesis process, 375 mg of aminolipid 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 value 993.88, Measured value 994.3.

[0338] Example 88: Synthesis of aminolipid compound 338 [ka]

[0339] Following a general synthesis process, 460 mg of aminolipid 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 value 1007.90, Measured value 1008.4.

[0340] Example 89: Synthesis of aminolipid compound 339 [ka]

[0341] Following a general synthesis process, 207 mg of aminolipid 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 value 1007.89, Measured value 1008.3.

[0342] Example 90: Synthesis of aminolipid compound 365 [ka]

[0343] Following a general synthesis process, 750 mg of aminolipid 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 value 1005.88, Measured value 1006.4.

[0344] Example 91: Synthesis of aminolipid compound 366 [ka]

[0345] Following a general synthesis process, 750 mg of aminolipid 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 value 1019.89, Measured value 1020.4.

[0346] Example 92: Synthesis of aminolipid compound 367 [ka]

[0347] Following a general synthesis process, 650 mg of aminolipid 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 value 949.81, Measured value 950.2.

[0348] Example 93: Synthesis of aminolipid compound 368 [ka]

[0349] Following a general synthesis process, 550 mg of aminolipid 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 value 963.83, Measured value 964.3.

[0350] Example 94: Synthesis of aminolipid compound 369 [ka]

[0351] Following a general synthesis process, 206 mg of aminolipid 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 value 979.86, Measured value 980.3.

[0352] Example 95: Synthesis of aminolipid compound 370 [ka]

[0353] Following a general synthesis process, 155 mg of aminolipid compound 370 was prepared as a pale yellow oil with a purity of 92.69% and a yield of 11% from M6 (1.0 g, 1.34 mmol) and 370-B (0.50 g, 1.94 mmol). 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 value 965.85, Measured value 966.3.

[0354] Example 96: Synthesis of aminolipid compound 371 [ka]

[0355] Following a general synthesis process, 300 mg of aminolipid 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 value 965.85, Measured value 966.2.

[0356] Example 97: Synthesis of aminolipid compound 372 [ka]

[0357] Following a general synthesis process, 150 mg of aminolipid 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 value 965.85, Measured value 966.3.

[0358] Example 98: Synthesis of aminolipid compound 373 [ka]

[0359] Following a general synthesis process, 296 mg of aminolipid 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 value 979.86, Measured value 980.3.

[0360] Example 99: Synthesis of aminolipid compound 374 [ka]

[0361] Following a general synthesis process, 405 mg of aminolipid 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 value 895.77, Measured value 896.2.

[0362] Example 100: Synthesis of aminolipid compound 375 [ka]

[0363] Following a general synthesis process, 977 mg of aminolipid 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 value 965.85, Measured value 966.6.

[0364] Example 101: Synthesis of aminolipid compound 376 [ka]

[0365] Following a general synthesis process, 380 mg of aminolipid 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 value 965.85, Measured value 966.3.

[0366] Example 102: Synthesis of aminolipid compound 377 [ka]

[0367] Following a general synthesis process, 468 mg of aminolipid 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 value 909.78, Measured value 910.2.

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

[0369] 1) Synthesis of (2-bromoethoxy)-tert-butyldimethylsilane (181-A) 2-bromoethanol (5g, 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, and the mixture was heated to room temperature and reacted at room temperature for 14 hours. The reaction mixture was quenched with saturated sodium bicarbonate aqueous solution and extracted twice with DCM (200 ml). The organic phases were combined, washed with 50 ml of saturated sodium chloride aqueous solution, dried on anhydrous sodium sulfate, and filtered. The filtrate was concentrated to obtain 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 hour, then cooled to -10°C. 181-A (9.57 g, 40 mmol, dissolved in 20 ml of acetonitrile) was slowly added dropwise through a 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 sodium chloride aqueous solution, dried on anhydrous sodium sulfate, and filtered. The filtrate was concentrated to obtain 7.8 g of crude 181-B, which was purified by silica gel column chromatography and eluted with EA:MeOH = 5:1 to obtain 3.98 g of 181-B as a pale yellow oil.

[0371] (2) Synthesis of aminolipid 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 neck flask and stirred at 0°C for 10 minutes. Then potassium carbonate (0.19 g, 1.41 mmol) was added, and M5 (0.99 g, 1.28 mmol, dissolved in 5 ml of THF) was added dropwise. After the dropwise addition was complete, the mixture was allowed to react for 30 minutes. Water (50 ml) and DCM (50 ml) were added to the reaction mixture and extracted. The organic phase was separated, and the aqueous phase was extracted twice with DCM (100 ml). The organic phases were combined, washed with saturated sodium chloride aqueous solution (50 ml), dried on anhydrous sodium sulfate, and filtered. The filtrate was concentrated, and the concentrate was purified by silica gel column chromatography. Elution with EA was performed, and 597 mg of 181-C was obtained as a pale yellow oil, with 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 necked flask, and TEA-3HF (863 mg, 5.36 mmol) was added dropwise while stirring at room temperature. The mixture was reacted with stirring for 1 hour. 10 ml of saturated ammonium chloride aqueous solution was added to the reaction mixture to quench it, and the mixture was 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 sodium chloride aqueous solution, dried on anhydrous sodium sulfate, and filtered. The filtrate was concentrated, and the concentrate was purified by silica gel column chromatography with elution at DCM:MeOH = 20:1 to obtain 236 mg of aminolipid 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 value 881.42, Measured value 882.2.

[0374] Example 104: Synthesis of aminolipid compound 187 [ka]

[0375] Following a general synthesis process, aminolipid compound 187-C was prepared from compound M5 (1.0 g, 1.29 mmol) and 187-B (0.53 g, 1.9 mmol). From 187-C, 382 mg of compound 187 was prepared as a colorless oil with a yield of 33.1% and a purity of 93.90%. 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 value 895.45, Measured value 896.2.

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

[0377] Aminolipid compound 118 can be prepared according to the synthesis method of compound 10 described in Chinese patent application CN107922364A (Example 9); 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 containing luciferase mRNA (Fluc mRNA) (1) Preparation: The Fluc mRNA stock solution, 0.2 M sodium acetate buffer, and DEPC water were added in predetermined amounts to a container and mixed thoroughly to obtain the aqueous phase. The aminolipid compounds, helper lipids, structural lipids, and PEG lipids of this disclosure were separately dissolved in absolute ethanol to prepare solutions at concentrations of 20 mg / mL, 10 mg / mL, 20 mg / mL, and 25 mg / mL, respectively. The above four solutions were pipetted and thoroughly mixed in a molar ratio of lipid:DSPC:CHO-HP:M-DMG-2000 of 48:10:40.5:1.5 to prepare an alcohol phase.

[0379] (2) Encapsulation: Using a microfluidic preparation device (MPE-L2), the aqueous phase and the 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-LNP).

[0380] (3) Dialysis: The product from step (2) was placed in a dialysis bag and placed in Tris Buffer-8% sucrose solution, which was then replaced to remove residual ethanol, uncomposed lipids, and other components. Dialysis was performed at room temperature under light-shielding conditions with magnetic agitation for 2 hours (the dialysate was replaced every hour).

[0381] (4) The product from step (3) was sterilized by passing it through a 0.22 μm microporous membrane and then packaged. Lipid nanoparticle formulations containing fluc mRNA were prepared with a fluc mRNA concentration of 0.2 μg / μL, a mass ratio of fluc mRNA to lipid of 1:10, a particle size of 80-130 nm, and an encapsulation efficiency of 85% or higher.

[0382] Experimental Example 2: Performance Evaluation of In Vivo Delivery of Lipid Nanoparticles Animal preparation: Female BALB / c mice aged 6-8 weeks were selected and housed in SPF-grade animal rooms. Animal experiments were conducted in strict compliance with national medical institution guidelines and animal ethics requirements.

[0383] In vivo delivery: Before injecting the test LNP formulation, the LNP formulation was gently and repeatedly inverted to thoroughly mix the prepared sample. The corresponding amount of the formulation sample was aspirated 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.

[0384] Six hours after injecting the LNP preparation, 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 by 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 body and various organs were observed using an In Vivo Imaging System (IVIS).

[0385] Table 4 shows the encapsulation efficiency of lipid nanoparticles containing luciferase mRNA (Fluc mRNA) using representative aminolipid compounds, and the resulting fluorescence expression intensity, with aminolipid compound 118 as a control.

[0386] [Table 4]

[0387] Table 5 shows the spleen delivery / total delivery ratio of fluorescence expression intensity induced by lipid nanoparticles containing luciferase mRNA (Fluc mRNA) encapsulated in aminolipid compound 118.

[0388] [Table 5]

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

[0390] Experimental Example 3: Evaluation of the delivery performance of lipid nanoparticles to lymph nodes Animal preparation: Female BALB / c mice aged 6-8 weeks were selected and housed in SPF-grade animal rooms. Animal experiments were conducted in strict compliance with national medical institution guidelines and animal ethics requirements.

[0391] In vivo delivery: Before injecting the test LNP formulation, the LNP formulation was gently and repeatedly inverted to thoroughly mix the prepared sample. A considerable amount of the formulation sample was aspirated 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 injecting the LNP preparation, 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 by 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 containing luciferase mRNA (Fluc mRNA) encapsulated in representative aminolipid compounds, with SM-102 as the control.

[0394] [Table 6]

[0395] As can be seen from Table 6, aminolipid compounds 250 and 268 exhibit superior delivery to lymph nodes compared to SM102, demonstrating 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 human erythropoietin mRNA (hEPO mRNA)-encapsulated lipid nanoparticle formulations were prepared with an hEPO mRNA concentration of 0.2 μg / μL, a mass ratio of hEPO mRNA to lipid of 1:10, a particle size of 90-130 nm, and an encapsulation efficiency of 90% or more.

[0397] Animal preparation: Female BALB / c mice aged 6-8 weeks were selected and housed in SPF-grade animal rooms. Animal experiments were conducted in strict compliance with national medical institution guidelines and animal ethics requirements.

[0398] In vivo delivery: Before injecting the test LNP formulation, the LNP formulation was gently and repeatedly inverted to thoroughly mix the prepared sample. A considerable amount of the formulation sample was aspirated into a 1 ml insulin syringe, and the LNP formulation was administered intravenously (IV) to 5 mice per formulation. Each mouse was injected with 300 μL of human erythropoietin mRNA (hEPO mRNA)-encapsulated lipid nanoparticle formulation.

[0399] Serum collection: 12 hours after injection, mouse blood was collected, placed in a tube without anticoagulants, allowed to coagulate naturally at room temperature for 30-60 minutes, then centrifuged at 3500 rpm for 10 minutes to obtain the supernatant, which was used as serum.

[0400] Alanine transaminase detection was performed according to the instructions for the kit (Nanjing Jiancheng Bioengineering Institute, catalog number C009-2-1), and a standard curve was created using the standard sample included in the kit. D-PBS was used in the experiment, which was purchased from Sangon Biotech (Shanghai) Co., Ltd. (catalog number E607009-0600).

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

[0402] Creating ALT standard curves: (1) Enzyme reaction: 5 μL of 0.1 mol / L phosphate buffer was sequentially mixed with 0, 2, 4, 6, 8, and 10 μL of 2 μmol / mL sodium pyruvate standard solution, then supplemented with matrix solution to make a total of 25 μL, and thoroughly mixed by repeatedly aspirating and dispensing with a pipette;

[0403] (2) Addition reaction: Add 20 μL of 2,4-dinitrophenylhydrazine solution to all reaction wells of (1), mix by aspirate and discharge, and then incubate in a 37°C incubator for 20 minutes;

[0404] (3) Development: Add 200 μL of 0.4 mol / L NaOH solution to all reaction wells of (2) to stop the reaction, mix by aspirate and discharge, and incubate 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 corresponding absolute OD value for each well was obtained 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 Karmen units 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, 20 μL of matrix solution was added to the corresponding sample well, and the mixture was mixed by repeatedly aspirating and spitting to avoid creating air bubbles, and then left to stand in a 37°C incubator for 30 minutes; (3) Addition reaction: 20 μL of 2,4-dinitrophenylhydrazine was added to all reaction wells of (2), mixed by aspirate and discharge, and then reacted in an incubator at 37°C for 20 minutes; (4) Development: Add 200 μL of 0.4 mol / L NaOH solution to all reaction wells of (3) to stop the reaction, mix by aspirate and discharge, and incubate at room temperature for 15 minutes. The OD value of each well was measured at a wavelength of 510 nm with a microplate reader; and, (5) Calculation of serum ALT enzyme activity: The absolute OD value of the corresponding sample well was obtained by subtracting the OD value of the control well from the OD value of the obtained sample well, and the ALT enzyme activity (carmen units) of the corresponding serum sample was calculated by substituting this value into the standard curve formula.

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

[0408] As can be seen from Figure 1, compared to commercially available ALC0315 and SM-102, representative aminolipid compounds show equivalent or lower ALT enzyme activity (carmen units), and 252, 255, 259, 260, 263, 264, 266, 267, 270, 272, and 273 show significantly lower ALT enzyme activity (carmen units), making them superior in terms of safety.

[0409] Experimental Example 5: IFN-γ Elispot Cell Immunotherapy 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, a mass ratio of IN002.5.1 mRNA to lipid of 1:10, a particle size of 80-130 nm, and an encapsulation efficiency of 90% or more.

[0410] The mRNA sequence IN002.5.1 is obtained by replacing all uracil (u) in SEQ ID NO.1 with N1-methylpseudridine. It is worth noting that the t (thymine) in the RNA sequence, SEQ ID NO.1, in the sequence listing is actually u (uracil) according to the WIPO standard ST.26 for nucleotide or amino acid sequence listings.

[0411] Animal preparation: Female BALB / c mice aged 6-8 weeks were selected and housed in SPF-grade animal rooms. Animal experiments were conducted in strict compliance with national medical institution guidelines and animal ethics requirements.

[0412] Immunization of mice: Before injecting the test LNP formulation, the LNP formulation was gently and repeatedly inverted to thoroughly mix the compound sample. A considerable amount of the formulation sample was drawn into a 1 ml insulin syringe, and the LNP formulation was injected into eight mice per formulation via intramuscular tail injection (IM). Each mouse was injected with 50 μL of the IN002.5.1 mRNA-encapsulated lipid nanoparticle formulation.

[0413] Spleen harvesting: On day 7 after immunization, 3-4 mice were selected from each LNP immunization group, euthanized, and their spleens were harvested using a super-clean bench.

[0414] Serum collection: On day 14 post-immunization, 150 μL of orbital blood was collected from 5 mice in each LNP immunization group, placed in anticoagulant-free tubes, allowed to coagulate spontaneously at room temperature for 30-60 minutes, then centrifuged at 3500 rpm for 10 minutes, and the supernatant was used as serum.

[0415] Erythropoiesis-24 Lymphocyte Isolation: Mouse spleens were removed in a superclean bench. 7 mL of mouse lymphocyte isolate was added to a 6-well cell culture plate. Mouse splenocytes were crushed with a syringe piston, and the splenocyte suspension was filtered through a cell screen and immediately transferred to a 15 mL centrifuge tube. 1000 μL of RPMI1640 medium was slowly added, separating the liquid interface. Centrifuge at 800 g with a horizontal rotor at room temperature for 30 minutes, resulting in clear stratification. The lymphocyte layer was aspirated, 10 mL of RPMI1640 medium was added, and the tube was inverted and washed. Cells were collected by centrifugation at 250 g at room temperature for 10 minutes, and erythrocytes were lysed. After erythrocyte lysis was complete, the supernatant was poured in, the cells were suspended in culture medium, and counted.

[0416] Stimulant addition and lymphocyte culture: Pre-coated plates were activated with RPMI-1640 medium containing 10% fetal bovine serum. After standing at room temperature for at least 30 minutes, the medium was removed and a cell suspension of adjusted concentration was added (100 μL / well). The medium used for resuspending the cells was used as a background negative control. 10 μL of positive stimulant working solution (PMA + ionomycin (dissolved in DPBS)) was added to the positive control well; 10 μL of the medium used for resuspending the cells was added to the negative control well; and a peptide library diluted with RPMI1640 was added to the experimental wells at 10 μL / well. After adding all samples and stimulants, the plates were capped and incubated in an incubator at 5% CO2, 37°C for 16–24 hours.

[0417] Post-culturing Erispot detection: Discard the cells and culture medium in the wells, and dissolve the wells in 200 μL / well of ice-cold deionized water at 4°C for 10 minutes. Shake the wells and wash five times with PBS buffer. Incubate the wells with 100 μL / well of 1 μg / ml detection antibody at room temperature for 2 hours; wash the plate five times with PBS, then incubate the wells with 100 μL / well of 1000-fold diluted enzyme-labeled avidin (Streptavidin-HRP) at room temperature for 1 hour. Wash the plate five times with PBS, then incubate in the dark at room temperature for 15 minutes with TMB substrate developer equilibrated to room temperature. Discard the developer, wash the front, back and base of the plate 3-5 times with deionized water, and stop development.

[0418] Spot counting on ELISPOT plates: The plates were left at room temperature in a cool, dark place and air-dried before the bottom was sealed. The plates were read using an enzyme-linked immunospot analyzer, and various spot parameters were recorded.

[0419] The results of an IFN-γ erythropoietic cell immunoassay using lipid nanoparticles in which IN002.5.1 mRNA was encapsulated with representative aminolipid compounds are shown in Figures 2, 3, and 4, with ALC0315 as the control.

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

[0421] Experimental Example 6: Humoral Immunity Test of Fully Binding IgG Antibodies Using the serum obtained in Experimental Example 5, a humoral immunoassay for antigen-specific IgG against the expression of IN002.5.1 mRNA was performed.

[0422] Preparation of test reagents: Cleaning solution: Take an appropriate amount of the coating solution, add Tween 20 to a final concentration of 0.05%, and mix thoroughly for later use. Blocking solution: Accurately weigh the BSA and add it to the washing solution to a 3% w / v concentration. Mix thoroughly for later use (use immediately after preparation). Sample Diluent: Accurately measure the BSA and add it to the washing solution to a 1% w / v concentration. Mix thoroughly for later use (use immediately after preparation).

[0423] Operating Procedure Coating: Dilute the coated protein with SARS-CoV-2 antigen protein (Acro, #SPN-C52He) in PBS buffer to the required concentration for the test and mix thoroughly for later use; 100 μL / well, seal with seal film and incubate overnight (16-20 hours) at 2-8°C or at 37°C for 2 hours. Plate washing: After incubation, perform mechanical washing three times with 300 μl / well of washing solution, then pat with clean paper and dry. Blocking: Add 250 μl / well of blocking solution to the ELISA plate, seal the plate with sealing film, and incubate at 37°C for 40-60 minutes. Plate washing: After blocking, wash three times in a washing machine with 300 μl / well of washing solution, then pat dry on clean paper. Sample preparation: The previously separated serum is vortexed and the IgG titer is measured (if storing in a -80°C refrigerator, dissolve it at 4°C beforehand). Serum sample dilution: Collect the isolated serum, determine the initial dilution factor (generally 300-3000 times) based on the difference in immunization time, and perform a total of eight gradient dilutions using this initial dilution factor; add the diluted serum to an ELISA plate at 100 μl / well and incubate at 37°C (incubation time varies depending on the test requirements). Plate washing: After incubation, the plate is washed three times by machine with 300 μl / well of washing solution, then patted with clean paper and dried. Enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody to a certain concentration with the sample diluent to 100 μl / well and incubate at 37°C (incubation time varies depending on the test requirements). Plate washing: After incubation, the plate is mechanically washed three times with 300 μl / well of washing solution, then patted with clean paper and dried. Development: Pre-equilibrium the TMB one-component developer to room temperature, add 100 μl / well to the plate, and incubate at room temperature in the dark. Stop: After development, add 50 μl / well of stop bath. Reading: Select a detection wavelength of 450 nm and a reference wavelength of 630 nm, and read and analyze the data using a microplate reader.

[0424] Calculation result Data analysis was performed using the SoftMax Pro microplate reader to obtain OD data. 450 The calculation was based on the values. The formula for calculating the cutoff value is as follows: Cutoff value = mean OD of negative serum solution 450 ×2.1 Note: OD of negative serum solution 450 If the average value is 0.05 or less, it will be calculated as 0.05. 450 If the average value exceeds 0.05, the actual OD 450 The calculation is performed as a value, and the cutoff value is set to three decimal places. Antibody titers are measured using the OD (Occurrence Disorder) between control serum and test serum. 450The average value was set to the maximum dilution rate corresponding to the cutoff value. The results of a total anti-IgG conjugated humoral immunoassay using lipid nanoparticles in which IN002.5.1 mRNA was encapsulated in representative aminolipid compounds are shown in Figures 5 and 6, with ALC00315 as the control. The test results are shown in Figures 5 and 6. From Figure 5, it can be seen that aminolipid compounds 269, 270, 271, 273, and 274 show superior humoral immune effects compared to commercially available ALC0315. From Figure 6, it can be seen that aminolipid compounds 263, 264, 265, and 266 show superior humoral immune effects compared to commercially available ALC0315.

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

[0426] In addition to those described herein, various modifications to this disclosure will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to be included in the attached claims.

Claims

1. Aminolipid compounds having the structure of formula (I) or their pharmaceutically acceptable salts or stereoisomers: 【Chemistry 1】 Z １ Z ２ Z ４ Each is an independent combination; Z ３ is -CH(OR ７ )-, -C(=O)O-, -OC(=O)-, or bond; Z ５ and Z ６ These are independently -C(=O)O- or -OC(=O)-; A １ 、 A ２ 、 and A ５ are 、 each independently a bond; A ３ A ６ , and A ７ Each of them is independent of C １ -C １２ Alkylene, carbon having 1, 2, 3, 4 or more double bonds ２ -C １２ It is an alkenylene; A ４ C １ -C １２ Alkylene, carbon having 1, 2, 3, 4 or more double bonds ２ -C １２ It is an alkenylene or bond; R １ and R ２ Each of them is independent of C １ -C １８ C having alkyl or 1, 2, 3, 4 or more double bonds ２ -C １８ Is it an alkenil; or R １ and R ２ These atoms, together with the nitrogen atoms to which they are bonded, form a 4- to 7-membered saturated heterocycle having an additional heteroatom of 0 independently selected from the nitrogen atoms and N, O, and S in the ring, wherein the heterocycle is C 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 They are optionally substituted with one, two, three or more substituents independently selected from; R ３ H, C １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ It is an alkenil; R ４ and R ５ Each of them is independent of C １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ It is Alkenil; and R ７ H, C １ -C １２ Alkyl or C having 1, 2, or 3 double bonds ２ -C １２ It is Alkenil.

2. Z ５ and Z ６ The aminolipid compound according to claim 1, wherein both are -C(=O)O-.

3. Z ３ The aminolipid compound according to claim 1, wherein is -C(=O)O- or -OC(=O)-.

4. Z ３ The equation is -C(=O)O-, where C(=O) is A ４ It is connected; R ３ C １ -C １８ Alkyl or C having 1, 2, 3, 4 or more double bonds ２ -C １８ The aminolipid compound according to claim 1, wherein the compound is an alkenyl.

5. A ４ Is it a combination, or A ４ C １ -C １０ The aminolipid compound according to claim 1, which is alkylene.

6. A ３ The aminolipid compound according to claim 1, wherein is a C1-C6 alkylene.

7. The aminolipid compound according to claim 1, wherein A3 is -(CH2)2-, -(CH2)3-, or -(CH2)4-.

8. A ６ and A ７ The aminolipid compound according to claim 1, wherein each of them is independently a C5-C10 alkylene.

9. The aminolipid compound according to claim 1, wherein A6 and A7 are each independently a C6-C9 alkylene.

10. R １ or R ２ The aminolipid compound according to claim 1, wherein the alkyl defined is a C1-C4 alkyl group.

11. The aminolipid compound according to claim 1, wherein the alkyl defined for R1 or R2 is a C1-C2 alkyl.

12. R １ and R ２ The aminolipid compound according to claim 1, wherein each of them is independently a C1-C4 alkyl group.

13. The aminolipid compound according to claim 1, wherein R1 and R2 are each independently a C1-C2 alkyl group.

14. R １ and R ２ together with the nitrogen atom to which they are attached form a 5- to 7-membered saturated heterocycle having in the ring the nitrogen atom and 0 additional heteroatoms independently selected from N, O, and S, and the heterocycle is 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 The amino lipid compound according to claim 1, optionally substituted with 1, 2, or 3 substituents independently selected from

15. R ３ The aminolipid compound according to claim 1, wherein the alkyl defined is a C1-C12 alkyl group.

16. The aminolipid compound according to claim 1, wherein the alkyl group defined for R3 is a C1-C10 alkyl group.

17. R ３ The alkenyl defined for has 1, 2, or 3 double bonds in C ２ -C １２ The aminolipid compound according to claim 1, wherein the compound is an alkenyl.

18. R ４ or R ５ The aminolipid compound according to claim 1, wherein the alkyl defined is a branched C8-C18 alkyl group.

19. The aminolipid compound according to claim 1, wherein the alkyl group defined for R4 or R5 is a branched C11-C18 alkyl group.

20. R ４ and R ５ The aminolipid compound according to claim 1, wherein each of them is independently a branched C8-C18 alkyl group.

21. The aminolipid compound according to claim 1, wherein R4 and R5 are each independently a branched C11-C18 alkyl group.

22. The aminolipid compound according to claim 1, having one of the following structures. Table 1

23. Lipid nanoparticles comprising the aminolipid compound described in claim 1.

24. Furthermore, the lipid nanoparticles according to claim 23 contain a biologically active ingredient.

25. The lipid nanoparticle according to claim 24, wherein the biologically active component is a nucleic acid.

26. A pharmaceutical composition comprising an aminolipid compound according to any one of claims 1 to 22 or lipid nanoparticles according to any one of claims 23 to 25, and a pharmaceutically acceptable carrier, diluent, or excipient.

27. Lipid nanoparticles according to any one of claims 23 to 25 for delivering active ingredients.

28. The lipid nanoparticle according to claim 27, wherein the active ingredient is nucleic acid.

29. A pharmaceutical product for use in nucleic acid introduction, gene therapy, protein replacement therapy, antisense therapy, interfering RNA therapy, or gene vaccination, comprising an aminolipid compound according to any one of claims 1 to 22 or lipid nanoparticles according to any one of claims 23 to 25.

30. A pharmaceutical product comprising the pharmaceutical composition described in Claim 26, for use in nucleic acid introduction, gene therapy, protein replacement therapy, antisense therapy, interfering RNA therapy, or gene vaccination.

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