A carbon-core cationic lipid

Abstract

Provided are a cationic lipid and a preparation method thereof, a lipid composition containing the cationic lipid, and an LNP-drug composition and a preparation thereof containing the lipid composition, especially an LNP-drug composition and a preparation thereof containing the cationic lipid, which have the characteristics of high delivery efficiency and high biocompatibility, thereby improving the therapeutic effect and preventive effect of the drug. The structure of the cationic lipid is shown in general formula (1), and the definitions of the symbols in the formula are as described in the text.

Description

Technical Field

[0001] The present invention belongs to the field of drug delivery, and particularly relates to a cationic lipid for a pharmaceutical carrier, and more particularly to a carbon-branched cationic lipid, a lipid composition, a lipid-drug composition, and their preparations and applications containing the cationic lipid. Background Art

[0002] Drug delivery strategies are generally divided into viral and non-viral delivery vectors, which have gradually matured in the past few decades. Although viral delivery vectors have a high cell transfection rate, due to the rapid clearance of existing antibodies, the generation of neutralizing antibodies against the vector, the limited size of the vector, and possible side effects, the development space of viral vectors in biomedical research and clinical applications is limited.

[0003] In recent years, lipid-based nano-delivery systems are the most widely used non-viral delivery vectors. Among them, LNPs (lipid nanoparticles) are the most popular lipid-based delivery vectors at present. Lipid nanoparticles usually include ionizable cationic lipids, cholesterol, phospholipids, and polyethylene glycolated lipids. Among them, ionizable lipids play a major role in protecting nucleic acids from nuclease degradation. In addition, auxiliary lipids, such as phospholipids and cholesterol, can improve the stability of the preparation and promote membrane fusion. Different from liposomes, LNPs have a micelle structure inside the particle core, showing better kinetic stability and a harder morphology. Large-scale commercial preparation methods, such as microfluidics, can obtain more uniform LNPs. LNPs can effectively deliver drugs (especially nucleic acid drugs). Ionizable cationic lipids have a nearly neutral charge at physiological pH, but carry a partial positive charge due to protonation at low pH, and can electrostatically bind to the negatively charged phosphate groups on nucleic acids, thereby effectively binding and transporting nucleic acid drugs.

[0004] In the field of drug delivery, cationic lipids have achieved certain success. For example, ALC-0315 disclosed in CN108368028A, SM102 disclosed in US9868692B2, and MC3 disclosed in CN102625696A are all cationic lipids with excellent performance. Among them, MC3 contains two hydrophobic fatty long tail chains and is an ionizable carbon nucleus that leads to the polar group tertiary amine; ALC-0315 contains four (2+2 type) hydrophobic fatty long tail chains and is a nitrogen nucleus that leads to the polar group hydroxyl; SM102 contains three (1+2 type) hydrophobic fatty long tail chains and is a nitrogen nucleus that leads to the polar group hydroxyl. Although these cationic lipids have achieved success, in order to enrich the types of cationic lipids, it is necessary to continue to develop cationic lipids and develop new cationic lipids that can be substituted or even have better effects. Summary of the Invention

[0005] To solve the above problems, the present invention provides a novel cationic lipid and its preparation method, a lipid composition containing the cationic lipid, a lipid drug composition and its preparation containing the lipid composition, and a liposome or lipid nanoparticle containing the lipid composition, especially an LNP-nucleic acid drug composition and its preparation containing the cationic lipid, which have the advantages of high delivery efficiency, low safety and toxicity, and high biocompatibility, and can improve the therapeutic and / or preventive effects of drugs.

[0006] The above object of the present invention is achieved by the following technical solutions:

[0007] An embodiment of the present invention provides a cationic lipid:

[0008] A cationic lipid, characterized in that the structure is shown in the general formula (1):

[0009]

[0010] Wherein, each occurrence of X is independently N or CR a , said R a is H or C 1-12 alkyl;

[0011] L 1 is -L c R, L c is a linking bond or a divalent linking group, and R is C 1-30 aliphatic hydrocarbon group; wherein, the aliphatic hydrocarbon group is alkyl, alkenyl or alkynyl;

[0012] L 2 is a linking bond or a divalent linking group;

[0013] L 5 is a linking bond or a divalent linking group;

[0014] L 3 and L 4 are each independently a linking bond or a divalent linking group;

[0015] B 1 and B 2 are each independently a linking bond or C 1-30 alkylene;

[0016] R 1 、R 2 are each independently C 1-30 aliphatic hydrocarbon group or Wherein, t is an integer from 0 to 12, t 1 、t 2 are each independently an integer from 0 to 5, t 3 、t 4Each independently is 0 or 1, and t 1 、t 2 、t 3 、t 4 are not all 0 at the same time; R e 、R f Each independently is any one of C 1-15 alkyl, C 2-15 alkenyl and C 2-15 alkynyl;

[0017] R 3 is a hydrogen atom, -R d 、-OR d 、C 3-6 carbocyclic group, nitrogen-containing heterocycle, -NR d R d 、-SR d 、-C(=O)R d 、-C(=O)OR d 、-OC(=O)R d 、-OC(=O)OR d or a functional group R 01 that can react with a biologically relevant substance; wherein, R d is C 1-12 alkyl;

[0018] or a pharmaceutically acceptable salt, tautomer, stereoisomer or solvate thereof.

[0019] The present invention also provides a lipid composition, and the embodiment is:

[0020] A lipid composition containing a cationic lipid having the structure shown in formula (1).

[0021] The present invention also provides a lipid pharmaceutical composition, and the embodiment is as follows:

[0022] A lipid pharmaceutical composition containing a lipid composition and a drug, and the lipid composition contains a cationic lipid having the structure shown in formula (1), and the drug is selected from any one of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs.

[0023] The present invention also provides a preparation of a lipid pharmaceutical composition, and the embodiment is as follows:

[0024] A preparation of a lipid pharmaceutical composition containing the aforementioned lipid pharmaceutical composition and a pharmaceutically acceptable diluent or excipient.

[0025] The present invention also provides a liposome or lipid nanoparticle, and the embodiment is as follows:

[0026] A liposome or lipid nanoparticle containing a lipid composition, wherein the lipid composition contains a cationic lipid having the structure shown in formula (1).

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention provides a new cationic lipid compound, enriching the types of cationic lipid compounds and providing more choices for the delivery of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides, protein drugs, etc.

[0029] The novel cationic lipid of the present invention contains four or five hydrophobic tail chains, and the arrangement of the four hydrophobic tail chains is of the "1 + 2 + 1" type or "2 + 2 + 1" type, with a looser arrangement and an increased cross-section in the tail region, which can produce a conical structure, helping to improve membrane instability and enhance endosomal escape, and promoting the release of drugs into the cytoplasm to play corresponding roles.

[0030] The novel cationic lipid of the present invention contains an ionizable tertiary amine structure and a polar short head group derived from a carbon core. Compared with the carbon core lipids in the prior art (such as MC3), it contains both an ionizable tertiary amine structure and a polar short head group, enhancing the binding of the ionizable cationic lipid to drugs (such as negatively charged nucleic acid drugs), thereby improving the drug delivery efficiency.

[0031] For the novel cationic lipid of the present invention, one fatty tail chain and the polar head group derived from the carbon core can be obtained simultaneously by the ring-opening of an epoxide, and the preparation process is simple and efficient.

[0032] The cationic liposome nucleic acid drug composition prepared from the novel cationic lipid of the present invention has high serum stability, strong gene complexing ability, high biocompatibility, and strong gene transfection effect, which helps to improve the gene therapy effect of drugs, especially the gene therapy effect of nucleic acid drugs.

[0033] The terminal of the novel cationic lipid of the present invention can also be conjugated with a fluorescent group or a targeting group, and the lipid composition and lipid drug composition prepared from the cationic lipid can have both fluorescent and targeting functions, further improving the treatment and / or diagnostic effect.

[0034] Embodiments

[0035] Term Explanation

[0036] In the present invention, unless otherwise specified, each term has the following meanings.

[0037] In the present invention, when the structure involved has isomers, any one of the isomers may be used without special designation. For example, for a structure with cis-trans isomers, it may be either the cis structure or the trans structure; for a structure with E / Z isomers, it may be either the E structure or the Z structure; when there is optical activity, it may be either the left-handed or the right-handed form.

[0038] In the present invention, the interpretation of a numerical range includes both the numerical range marked with a short dash (such as 1-6) and the numerical range marked with a wavy line (such as 1~6). In the present invention, unless otherwise specified, an integer range marked in interval form can represent the set of all integers within that range, and the range includes both endpoints. For example, the integer range 1-6 represents the set consisting of 1, 2, 3, 4, 5, and 6. The numerical ranges in the present invention include, but are not limited to, numerical ranges represented by integers, non-integers, percentages, and fractions, and unless otherwise specified, both endpoints are included.

[0039] In the present invention, the numerical values involving "about" or "around" generally refer to a numerical range of ±10%, and in some cases, it can be enlarged to ±15%, but not exceeding ±20%, based on the preset numerical value. For example, if the molar percentage of steroid lipids in the total lipids in a solution containing a solvent is about 40%, it is generally considered to include the case where the molar percentage of steroid lipids is 30%-50%.

[0040] In the present invention, "pharmaceutically acceptable salts" refer to salts of the compounds of the present invention that are recognized for use in animals and, more particularly, in humans, including salts formed from the compounds represented by formula (1) and inorganic or organic acids. For example, see S.M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci. 1977, 66, 1-19. Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid, etc.; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, etc. For example, HCl (or hydrochloric acid), HBr (or hydrobromic acid solution), methanesulfonic acid, sulfuric acid, tartaric acid or fumaric acid can be used to form pharmaceutically acceptable salts with the compounds represented by formula (1).

[0041] In the present invention, "solvate" refers to a complex formed by combining a compound of formula (1) or a pharmaceutically acceptable salt thereof and a solvent (such as ethanol or water). It should be understood that any solvate of a compound of formula (1) used in the treatment of a disease or disorder, although it may provide different properties (including pharmacokinetic properties), once absorbed into the subject, the compound of formula (1) will be obtained, such that the use of the compound of formula (1) respectively encompasses the use of any solvate of the compound of formula (1). It should be further understood that the compound of formula (1) or a pharmaceutically acceptable salt thereof can be separated in the form of a solvate, and thus any such solvate is included within the scope of the present invention. For example, the compound of formula (1) or a pharmaceutically acceptable salt thereof can exist in an unsolvated form and in a solvated form formed with a pharmaceutically acceptable solvent (such as water, ethanol, etc.).

[0042] In the present invention, unless otherwise specified, the terms "comprising", "including" and "containing" and similar expressions shall be interpreted in an open and inclusive sense as "including but not limited to" in this specification and the claims.

[0043] In the present invention, for two or more objects, "each independently preferably", when there are multiple levels of preference, it is not required that they are all selected from the same level of preferred groups. One can be a preference in a large range, one can be a preference in a small range, one can be the maximum range, and the other can be any kind of preference, or they can be selected from the same level of preference.

[0044] In the present invention, for the "divalent linking group", such as alkylene, alkylidene, arylene, amide bond, etc., without special limitation, when connecting other groups, either of the two connecting ends can be selected. For example, when using an amide bond as the divalent linking group between C-CH 2 CH 2 - and -CH 2 -D, it can be C-CH 2 CH 2 -C(=O)NH-CH 2 -D or C-CH 2 CH 2 -NHC(=O)-CH 2 -D.

[0045] In the structural formula of the present invention, when the end group of the linking group is prone to confusion with the substituents contained in the linking group, is used to mark the positions where other groups are connected in the linking group. For example, in the structural formula , the is used to mark the two positions where other groups are connected in the divalent linking group. The aforementioned two structural formulas respectively represent -CH(CH 2 CH 2 CH 3 ) 2 -, -CH 2 CH 2 CH(CH 3 ) 2 -CH 2 CH 2 -.

[0046] In the present invention, the range of the number of carbon atoms in the group is marked in subscript form at the subscript position of C, indicating the number of carbon atoms in the group. For example, C 1-12 represents "having 1 to 12 carbon atoms", and C 1-30 represents "having 1 to 30 carbon atoms". "Substituted C 1-12 alkyl" refers to the group obtained by substituting the hydrogen atoms of C 1-12 alkyl. "C 1-12 substituted alkyl" refers to the group having 1 - 12 carbon atoms obtained after substituting the hydrogen atoms of the alkyl. For another example, when a group can be selected from C 1-12 alkylene, it can be selected from any alkylene with the number of carbon atoms within the range indicated by the subscript, that is, it can be selected from C 1, C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 , C 12 Any one of the alkylene groups. In the present invention, unless otherwise specified, subscripts marked in the form of an interval represent any integer selected from within the range, and the range includes both endpoints.

[0047] In the present invention, the heteroatom is not particularly limited and includes, but is not limited to, O, S, N, P, Si, F, Cl, Br, I, B, etc.

[0048] In the present invention, the heteroatom used for substitution is referred to as a "substituting atom", and any group used for substitution is referred to as a "substituent".

[0049] In the present invention, "substituted" means that at least one hydrogen atom in any group (for example, an aliphatic hydrocarbon group, a hydrocarbon group, an alkyl group or an alkylene group) is replaced by a bond connected to a non-hydrogen atom, and the non-hydrogen atom is, for example, but not limited to: halogen atoms such as F, Cl, Br and I; an oxo group (=O); a hydroxyl group (-OH); a hydrocarbyloxy group (-OR d , where R d is C 1-12 alkyl); a carboxyl group (-COOH); an amine group (-NR c R c , where two R c are each independently H, C 1-12 alkyl); C 1-12 alkyl and cycloalkyl. In some embodiments, the substituent is C 1-12 alkyl. In other embodiments, the substituent is cycloalkyl. In other embodiments, the substituent is a halogenated group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is a hydrocarbyloxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0050] In the present invention, "optional" or "optionally" (for example, optionally substituted) means that the described subsequent event or situation may or may not occur, and the description includes instances where the event or situation occurs and instances where the event or situation does not occur. For example, "optionally substituted hydrocarbon group" means that the hydrocarbon group may or may not be substituted, and the description includes substituted hydrocarbon groups and unsubstituted hydrocarbon groups.

[0051] In the present invention, the "carbon chain linking group" refers to a linking group in which all the main chain atoms are carbon atoms, and the side chain part allows heteroatoms or groups containing heteroatoms to replace the hydrogen atoms of the main chain carbon. When the "main chain atom" is a heteroatom, it is also called a "main chain heteroatom", such as A-S-CH 2 -B, A-O-CH 2 -B, (the atomic interval is denoted as 4) is regarded as containing a main chain heteroatom. The carbon chain linking group can be divided into an alkylene group and a carbon chain linking group with a heteroatom-containing side group; the heteroatom-containing side group includes but is not limited to oxo (=O), thio (=S), amino (linked to the main chain carbon through a carbon-nitrogen double bond), an oxahydrocarbyl group in the form of an ether bond, a thiahydrocarbyl group in the form of a thioether bond, a nitrogen-containing hydrocarbyl group in the form of a tertiary amino group, etc. The "carbon chain linking group" has a main chain entirely composed of carbon atoms, and the side group of the carbon chain is allowed to contain heteroatoms. That is, it is formed by connecting methylene groups or substituted methylene groups. The substituted methylene group can be substituted by a monovalent substituent, two monovalent substituents or a divalent substituent (such as divalent oxygen, such as forming a three-membered ring together with the divalent methylene )). The substituted methylene group can be one hydrogen atom substituted (such as -CH(CH 3 ))-), or two hydrogen atoms can be substituted separately (such as -(CH 3 )C(OCH 3 ))-), or two hydrogen atoms can be substituted simultaneously (such as carbonyl, thiocarbonyl, -C(=NH)-, -C(=N + H 2 ))-), or it can also be a cyclic side group (such as the atomic interval is denoted as 1).

[0052] In the present invention, for a compound or a group, it can be substituted and hybridized simultaneously. For example, a nitrobenzene group substitutes a hydrogen atom, or another example, -CH 2 -CH 2 -CH 2 - is replaced by -CH 2 -S-CH(CH 3 ))-. In the present invention, the "linking bond" is a connecting part that only plays a connecting role and does not contain any atoms. When a certain group is defined as a linking bond, it means that this group can be absent.

[0053] In the present invention, "each independently at each occurrence" not only means that in different groups, they can each independently be any option in the definition, but also means that when they appear at different positions in the same group, they can also each independently be any option in the definition. For example, -Z-L 6-Z- in, "Z is each independently, every time it appears, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-, -C(=O)NR c -, -NR c C(=O)NR c -, -OC(=O)NR c -, -NR c C(=O)O-, -SC(=O)NR c -, and -NR c C(=O)S- any one of them, where R c is each independently, every time it appears, a hydrogen atom or C 1-12 alkyl", in the "-Z-L 6 -Z-" group, the two Z groups can be the same or different, in the group "-NR c C(=O)NR c -", the two R c can be the same or different, each independently being a hydrogen atom or C 1-12 alkyl.

[0054] The "group" in the present invention contains at least 1 atom, referring to a radical formed by a compound losing one or more atoms. Relative to a compound, the group formed after losing a partial group is also called a residue. The valence state of the group is not particularly limited, and by way of example, it can be classified into monovalent groups, divalent groups, trivalent groups, tetravalent groups,..., 100-valent groups, etc. Among them, groups with a valence state greater than or equal to 2 are collectively called linking groups. A linking group can also contain only one atom, such as an oxygen group or a sulfur group.

[0055] In the present invention, "hydrocarbon" refers to a hydrocarbon compound composed of carbon atoms and hydrogen atoms.

[0056] In the present invention, according to the type of hydrocarbon group, hydrocarbons are divided into two types: aliphatic hydrocarbons and aromatic hydrocarbons. A hydrocarbon that does not contain a benzene ring or any one of the structures of a benzene ring substituted by a hydrocarbon group is defined as an aliphatic hydrocarbon. A hydrocarbon that contains at least one benzene ring or a benzene ring substituted by a hydrocarbon group is defined as an aromatic hydrocarbon. And aromatic hydrocarbons can contain an aliphatic hydrocarbon group structure, such as toluene, diphenylmethane, indane, etc.

[0057] In the present invention, according to the saturation situation, hydrocarbons are divided into two types: saturated hydrocarbons and unsaturated hydrocarbons. All aromatic hydrocarbons are unsaturated hydrocarbons. Saturated aliphatic hydrocarbons are also called alkanes. The degree of unsaturation of unsaturated aliphatic hydrocarbons is not particularly limited. By way of example, it includes but is not limited to alkenes (containing double bonds), alkynes (containing triple bonds), dienes (conjugated double bonds), etc. When the aliphatic hydrocarbon part in an aromatic hydrocarbon is a saturated structure, it is also called an arylalkane, such as toluene.

[0058] In the present invention, there is no particular limitation on the structure of the hydrocarbon, which may be a straight-chain structure without side groups, a branched-chain structure with side groups, a cyclic structure, a dendritic structure, a comb structure, a hyperbranched structure, etc. In the absence of a specific definition, a straight-chain structure without side groups, a branched-chain structure with side groups, and a cyclic structure are preferably selected, corresponding to straight-chain hydrocarbons, branched-chain hydrocarbons, and cycloalkanes, respectively. Among them, hydrocarbons without a cyclic structure are collectively referred to as open-chain hydrocarbons, including but not limited to a straight-chain structure without side groups and a branched-chain structure with side groups. Open-chain hydrocarbons belong to aliphatic hydrocarbons. Therefore, straight-chain hydrocarbons can also be referred to as straight-chain aliphatic hydrocarbons. Branched-chain hydrocarbons can also be referred to as branched-chain aliphatic hydrocarbons.

[0059] In the present invention, a compound formed by substituting a carbon atom at any position in a hydrocarbon with a heteroatom is collectively referred to as a hetero-hydrocarbon.

[0060] In the present invention, the "hydrocarbyl group" refers to a residue formed after a hydrocarbon loses at least one hydrogen atom. According to the number of hydrogen atoms lost, it can be divided into monovalent hydrocarbyl groups (losing one hydrogen atom), divalent hydrocarbyl groups (losing two hydrogen atoms, also known as alkylene groups), trivalent hydrocarbyl groups (losing three hydrogen atoms), and so on. By analogy, when losing n hydrogen atoms, the valence state of the formed hydrocarbyl group is n. In the absence of a specific designation, the hydrocarbyl group in the present invention specifically refers to a monovalent hydrocarbyl group. Unless otherwise expressly stated in this specification, the hydrocarbyl group is optionally substituted.

[0061] In the present invention, there is no particular limitation on the source of the hydrocarbyl group. For example, it may be derived from aliphatic hydrocarbons or aromatic hydrocarbons, may also be derived from saturated hydrocarbons or unsaturated hydrocarbons, may also be derived from straight-chain hydrocarbons, branched-chain hydrocarbons or cycloalkanes, and may also be derived from hydrocarbons or hetero-hydrocarbons, etc. From the perspective of saturation, for example, it may be derived from alkanes, alkenes, alkynes, dienes, etc.; for cycloalkanes, for example, it may be derived from alicyclic hydrocarbons or aromatic hydrocarbons, monocyclic hydrocarbons or polycyclic hydrocarbons; for heterocyclic hydrocarbons, for example, it may be derived from aliphatic heterocyclic hydrocarbons or aromatic heterocyclic hydrocarbons.

[0062] In the present invention, the "aliphatic hydrocarbyl group" refers to a residue formed after an aliphatic hydrocarbon loses at least one hydrogen atom. In the absence of a specific designation, the aliphatic hydrocarbyl group in the present invention specifically refers to a monovalent aliphatic hydrocarbyl group. The aliphatic hydrocarbyl group includes saturated aliphatic hydrocarbyl groups and unsaturated aliphatic hydrocarbyl groups. Unless otherwise expressly stated in this specification, the aliphatic hydrocarbyl group is optionally substituted.

[0063] In the present invention, the "alkyl group" refers to a hydrocarbyl group formed from an alkane. In the absence of a specific designation, it refers to a hydrocarbyl group formed by losing a hydrogen atom at any position, which may be straight-chain or branched-chain, and may be substituted or unsubstituted. Specifically, for example, propyl refers to either n-propyl or isopropyl, and propylene refers to any one of 1,3-propylene, 1,2-propylene, and isopropylene. Unless otherwise expressly stated in this specification, the alkyl group is optionally substituted.

[0064] In the present invention, the "unsaturated hydrocarbon group" refers to a hydrocarbon group formed by an unsaturated hydrocarbon losing a hydrogen atom. The hydrocarbon group formed by an unsaturated hydrocarbon losing a hydrogen atom on an unsaturated carbon can be divided into alkenyl, alkynyl, diene-based groups, etc., specifically such as propenyl and propynyl. The hydrocarbon group formed by an unsaturated hydrocarbon losing a hydrogen atom on a saturated carbon is called an olefin-based group, an alkyne, a diene-based group, etc. according to the different unsaturated bonds, specifically such as allyl and propargyl.

[0065] In the present invention, the "alkenyl" or "alkenyl group" means a substituted or unsubstituted straight-chain or branched-chain alkenyl group including two or more carbon atoms (for example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon double bond. The label "C 2-15 alkenyl" means a substituted or unsubstituted straight-chain or branched-chain alkenyl group including 2-15 carbon atoms and at least one carbon-carbon double bond, that is, the alkenyl group can include one, two, three, four or more carbon-carbon double bonds. Unless otherwise specifically stated, the alkenyl groups described herein refer to both unsubstituted and substituted alkenyl groups. Unless otherwise explicitly stated in this specification, the alkenyl group is optionally substituted.

[0066] In the present invention, the "alkynyl" or "alkynyl group" means an optionally substituted straight-chain or branched-chain hydrocarbon including two or more carbon atoms (for example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon triple bond. The label "C 2-15 alkynyl" means a substituted or unsubstituted straight-chain or branched-chain alkynyl group including 2-15 carbon atoms and at least one carbon-carbon triple bond. The alkynyl group can include one, two, three, four or more carbon-carbon triple bonds. Unless otherwise specifically stated, the alkynyl groups described herein refer to both unsubstituted and substituted alkynyl groups. Unless otherwise explicitly stated in this specification, the alkynyl group is optionally substituted.

[0067] In the present invention, the "alkylene" or "alkylene chain" refers to a straight-chain or branched-chain divalent hydrocarbon chain that connects the remaining part of the molecule to a radical group, which consists only of carbon and hydrogen and is saturated or unsaturated. For example, an alkylene group (C 1-24 alkylene) having one to twenty-four carbon atoms, an alkylene group (C 1-12 alkylene) having one to twelve carbon atoms, specifically, for example, methylene, ethylene, propylene, n-butylene, vinylidene, propenylene, n-butenylene, propynylene, n-butynylene, etc. Unless otherwise explicitly stated in this specification, the alkylene group is optionally substituted.

[0068] In the present invention, "alkylene" refers to divalent alkyl, including acyclic alkylene and divalent cycloalkyl. Acyclic alkylene refers to divalent alkyl without a cyclic structure, and divalent cycloalkyl refers to divalent alkyl with a cyclic structure. Unless otherwise specifically stated in this specification, alkylene is optionally substituted.

[0069] In the present invention, the aliphatic hydrocarbon derivative is preferably an ether-derivatized aliphatic hydrocarbon, an aliphatic hydrocarbon derivative containing 1-2 ether bonds, and more preferably an aliphatic hydrocarbon derivative containing 2 ether bonds.

[0070] In the present invention, "molecular weight" characterizes the mass of a compound molecule. When not specifically stated, the measurement unit of "molecular weight" is Dalton, Da.

[0071] In the present invention, for percentages, "about" generally means ±0.5%.

[0072] In the present invention, "stable existence" and "degradability" of a group are relative concepts. For detailed examples of groups with stable existence and degradable groups, see paragraphs

[0134] -

[0145] in CN113402405A.

[0073] In the present invention, "hydroxy protecting group" includes all groups that can be used as common protecting groups for hydroxyl groups. Hydroxy protecting groups are preferably alkanoyl groups (such as acetyl, tert-butylcarbonyl), aralkanoyl groups (such as benzoyl), benzyl, trityl, trimethylsilyl, tert-butyldimethylsilyl, allyl, acetal group or ketal group. The removal of acetyl is generally carried out under basic conditions, and the most commonly used are ammonolysis with NH 3 / MeOH and methanolysis catalyzed by methoxide anion; benzyl can be easily removed by palladium-catalyzed hydrogenolysis at room temperature in a neutral solution, and can also be reductively cleaved with sodium metal in ethanol or liquid ammonia; trityl is generally removed by catalytic hydrogenolysis; trimethylsilyl is usually removed using a fluoride ion-containing reagent (such as tetrabutylammonium fluoride / anhydrous THF, etc.); tert-butyldimethylsilyl ether is relatively stable and can withstand the ester hydrolysis conditions of alcoholic potassium hydroxide and mild reduction conditions (such as Zn / CH 3 OH, etc.), and can be removed with fluoride ions (such as Bu 4 N + F - ) in a tetrahydrofuran solution, or can also be removed with acetic acid containing water at room temperature.

[0074] In the present invention, "carboxy protecting group" refers to a protecting group that can be converted into a carboxy group through hydrolysis and deprotection reaction of the carboxy protecting group. Carboxy protecting groups are preferably alkyl groups (such as methyl, ethyl, tert-butyl) or aralkyl groups (such as benzyl), and more preferably tert-butyl (tBu), methyl (Me) or ethyl (Et).

[0075] In the present invention, the "protected carboxyl group" refers to the group formed after the carboxyl group is protected by a suitable carboxyl protecting group, preferably methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, or benzyloxycarbonyl. The carboxyl protecting group can be removed by hydrolysis under the catalysis of an acid or a base, and occasionally can also be removed by a pyrolysis reaction. For example, the tert-butyl group can be removed under mild acidic conditions, and the benzyl group can be removed by hydrogenolysis. The reagent for removing the carboxyl protecting group is selected from TFA, H 2 O, LiOH, NaOH, KOH, MeOH, EtOH, and combinations thereof, preferably the combination of TFA and H 2 O, the combination of LiOH and MeOH, or the combination of LiOH and EtOH. The protected carboxyl group is deprotected to produce the corresponding free acid, and the deprotection is carried out in the presence of a base, and the base and the free acid formed by the deprotection form a pharmaceutically acceptable salt.

[0076] In the present invention, the "amino protecting group" includes all groups that can be used as protecting groups for a normal amino group, such as aryl C 1-6 alkyl, C 1-6 alkoxy C 1-6 alkyl, C 1-6 alkoxycarbonyl, aryloxycarbonyl, C 1-6 alkylsulfonyl, arylsulfonyl, or silyl, etc. The amino protecting group is preferably Boc (tert-butoxycarbonyl), Moz (p-methoxybenzyloxycarbonyl), or Fmoc (9-fluorenylmethyloxycarbonyl). The reagent for removing the amino protecting group is selected from TFA, H 2 O, LiOH, MeOH, EtOH, and combinations thereof, preferably the combination of TFA and H 2 O, the combination of LiOH and MeOH, or the combination of LiOH and EtOH. The reagent for removing the Boc protecting group is TFA or HCl / EA; preferably TFA. The deprotecting agent used for the reaction of removing the Fmoc protecting group is a solution of N,N-dimethylformamide (DMF) containing 20% piperidine.

[0077] In the present invention, a "cation" means that the corresponding structure is permanently or non-permanently positively charged in response to certain conditions (such as pH). Thus, cations include both permanent cations and cationizable ones. A permanent cation means that the corresponding compound, group or atom is positively charged at any pH value or hydrogen ion activity in its environment. Typically, the positive charge is generated due to the presence of a quaternary nitrogen atom. When a compound carries multiple such positive charges, it can be called a permanent cation. Cationizable means that a compound, group or atom is positively charged at a lower pH and uncharged at a higher pH in its environment. Additionally, in a non-aqueous environment where the pH value cannot be measured, a cationizable compound, group or atom is positively charged at a high hydrogen ion concentration and uncharged at a low hydrogen ion concentration or activity. It depends on the individual properties of the cationizable or polycationizable compound, especially the pKa of the corresponding cationizable group or atom, at which it is charged or uncharged at the said pH or hydrogen ion concentration. In a dilute aqueous environment, the so-called Henderson-Hasselbalch equation can be used to estimate the fraction of the cationizable compound, group or atom that is positively charged, which is well known to those skilled in the art. For example, in some embodiments, if a compound or moiety is cationizable, preferably, it is positively charged at a pH value of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, more preferably at a pH value equal to or lower than 9, equal to or lower than 8, equal to or lower than 7, and most preferably at a physiological pH value (such as about 7.3 to 7.4), i.e., under physiological conditions, especially under the physiological conditions of cells in vivo. In other embodiments, preferably, the cationizable compound or moiety is mainly neutral at a physiological pH value (such as about 7.0 - 7.4), but becomes positively charged at a lower pH value. In some embodiments, the preferred range of the pKa of the cationizable compound or moiety is about 5 to about 7.

[0078] In the present invention, "cationic lipid" refers to a lipid that contains an overall positive charge or an ionizable lipid. In addition to those shown in the general structural formula (1) of the present invention, cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 3-(dilauryldimethylammonio)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(dilauryldimethylammonio)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-bis(tridecyl)-15,18,21,24-tetraaza-tritriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino)butyric acid heptatriaconta-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), and 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), nonadec-9-yl 8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate) (SM102), any one of them and their mixtures.

[0079] In the present invention, "PEGylated lipid" refers to a molecule that contains a lipid moiety and a polyethylene glycol moiety.

[0080] In the present invention, "neutral lipid" refers to any one of many lipid substances that exist in a non-charged or neutral zwitterionic form at a selected pH, preferably a phospholipid, which can be synthetic or of natural origin.

[0081] In the present invention, "steroid lipid" is a steroid or a steroid analogue.

[0082] In the present invention, a variant form refers to a structural form that can be transformed into a target reactive group through any one of the following chemical change processes: oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, deprotonation, substitution, deprotection, changing the leaving group, etc.

[0083] In the present invention, the "variant form of a reactive group" refers to a form that remains active (is still a reactive group) after undergoing at least one chemical change process such as oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, deprotonation, substitution, deprotection, change of leaving group, etc., or an inactive form after being protected.

[0084] In the present invention, "micro-modification" refers to a chemical modification process that can be completed through a simple chemical reaction process. The simple chemical reaction process mainly refers to chemical reaction processes such as deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and transformation of leaving groups. The "micro-variant form" corresponds to "micro-modification" and refers to a structural form that can form a target reactive group after undergoing simple chemical reaction processes such as deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and transformation of leaving groups. The transformation of the leaving group, such as the transformation from an ester form to an acyl chloride form.

[0085] In the present invention, the "N / P ratio" refers to the molar ratio of ionizable nitrogen atoms in the cationic lipid to phosphoric acid in the nucleic acid.

[0086] In the present invention, "nucleic acid" refers to DNA or RNA or a modified form thereof.

[0087] In the present invention, "RNA" refers to ribonucleic acid that may be naturally occurring or non-naturally occurring. For example, RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA may include a cap structure, chain-terminating nucleosides, stem-loops, polyadenylation sequences, and / or polyadenylation signals. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translating mRNA encoding a specific polypeptide, such as translating mRNA inside mammalian cells in vivo, can produce the encoded polypeptide. RNA may be selected from the non-limiting group consisting of: small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single-stranded guide RNA (sgRNA), self-amplifying RNA (saran), circular RNA (circRNA), cas9 mRNA, and mixtures thereof.

[0088] In the present invention, FLuc mRNA can express luciferase protein, which emits bioluminescence in the presence of luciferin substrate, so FLuc is commonly used in mammalian cell culture to measure gene expression and cell viability.

[0089] In the present invention, the methods for determining the expression level of a target gene include, but are not limited to, dot blot, northern blot, in situ hybridization, ELISA, immunoprecipitation, enzymatic action, and phenotypic determination.

[0090] In the present invention, "transfection" refers to the introduction of a substance (such as RNA) of one species into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0091] In the present invention, an "antigen" refers to a substance that can be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, for example, by forming antibodies and / or antigen-specific T cells as part of the adaptive immune response. Typically, an antigen can be or can contain a peptide or protein that can be presented by MHC to T cells. In the context of the present invention, an antigen can be the translation product of the provided nucleic acid molecule (preferably mRNA as defined herein). In this context, fragments, variants, and derivatives of peptides and proteins that contain at least one epitope are also understood as antigens.

[0092] In the present invention, "delivery" refers to the provision of an entity to a target. For example, delivering a drug and / or a therapeutic agent and / or a prophylactic agent to a subject, which is a tissue and / or cell of a human and / or other animal.

[0093] In the present invention, a "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle administered together with a therapeutic agent, and is suitable for contacting tissues of humans and / or other animals within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic reaction or other problems or complications corresponding to a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is an exemplary carrier. Normal saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, especially for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, etc. The composition may also optionally contain small amounts of wetting agents, emulsifying agents or pH buffering agents. Oral preparations may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate, etc. Specifically, for example, excipients include, but are not limited to, anti-adhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), demulcents, emulsifiers, fillers (diluents), film formers or coatings, flavoring agents, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweetening agents, and water for hydration. More specifically, excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, croscarmellose sodium, crospovidone, citric acid, cross-linked polyvinylpyrrolidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, phenylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, xylitol.

[0094] The pharmaceutical compositions of the present invention can act systemically and / or locally. For this purpose, they can be administered by suitable routes, such as by injection (e.g., intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including infusion) or transdermal administration; or by oral, buccal, nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation. For these routes of administration, the pharmaceutical compositions of the present invention can be administered in suitable dosage forms. The dosage forms include, but are not limited to, tablets, capsules, lozenges, troches, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

[0095] In the present invention, a "vaccine" is a prophylactic or therapeutic material that provides at least one antigen or antigenic function. The antigen or antigenic function can stimulate the adaptive immune system of the body to provide an adaptive immune response.

[0096] In the present invention, "treatment" refers to the treatment and care of a patient to combat a disease, disorder or condition, and is intended to include delaying the progression of the disease, disorder or condition, alleviating or mitigating symptoms and complications, and / or curing or eliminating the disease, disorder or condition. The patient to be treated is preferably a mammal, especially a human. Detailed Description of the Invention

[0098] One embodiment of the present invention is as follows:

[0099] 1.1. A cationic lipid, characterized in that its structure is shown in the general formula (1):

[0100]

[0101] Wherein, each occurrence of X is independently N or CR a , said R a is H or C 1-12 alkyl;

[0102] L 1 is -L c R, L c is a linking bond or a divalent linking group, and R is C 1-30 aliphatic hydrocarbon group; wherein, the aliphatic hydrocarbon group is an alkyl group, an alkenyl group or an alkynyl group;

[0103] L 2 is a linking bond or a divalent linking group;

[0104] L 5 is a linking bond or a divalent linking group;

[0105] L 3 and L 4 are each independently a linking bond or a divalent linking group;

[0106] B 1 and B 2 are each independently a linking group or C 1-30 alkylene;

[0107] R 1 、R 2 are each independently C 1-30 aliphatic hydrocarbon group or wherein, t is an integer from 0 to 12, t 1 、t 2 are each independently an integer from 0 to 5, t 3 、t 4 are each independently 0 or 1, and t 1 、t 2 、t 3 、t 4 are not simultaneously 0; R e 、R f are each independently C 1-15 alkyl, C 2-15 alkenyl and C 2-15 alkynyl, any one of them;;

[0108] R 3 is a hydrogen atom, -R d 、-OR d 、C 3-6 carbocyclic group, nitrogen-containing heterocycle, -NR d R d 、-SR d 、-C(=O)R d 、-C(=O)OR d 、-OC(=O)R d 、-OC(=O)OR d or a functional group R that can react with a biologically relevant substance 01 ; wherein, R d is C 1-12 alkyl;

[0109] or a pharmaceutically acceptable salt, tautomer, stereoisomer or solvate thereof.

[0110] 1.1.1.X

[0111] In the present invention, each occurrence of X is independently N or CR a , wherein, R a is H or C 1-12 alkyl.

[0112] 1.1.2.L 1 、L 2 、L 3 、L 4 、L5 、L 6 、L 7 、Z

[0113] In the present invention, the structures of L 1 、L 2 、L 3 、L 4 、L 5 、L 6 、L 7 、Z are not particularly limited and each independently includes but is not limited to a straight-chain structure, a branched-chain structure or a structure containing a ring structure.

[0114] In the present invention, the number of non-hydrogen atoms of L 1 、L 2 、L 3 、L 4 、L 5 、L 6 、L 7 、Z is not particularly limited and each independently preferably has 1 to 50 non-hydrogen atoms; more preferably 1 to 20 non-hydrogen atoms; still more preferably 1 to 10 non-hydrogen atoms. The non-hydrogen atoms are carbon atoms or heteroatoms. The heteroatoms include but are not limited to O, S, N, P, Si, B, etc. When the number of non-hydrogen atoms is 1, the non-hydrogen atom can be a carbon atom or a heteroatom. When the number of non-hydrogen atoms is greater than 1, the types of non-hydrogen atoms are not particularly limited; it can be 1 type, or 2 types or more; when the number of non-hydrogen atoms is greater than 1, it can be any combination of carbon atoms with carbon atoms, carbon atoms with heteroatoms, or heteroatoms with heteroatoms.

[0115] In the present invention, two identical or different reactive groups can react to form a divalent linking group. The reaction conditions are related to the type of the divalent linking group formed by the reaction and can adopt the existing publicly disclosed technologies. For example: an amino group reacts with an active ester, a formic acid active ester, a sulfonic acid ester, an aldehyde, an α,β-unsaturated bond, a carboxylic acid group, an epoxide, an isocyanate, or an isothiocyanate to obtain a divalent linking group such as an amide group, a urethane group, an amino group, an imine group (which can be further reduced to a secondary amino group), an amino group, an amide group, an amino alcohol, a urea bond, or a thiourea bond; a mercapto group reacts with a compound containing an active ester, a formic acid active ester, a sulfonic acid ester, a mercapto group, maleimide, an aldehyde, an α,β-unsaturated bond, a carboxylic acid group, iodoacetamide, or an acid anhydride to obtain a divalent linking group such as a thioester group, a thiocarbonate group, a thioether, a disulfide, a thioether, a thiohemiacetal, a thioether, a thioester, a thioether, or an imide; an unsaturated bond reacts with a mercapto group to obtain a thioether group; a carboxyl group or an acyl halide reacts with a mercapto group or an amino group to obtain a thioester group, an amide group, etc.; a hydroxyl group reacts with a carboxyl group, an isocyanate, an epoxide, or a chloromethoxy group to obtain a divalent linking group such as an ester group, a carbamate group, an ether bond, or a carbonate group; a carbonyl group or an aldehyde group reacts with an amino group, a hydrazine, or a hydrazide to obtain a divalent linking group such as an imine bond, a hydrazone, or an acylhydrazone; click chemical reactions occur between reactive groups such as azide, alkynyl, alkenyl, mercapto, azide, diene, maleimide, 1,2,4-triazoline-3,5-dione, dithioester, hydroxylamine, hydrazide, acrylate, allyloxy, isocyanate, or tetrazole to generate various divalent linking groups including but not limited to structures such as triazole, isoxazole, or thioether bond.

[0116] L 1 、L 2 、L 3 、L 4 、L 5 、L 6 、L 7 There is no particular limitation on the stability of L, L, L, L, L, L, L, or Z. Any one of the divalent linking groups or any divalent linking group formed by any one and an adjacent heteroatom group is independently a linking group STAG that can stably exist or a linking group DEGG that can be degraded.

[0117] 1.1.2.1.L 1

[0118] In the present invention, L 1 is -L c R, and L c is a linking bond or a divalent linking group, and R is a C 1-30 aliphatic hydrocarbon group, where the aliphatic hydrocarbon group is an alkyl group, an alkenyl group, or an alkynyl group.

[0119] In a specific embodiment of the present invention, L c is preferably selected from a linking bond, -(CH 2 ) tm -, -(CH2 ) tm Z-, -Z(CH 2 ) tm -, -(CH 2 ) tm Z(CH 2 ) tm - and -(CH 2 ) tm Z(CH 2 ) tm Any one of Z-, where tm is independently an integer from 1 to 12 each time it appears; Z is independently -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-, -C(=O)NR c -, -NR c C(=O)NR c- , -OC(=O)NR c -, -NR c C(=O)O-, -SC(=O)NR c - and -NR c C(=O)S-, where R c is independently H or C 1-12 alkyl each time it appears; L c is preferably a linking bond, -(CH 2 ) tm -, -(CH 2 ) tm O-, -(CH 2 ) tm C(=O)-, -(CH 2 ) tm C(=O)O-, -(CH 2 ) tm OC(=O)-, -(CH 2 ) tm C(=O)NH-, -(CH 2 ) tm NHC(=O)-, -(CH 2 ) tm OC(=O)O-, -(CH 2 ) tm NHC(=O)O-, -(CH 2 ) tm OC(=O)NH-, -(CH 2 ) tm NHC(=O)NH-, -(CH 2 )tm O(CH 2 ) tm -、-(CH 2 ) tm C(=O)(CH 2 ) tm -、-(CH 2 ) tm C(=O)O(CH 2 ) tm -、-(CH 2 ) tm OC(=O)(CH 2 ) tm -、-(CH 2 ) tm C(=O)NH(CH 2 ) tm -、-(CH 2 ) tm NHC(=O)(CH 2 ) tm -、-(CH 2 ) tm OC(=O)O(CH 2 ) tm -、-(CH 2 ) tm NHC(=O)O(CH 2 ) tm -、-(CH 2 ) tm OC(=O)NH(CH 2 ) tm - and -(CH 2 ) tm NHC(=O)NH(CH 2 ) tm - any one of; more preferably a linking bond, -(CH 2 ) tm O(CH 2 ) tm -、-(CH 2 ) tm C(=O)O(CH 2 ) tm - and -(CH 2 ) tm OC(=O)(CH 2 ) tm - any one of.

[0120] In a specific embodiment of the present invention, preferably R is any one of a linear alkyl, a branched alkyl, a linear alkenyl, a branched alkenyl, a linear alkynyl and a branched alkynyl; preferably a linear alkyl or a linear alkenyl; more preferably C1-25 a linear alkyl group; more preferably C 1-17 a linear alkyl group, specifically any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, and heptadecyl.

[0121] 1.1.2.2.L 2

[0122] In the present invention, L 2 is a linking bond or a divalent linking group.

[0123] In a specific embodiment of the present invention, L 2 is a linking bond, -(CH 2 ) m -, -(CH 2 ) m C(=O)-, -C(=O)(CH 2 ) m -, and -C(=O)(CH 2 ) m C(=O)-, where m is an integer from 1 to 4.

[0124] 1.1.2.3.L 3 , L 4

[0125] In the present invention, L 3 and L 4 are each independently a linking bond or a divalent linking group.

[0126] In a specific embodiment of the present invention, preferably L 3 , L 4 is a linking bond, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, -O(CR c R c ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-, -C(=O)NR c -, -NR c C(=O)NR c -, -OC(=O)NR c , -NR c C(=O)O-, -SC(=O)NR c -, and -NR c C(=O)S-, where R c is each independently a hydrogen atom or C 1-12An alkyl group, and s is 2, 3 or 4.

[0127] In a specific embodiment of the present invention, L is more preferably 3 , L 4 is one of the following cases:

[0128] Case (1): One of L 3 , L 4 is a linking bond, and the other is -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, -O(CR c R c ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-, -C(=O)NR c -, -N Rc C(=O)NR c -, -OC(=O)NR c -, -NR c C(=O)O-, -SC(=O)NR c - and -NR c C(=O)S-;

[0129] Case (2): Both L 3 , L 4 are linking bonds;

[0130] Case (3): L 3 , L 4 are each independently selected from -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, -O(CH 2 ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH- and -NHC(=O)S-.

[0131] In a specific embodiment of the present invention, L 3 , L 4 are each independently selected from any one of -OC(=O)-, -C(=O)O- and -OC(=O)O-.

[0132] In a specific embodiment of the present invention, L 3 , L 4 One of them is -OC(=O)O-, and the other is -OC(=O)- or -C(=O)O-.

[0133] In a specific embodiment of the present invention, L is more preferably 3 and L 4 are both -OC(=O)- or both -C(=O)O- or both -OC(=O)O-.

[0134] 1.1.2.4.L 5

[0135] In the present invention, L 5 is a linking bond or a divalent linking group.

[0136] In a specific embodiment of the present invention, L is preferably 5 a divalent linking group selected from any one, any two, or any combination of two or more of L 6 , L 7 , and the divalent linking groups formed by any combination of two or more of the divalent linking groups of Z; more preferably, L 5 is -L 6 -, -L 6 -Z-, -Z-L 6 -, -Z-L 6 -Z-, -L 6 -Z-L 7 -, -Z-L 6 -Z-L 7 -, -L 6 -Z-L 7 -Z-, -Z—L 6 -Z—L 7 -Z- and -L 6 -Z-L 7 -Z-L 6 -Z-; wherein, the L 6 , L 7 are carbon chain linking groups, each independently being -(CR a R b ) t -(CR a R b ) o -(CR a R b ) p -, where t, o, and p are each independently integers from 0 to 12, and t, o, and p are not all 0, and R a and R b are each independently a hydrogen atom or a C 1-12 alkyl group each time they appear; each occurrence of Z is independently -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NRc C(=O)-, -C(=O)NR c -, -NR c C(=O)NR c -, -OC(=O)NR c -, -NR c C(=O)O-, -SC(=O)NR c - and -NR c C(=O)S- any one of them, wherein, R c is independently H or C 1-12 alkyl each time it appears.

[0137] In a specific embodiment of the present invention, more preferably L 5 is a linking bond, -(CH 2 ) tn Z-, -Z(CH 2 ) tn -, -Z(CH 2 ) tn Z-, -(CH 2 ) tn Z(CH 2 ) tn Z- and -Z(CH 2 ) tn Z(CH 2 ) tn Z- any one of them, wherein, tn is independently an integer from 1 to 12 each time it appears; more preferably a linking bond, -(CH 2 ) tn O-, -(CH 2 ) tn C(=O)O-, -(CH 2 ) tn OC(=O)-, -(CH 2 ) tn NHC(=O)-, -(CH 2 ) tn OC(=O)O-, -(CH 2 ) tn NHC(=O)O-, -O(CH 2 ) tn O-, -C(=O)O(CH 2 ) tn C(=O)O-, -OC(=O)(CH 2 ) tn OC(=O)-, -C(=O)O(CH 2 ) tn OC(=O)-, -OC(=O)(CH 2 ) tnC(=O)O-, -OC(=O)O(CH 2 ) tn OC(=O)O-, -NHC(=O)O(CH 2 ) tn NHC(=O)O-, -OC(=O)NH(CH 2 ) tn NHC(=O)O-, -C(=O)(CH 2 ) tn O-, -C(=O)(CH 2 ) tn C(=O)O-, -C(=O)(CH 2 ) tn OC(=O)-, -C(=O)(CH 2 ) tn OC(=O)O-, -C(=O)(CH 2 ) tn NHC(=O)O- and -C(=O)(CH 2 ) tn C(=O)(CH 2 ) tn NHC(=O)O-; more preferably a linking bond, -C(=O)O-, -(CH 2 ) tn O-, -(CH 2 ) tn C(=O)O-, -(CH 2 ) tn O-, -C(=O)(CH 2 ) tn O-, -C(=O)(CH 2 ) tn C(=O)O-, -C(=O)(CH 2 ) tn NHC(=O)O- and -C(=O)(CH 2 ) tn C(=O)(CH 2 ) tn NHC(=O)O-.

[0138] 1.1.3.B 1 、B 2

[0139] In the present invention, B 1 、B 2 are each independently a linking bond or C 1-30 alkylene.

[0140] In a specific embodiment of the present invention, B 1 、B2 Preferably, each is independently a linking bond or a C 1-20 alkylene group; more preferably, B 1 and B 2 are any one of the following cases:

[0141] Case (1): B 1 and B 2 are each independently a C 1-20 alkylene group, specifically B 1 and B 2 are each independently any one of methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene, and icosylene; more preferably B 1 and B 2 are each independently a C 2-10 alkylene group;

[0142] Case (2): One of B 1 and B 2 is a linking bond, and the other is a C 1-20 alkylene group.

[0143] Case (3): Both B 1 and B 2 are linking bonds.

[0144] 1.1.4.R 1 and R 2 and R, R c

[0145] 1.1.4.1.R 1 and R 2

[0146] In the present invention, R 1 and R 2 are each independently a C 1-30 aliphatic hydrocarbon group or

[0147] In a specific embodiment of the present invention, R 1 and R 2 are each independently a C 1-30 linear aliphatic hydrocarbon group, a C 1-30 branched aliphatic hydrocarbon group or

[0148] In a specific embodiment of the present invention, the linear aliphatic hydrocarbon group is a linear alkyl group, a linear alkenyl group or a linear alkynyl group; more preferably a C 1-25 linear aliphatic hydrocarbon group, more preferably a C5-17 Linear aliphatic hydrocarbon group.

[0149] In a specific embodiment of the present invention, the branched aliphatic hydrocarbon group is a branched alkyl group, a branched alkenyl group or a branched alkynyl group, represented as

[0150] In a specific embodiment of the present invention, R 1 , R 2 are each independently

[0151] wherein t is an integer from 0 to 12, t 1 , t 2 are each independently integers from 0 to 5, t 3 , t 4 are each independently 0 or 1, and t 1 , t 2 , t 3 , t 4 are not simultaneously 0; R e , R f are each independently any one of C 1 -C 15 alkyl, C 2 -C 15 alkenyl and C 2 -C 15 alkynyl; R e , R f are more preferably each independently selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, vinyl, propenyl, allyl, butenyl, butenyl, pentenyl, pentenyl, hexenyl, hexenyl, heptenyl, heptenyl, octenyl, octenyl, nonenyl, nonenyl, decenyl, decenyl, ethynyl, propynyl, propargyl, butynyl, butynyl, pentynyl, pentynyl, hexynyl, hexynyl, heptynyl, heptynyl, octynyl, octynyl, nonynyl, nonynyl, decynyl and decynyl; more preferably each independently selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl.

[0152] In a specific embodiment of the present invention, R 1 , R 2 are preferably any one of the following cases:

[0153] Case (1): One of R 1 , R 2 is a linear aliphatic hydrocarbon group and the other is a C 1-30 branched aliphatic hydrocarbon group; 1-30

[0154] Case (2): R 1 and R 2 are each independently a C 1-30 branched-chain aliphatic hydrocarbon group

[0155] Case (3): R 1 and R 2 are each independently

[0156] Case (4): One of R 1 and R 2 is a C 1-30 straight-chain aliphatic hydrocarbon group or a C 1-30 branched-chain aliphatic hydrocarbon group and the other is

[0157] In a more specific embodiment of the present invention, R 1 and R 2 are further preferably each independently selected from any one of the following structures:

[0158]

[0159]

[0160] 1.1.4.2.R

[0161] In the present invention, R is a C 1-30 aliphatic hydrocarbon group.

[0162] In a specific embodiment of the present invention, R is a straight-chain alkyl group, a branched-chain alkyl group, a straight-chain alkenyl group, a branched-chain alkenyl group, a straight-chain alkynyl group or a branched-chain alkynyl group; preferably a straight-chain alkyl group or a straight-chain alkenyl group; more preferably a C 1-25 straight-chain alkyl group; more preferably a C 1-17 straight-chain alkyl group, specifically any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl and heptadecyl.

[0163] 1.1.4.3.R c

[0164] In the present invention, each occurrence of R c is independently H or a C 1-12 alkyl group.

[0165] In a specific embodiment of the present invention, R c is preferably a hydrogen atom or a C 1-8 alkyl group; more preferably a hydrogen atom or a methyl group.

[0166] 1.1.5.R 3

[0167] In the present invention, R 3 is, each time it appears, independently a hydrogen atom, -R d , -OR d , C 3-6 carbocyclic group, nitrogen-containing heterocyclic group, -NR d R d , -SR d , -C(=O)R d , -C(=O)OR d , -OC(=O)R d , -OC(=O)OR d or a functional group R that can react with a biologically relevant substance 01 ; wherein, R d is, each time it appears, independently C 1-12 alkyl group.

[0168] In a specific embodiment of the present invention, preferably R 3 is, each time it appears, independently a hydrogen atom, an alkyl group, an alkoxy group, -C(=O)OR d , -OC(=O)R d , -OC(=O)OR d , epoxy group, alcoholic hydroxyl group, protected alcoholic hydroxyl group, mercapto group, protected mercapto group, carboxyl group, protected carboxyl group, amino group, protected amino group, aldehyde group, protected aldehyde group, active ester group, carbonate group, carbamate group, isocyanate group, isothiocyanate group, succinimidyl group, maleimidyl group, protected maleimidyl group, dimethylamino group, alkenyl group, alkenoate group, azide group, cyano group, dithiopyridyl group, α-haloacetyl alkynyl group, alkynyl group, folic acid group, rhodamine group, biotin group, monosaccharide group, polysaccharide group, any one of them, wherein, R d is, each time it appears, independently C 1-12 alkyl group.

[0169] In a specific embodiment of the present invention, R 3 is, each time it appears, independently a functional group R that can react with a biologically relevant substance 01 , and the R 01 is a functional group with therapeutic targeting property; preferably a residue of any one of folic acid and N-acetylgalactosamine or a residue of any one of their functional derivatives; more preferably any one of the following structures:

[0170]

[0171] 1.1.6.-L 5 -R 3 Fragment

[0172] In a specific embodiment of the present invention, the L described in part 1.1.2.4 5 and the R described in part 1.1.5 3 form -L 5 -R 3 Each independently selects any one of the following structures each time it appears:

[0173]

[0174] 1.1.7. Structural formula examples

[0175] In a specific embodiment of the present invention, according to X described in 1.1.1, the structure of the cationic lipid of the present invention is shown in formula (2):

[0176]

[0177] Wherein, L 1 , L 2 , L 3 , L 4 , L 5 , B 1 , B 2 , R 1 , R 2 and R 3 have the same definitions as those described in general formula (1), which will not be elaborated here.

[0178] More preferably, the structure of the cationic lipid satisfies any one of the following general formulas:

[0179] Wherein, s1 is 0, 1, 2, 3, or 4; L 2 , L 5 , B 1 , B 2 , R, R 1 , R 2 and R 3 have the same definitions as those described in general formula (1), which will not be elaborated here.

[0180] 1.1.8. Specific structural examples

[0181] In a specific embodiment of the present invention, the structure of the preferred cationic lipid includes but is not limited to any one of the following structures:

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] 2. Preparation of Cationic Lipids

[0190] 2.1 Preparation Method

[0191] In a specific embodiment of the present invention, the cationic lipid shown in formula (2) is prepared by the following method.

[0192] Step 1: Obtaining of small molecule raw material primary amine derivatives 1-3 and 1-6 capable of reacting with amino or secondary amino groups: They can be obtained by purchase or by self-synthesis. Self-synthesis can be carried out by the following route, where F 1 and F 2 are reactive groups capable of reacting to form divalent linker L 3 ; F 3 and F 4 are reactive groups capable of reacting to form divalent linker L 4 ; It is worth mentioning that the amino group in 1-1 can also be a protected amino group, and the naked amino group-containing 1-3 can be obtained by subsequent deprotection; F N in 1-4 is a reactive group capable of reacting with amino or secondary amino groups and its protected form, preferably -OMs, -OTs, -CHO, -F, -Cl or -Br;

[0193] Step 2: Obtaining of secondary amine derivative 1-8: After the ring-opening reaction of the primary amine derivative with epoxide 1-7, a secondary amine derivative 1-8 containing a polar group hydroxyl and L1 is obtained, where tp is 1, 2 or 3, and R a is independently H or C 1-12 alkyl each time it appears, that is, when tp is 2 or 3, two or three R a can be the same or different from each other; The epoxy group in epoxide 1-7 is preferably epoxyethyl, epoxypropyl, epoxybutyl, substituted epoxyethyl, substituted epoxypropyl or substituted epoxybutyl;

[0194] Step 3. Obtaining the compound represented by formula (2'): An alkylation reaction is carried out between the secondary amine derivative 1-8 and the small molecule 1-6 to obtain the compound represented by the general formula (2'), where, corresponding to L 2 ;

[0195] Step 4. Obtaining the final product represented by formula (2): When -L 5 -R 3 in formula (2) is a hydroxyl group, the compound represented by formula (2') is the compound represented by formula (2); when -L 5 -R 3 in formula (2) is not a hydroxyl group, the hydroxyl group at the polar head of formula (2') needs to be end-linearly functionalized to obtain the compound represented by formula (2);

[0196] wherein, B 1 、B 2 、L 1 、L 2 、L 3 、L 4 、L 5 、R 1 、R 2 、R 3 are the same as those described in formula (2), and will not be elaborated here.

[0197] Step 1

[0198]

[0199] Step 2

[0200]

[0201] Step 3

[0202]

[0203] Step 4

[0204]

[0205] In a specific embodiment of the present invention, the specific operation steps of the foregoing preparation method are as follows:

[0206] Step 1. React the small molecule A-1 with the small molecule A-2 to generate a primary amine derivative A-3 containing an ester bond, with an amino group at one end and R 2 at the other end; React the small molecule A-1' (R 1 -OH) with the small molecule A-2' to generate a compound containing an ester bond, with a bromo group at one end and R 1Small molecule intermediate A-3’ wherein, t is an integer from 1 to 12;

[0207] Step 2. A primary amine derivative A-3 reacts with an epoxide to undergo a ring-opening reaction to form a secondary amine derivative A-5 containing a divalent linking group and a naked hydroxyl group wherein, tp is 2, 3 or 4;

[0208] Step 3. One molecule of the small molecule intermediate A-3’ undergoes an alkylation reaction with the secondary amine derivative A-5 to obtain a compound A-6

[0209] Step 4. The compound A-6 is coupled with a small molecule A-7 containing a reactive group to obtain a cationic lipid derivative A-8 or A-8’; wherein, the carboxyl group of A-7 can react with the hydroxyl group of the compound A-6 to form a branched central carbon atom and a divalent linking group ester group;

[0210] When R 3’ is equal to R 3 , the obtained structure A-8’ is the structure corresponding to the general formula (2);

[0211] When R 3’ is not equal to R 3 , A-8’ is subjected to terminal micro-modification to obtain the structure of A-8 corresponding to the general formula (2); the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, changing the leaving group;

[0212] When -L 5 -R 3 in the formula (2) is a hydroxyl group, the compound A-6 obtained in Step 3 is the structure corresponding to the general formula (2);

[0213] wherein, the definitions of R 1 , R 2 and R 3 are the same as those described in the general formula (2), and will not be elaborated here.

[0214] Step 1

[0215]

[0216] Step 2

[0217]

[0218] Step 3

[0219]

[0220] Step Four

[0221]

[0222] The above-mentioned various small molecule raw materials A-1, A-2, A-3, A-1’, A-2’, A-3’, A-4, A-7, etc. can be obtained by purchase or by self-synthesis. For example, in Example 1, the small molecule A-3’ is It can be obtained by self-synthesis using as the raw material.

[0223] In the reaction raw materials A-3 and A-3’ in the above preparation method, R 2 and R 1 can each independently be an etherified aliphatic hydrocarbon derivative residue wherein, each time t appears, it is independently an integer from 0 to 12; Re and Rf are each independently C 1 -C 15 alkyl, C 2 -C 15 alkenyl and C 2 -C 15 alkynyl, any one of them. More specifically, A-3 and A-3’ can be It can be obtained by purchase or by self-synthesis. When self-synthesizing, aldol addition can be used. For example, one molecule of reacts with two molecules of R e -OH to obtain At this time, R e and R f are the same; A-3 and A-3’ can also be It can be obtained by purchase or by self-synthesis. When self-synthesizing, it can be obtained by reacting with a related alkylating reagent. The alkylating reagent is preferably a halide, such as S2-4 in Example 4 can be obtained by reacting one molecule of glycerol with a TBS-protected hydroxyl group and two molecules of bromohexane followed by deprotection.

[0224] 2.2. Description of Related Raw Materials and / or Steps in the Preparation Process

[0225] 2.2.1. Protection and Deprotection of Related Groups Involved in the Reaction Process

[0226] In the present invention, the reaction process also involves the "protection" and "deprotection" processes of related groups. To prevent the functional group from affecting the reaction, the functional group is usually protected. Moreover, when there are two or more functional groups, only the target functional group is selectively reacted, so other functional groups are protected. The protecting group not only stably protects the target functional group, but also needs to be easily removed as required. Therefore, in organic synthesis, it is important to deprotect only the protecting group bonded to the specified functional group under appropriate conditions.

[0227] In the present invention, the definitions of "carboxyl protecting group" and "amino protecting group" are the same as those in the "Term Explanation" section, and will not be elaborated here.

[0228] In the present invention, the hydroxyl group protected by the hydroxyl protecting group is not particularly limited. For example, it can be a hydroxyl group such as an alcohol hydroxyl group or a phenolic hydroxyl group. Among them, the amino group of the amino protecting group is not particularly limited. For example, it can be from a primary amine, a secondary amine, a hydrazine, an amide, etc. The amino group in the present invention is not particularly limited, including but not limited to a primary amino group, a secondary amino group, a tertiary amino group, and a quaternary ammonium ion.

[0229] In the present invention, the deprotection of the protected hydroxyl group is related to the type of the hydroxyl protecting group. The type of the hydroxyl protecting group is not particularly limited. Taking the protection of the terminal hydroxyl group with a benzyl group, a silyl ether, an acetal, or a tert-butyl group as an example, the corresponding deprotection methods are as follows:

[0230] A: Deprotection of the benzyl protecting group

[0231] The deprotection of the benzyl protecting group can be achieved by the hydrogenation of a hydrogenation reducing agent and a hydrogen donor. The water content in this reaction system should be less than 1% for the reaction to proceed smoothly.

[0232] The hydrogenation reduction catalyst is not limited. Palladium and nickel are preferred, but the carrier is not limited. Alumina or carbon is preferred, and carbon is more preferred. The amount of palladium used is 1 to 100 wt% of the compound containing the protected hydroxyl group, preferably 1 to 20 wt% of the compound containing the protected hydroxyl group.

[0233] The reaction solvent is not particularly limited as long as both the raw material and the product can be dissolved. However, methanol, ethanol, ethyl acetate, tetrahydrofuran, and acetic acid are preferred; methanol is more preferred. The hydrogen donor is not particularly limited, but hydrogen, cyclohexene, 2-propanol, ammonium formate, etc. are preferred. The reaction temperature is preferably 25 to 40 °C. The reaction time is not particularly limited. The reaction time is negatively correlated with the amount of the catalyst used, and is preferably 1 to 5 hours.

[0234] B: Deprotection of the acetal and ketal protecting groups

[0235] For the acetal or ketal compounds used for such hydroxyl protection, ethyl vinyl ether, tetrahydropyran, acetone, 2,2-dimethoxypropane, benzaldehyde, etc. are preferred. The deprotection of such acetal and ketal protecting groups is achieved under acidic conditions, and the solution pH is preferably 0 to 4. The acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, nitric acid are preferred, and hydrochloric acid is more preferred. The reaction solvent is not particularly limited as long as it can dissolve the reactants and products, and water is preferred. The reaction temperature is preferably 0 to 30 °C.

[0236] C: Deprotection of silyl ether protecting groups

[0237] Compounds used for such hydroxyl protection include trimethylsilyl ether, triethylsilyl ether, dimethyl tert-butylsilyl ether, tert-butyldiphenylsilyl ether, etc. The deprotection of such silyl ether protecting groups is carried out by a fluoride ion-containing compound, preferably tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid, potassium fluoride, and tetrabutylammonium fluoride and potassium fluoride are more preferred. The dosage of the fluorine-containing reagent is 5 to 20 times the molar equivalent of the protected hydroxyl group, preferably 8 to 15 times the initiator. If the dosage of fluorine is less than 5 times the molar equivalent of the protected hydroxyl group, incomplete deprotection will occur; when the dosage of the deprotection reagent is greater than 20 times the molar equivalent of the protected hydroxyl group, the excess reagent or compound will cause trouble in purification and may be mixed into subsequent steps, thus causing side reactions. The reaction solvent is not particularly limited as long as it can dissolve the reactants and products, and an aprotic solvent is preferred, and tetrahydrofuran and dichloromethane are more preferred. The reaction temperature is preferably 0 to 30 °C. When the temperature is lower than 0 °C, the reaction rate is slow and the protecting group cannot be completely removed.

[0238] D: Deprotection of tert-butyl protecting groups

[0239] The deprotection of tert-butyl is carried out under acidic conditions, and the solution pH is preferably 0 to 4. The acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, nitric acid are preferred, and hydrochloric acid is more preferred. The reaction solvent is not particularly limited as long as it can dissolve the reactants and products, and water is preferred. The reaction temperature is preferably 0 to 30 °C.

[0240] 2.2.2. Alkylation reaction

[0241] The alkylation reaction of the present invention is preferably a reaction based on the alkylation of hydroxyl, mercapto or amino groups, corresponding to the formation of ether bonds, thioether bonds, secondary amino or tertiary amino groups in turn. Examples are as follows:

[0242] 2.2.2.1. Alkylation of substrate alcohols with sulfonates and halides

[0243] In the presence of a base, an ether intermediate is obtained by nucleophilic substitution of a substrate alcohol with a sulfonate derivative or a halide. Among them, the molar equivalent of the sulfonate or halide is 1 to 50 times that of the substrate alcohol, preferably 1 to 5 times. When the molar equivalent of the sulfonate or halide is less than 1 times the molar equivalent of the substrate alcohol, the reaction substitution is incomplete and difficult to purify. When the molar equivalent of the sulfonate or halide is greater than 50 times that of the substrate alcohol, the excess reagent causes trouble in purification, may be mixed into subsequent steps, resulting in an increase in side reactions in the next step and increasing the purification difficulty.

[0244] The obtained product is a mixture of an ether intermediate and excess sulfonate and halide, which can be purified by anion exchange resin, osmosis, ultrafiltration, etc. Among them, there is no particular limitation on the anion exchange resin, as long as the target product can undergo ion exchange and adsorption on the resin. Ion exchange resins of tertiary amines or quaternary ammonium salts with skeletons such as dextran, agarose, polyacrylate, polystyrene, and polydiphenylethylene are preferred. There is no limitation on the solvent for osmosis and ultrafiltration. Generally, water or an organic solvent can be used. There is no particular limitation on the organic solvent as long as the product can dissolve in it. Dichloromethane, chloroform, etc. are preferred.

[0245] The reaction solvent is not limited, and aprotic solvents such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide, or dimethylacetamide are preferred, and dimethylformamide, dichloromethane, dimethyl sulfoxide, or tetrahydrofuran are more preferred.

[0246] The base includes an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole, or diisopropylethylamine) or an inorganic base (such as sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium carbonate, or potassium hydroxide). Organic bases are preferred, and triethylamine and pyridine are more preferred. The molar amount of the base is 1 to 50 times the molar equivalent of the sulfonate or halide, preferably 1 to 10 times, and more preferably 3 to 5 times.

[0247] 2.2.2.2. Alkylation of the substrate amine with a sulfonate or halide

[0248] A. Alkylation of the substrate amine with a sulfonate or halide

[0249] In the presence of a base, an amine intermediate is obtained by nucleophilic substitution of a substrate amine with a sulfonate derivative or a halide. Among them, the molar equivalent of the sulfonate or halide is 1 to 50 times that of the substrate amine, preferably 1 to 5 times. When the molar equivalent of the sulfonate or halide is less than 1 times the molar equivalent of the substrate amine, the reaction substitution is incomplete and difficult to purify. When the molar equivalent of the sulfonate or halide is greater than 50 times that of the substrate amine, the excess reagent causes trouble in purification, may be mixed into subsequent steps, resulting in an increase in side reactions in the next step and increasing the purification difficulty.

[0250] The resulting product is a mixture of amine intermediates and excess sulfonates and halides, which can be purified by column chromatography, anion exchange resin, dialysis, ultrafiltration, etc. Among them, there is no particular limitation on the anion exchange resin, as long as the target product can undergo ion exchange and adsorption on the resin. Ion exchange resins of tertiary amines or quaternary ammonium salts with skeletons such as dextran, agarose, polyacrylate, polystyrene, and polydiphenylethylene are preferred. There is no limitation on the solvents for dialysis and ultrafiltration. Generally, water or organic solvents can be used. There is no particular limitation on the organic solvents, as long as the product can dissolve in them. Dichloromethane, chloroform, etc. are preferred.

[0251] The selection of "reaction solvent" and "base" is the same as that in the part of "alkylation of substrate alcohol with sulfonate" and "halide", and will not be elaborated here.

[0252] 2.2.2.3. Alkylation reaction of substrate amine with aldehyde derivatives

[0253] After obtaining the imine intermediate by reacting the substrate amine with the aldehyde derivative, the amine intermediate is obtained under the action of a reducing agent. Among them, the molar equivalent of the aldehyde derivative is 1 to 20 times that of the substrate amine, preferably 1 to 2 times, and more preferably 1 to 1.5 times. When the molar equivalent of the aldehyde derivative is greater than 20 times that of the substrate amine, the excess reagent brings trouble to purification and may be mixed into subsequent steps, increasing the purification difficulty. When the molar equivalent of the aldehyde derivative is less than 1 time that of the substrate amine, the reaction is incomplete, increasing the purification difficulty. Among them, the reaction product can be purified by means of cation exchange resin, dialysis, ultrafiltration, etc. There is no particular limitation on the said cation exchange resin, as long as it can achieve the separation effect by exchanging with quaternary ammonium cations. There is no limitation on the solvents for dialysis and ultrafiltration. Generally, water or organic solvents can be used. There is no particular limitation on the organic solvents, as long as the product can dissolve in them. Dichloromethane, chloroform, etc. are preferred.

[0254] The reaction solvent is not limited, and organic solvents such as methanol, ethanol, water, toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide are preferred; water and methanol are more preferred.

[0255] There is no particular limitation on the reducing agent, as long as it can reduce the imine to an amine. Sodium borohydride, lithium aluminum hydride, sodium cyanoborohydride, Zn / AcOH, etc. are preferred, and sodium cyanoborohydride is more preferred. Generally, the dosage of the reducing agent is 0.5 to 50 times the amount of substance of the aldehyde derivative, and more preferably 1 - 10 times.

[0256] 2.2.3. Terminal linear functionalization

[0257] The method for terminal linear functionalization is not particularly limited and is related to the type of the final functional group or its protected form. For the linear functionalization of the terminal hydroxyl group, that is, starting from the terminal hydroxyl group of the compound corresponding to formula (2’) or compound A-6, other functional groups or their protected form -L are obtained through functionalization. 5 -R 3 , and the specific preparation method is as described in paragraphs

[0960] to

[1205] of document CN104530417A.

[0258] In the present invention, the raw materials used in each preparation method can be obtained by purchase or self-synthesis.

[0259] In the present invention, the intermediates and end products prepared can be purified by purification methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, anti-precipitation, thin film dialysis or supercritical extraction, etc. For the characterization and confirmation of the structure and molecular weight of the end product, characterization methods including but not limited to nuclear magnetic resonance, electrophoresis, ultraviolet-visible spectrophotometer, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism, mass spectrometry, etc. can be used.

[0260] 3.1. Lipid composition

[0261] In the present invention, a lipid composition contains any one of the cationic lipids having the structure as shown in the general formula (1) described above.

[0262] In a specific embodiment of the present invention, preferably, in addition to containing the cationic lipid having the structure as shown in the general formula (1), the lipid composition further contains one or more of phospholipids, steroid lipids and polyethylene glycolated lipids, selected from any one of the following situations:

[0263] Situation (1): further contains phospholipids;

[0264] Situation (2): further contains steroid lipids;

[0265] Situation (3): further contains polyethylene glycolated lipids;

[0266] Situation (4): further contains phospholipids and steroid lipids;

[0267] Situation (5): further contains phospholipids and polyethylene glycolated lipids;

[0268] Situation (6): further contains steroid lipids and polyethylene glycolated lipids;

[0269] Situation (7): further contains phospholipids, steroid lipids and polyethylene glycolated lipids;

[0270] More preferably, it further contains three lipids, namely phospholipids, steroid lipids and polyethylene glycolated lipids, simultaneously.

[0271] In a specific embodiment of the present invention, the phospholipid in the lipid composition is preferably 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-doundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dioleoyl phosphatidylserine (DOPS), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE), any one of them and their compositions.

[0272] In a specific embodiment of the present invention, the steroid lipid in the lipid composition is preferably any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and their combinations.

[0273] In a specific embodiment of the present invention, the polyethylene glycolated lipid in the lipid composition is preferably polyethylene glycol-1,2-dimyristoyl glycerol (PEG-DMG), polyethylene glycol-distearoyl phosphatidylethanolamine (PEG-DSPE), PEG-cholesterol, polyethylene glycol-diacylglycerol (PEG-DAG), polyethylene glycol-dialkoxypropyl (PEG-DAA), specifically including polyethylene glycol 500-dipalmitoyl phosphatidylcholine, polyethylene glycol 2000-dipalmitoyl phosphatidylcholine, polyethylene glycol 500-stearoyl phosphatidylethanolamine, polyethylene glycol 2000-distearoyl phosphatidylethanolamine, polyethylene glycol 500-1,2-dioleoyl phosphatidylethanolamine, polyethylene glycol 2000-1,2-dioleoyl phosphatidylethanolamine and polyethylene glycol 2000-2,3-dimyristoyl glycerol (PEG-DMG) and their combinations.

[0274] In a specific embodiment of the present invention, the polyethylene glycolated lipid in the lipid composition is preferably any one of the following structures and their combinations:

[0275]

[0276]

[0277] wherein, n 1 is an integer from 25 to 300, more preferably n 1 is any one of 44, 45, 46, 47, 48.

[0278] In a specific embodiment of the present invention, it is preferred that any one of the aforementioned lipid compositions contains 20-80% of the cationic lipid shown in formula (1), 5-15% of phospholipid, 25-55% of steroid lipid and 0.5-10% of polyethylene glycolated lipid, and the percentages are the molar percentages of each lipid in the total lipid in the solution containing the solvent.

[0279] In a specific embodiment of the present invention, it is preferred that in any one of the aforementioned lipid compositions, the molar percentage of the cationic lipid in the total lipid in the solution containing the solvent is 30-65%; more preferably it is any one of about 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 55%.

[0280] In a specific embodiment of the present invention, preferably in any of the aforementioned lipid compositions, the molar percentage of phospholipids in the total lipids in the solution containing the solvent is about 7.5 - 13%; more preferably, it is any one of about 8%, 9%, 10%, 11%, 12%.

[0281] In a specific embodiment of the present invention, preferably in any of the aforementioned lipid compositions, the molar percentage of steroid lipids in the total lipids in the solution containing the solvent is 35 - 50%, and more preferably, it is any one of about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.

[0282] In a specific embodiment of the present invention, preferably in any of the aforementioned lipid compositions, the molar percentage of polyethylene glycolated lipids in the total lipids in the solution containing the solvent is 0.5 - 5%; preferably 1 - 3%; more preferably, it is any one of about 1.5%, 1.6%, 1.7%, 1.8%, 1.9%.

[0283] 3.2. Preparation of Lipid Compositions

[0284] In the present invention, the lipid composition can be prepared by the following methods, including but not limited to ethanol injection method, microfluidic method, T-tube mixing method, and membrane extrusion method, preferably the ethanol injection method and the microfluidic method.

[0285] 4. Lipid Drug Compositions and Their Preparations

[0286] 4.1. Lipid Drug Compositions

[0287] In one embodiment of the present invention, a lipid drug composition contains any of the lipid compositions described in Section 3.1 above and a drug. Among them, the lipid composition contains any of the cationic lipids having any of the structures shown in the general formula (1) above, and the drug is selected from any one of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs, or protein drugs. In a specific embodiment of the present invention, in the lipid drug composition, the nucleic acid drugs are selected from any one of RNA, DNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir, and ribozyme, and the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA, and siRNA; preferably, the nucleic acid drugs are any one of DNA, mRNA, miRNA, and siRNA.

[0288] In a specific embodiment of the present invention, the lipid drug composition is preferably used as a drug and is selected from any one of the following drugs: anti-tumor agents, antiviral agents, antifungal agents, and vaccines.

[0289] In a specific embodiment of the present invention, it is preferred that the N / P ratio of the lipid composition to the nucleic acid is (0.1-100):1, more preferably (0.2-30):1, and most preferably (0.5-20):1.

[0290] 4.2. Lipid drug composition preparation

[0291] In a specific embodiment of the present invention, the drug in the lipid drug composition is a nucleic acid drug, and the working solution of the lipid drug composition preparation is deionized water, ultrapure water, phosphate buffer solution or physiological saline, more preferably phosphate buffer solution or physiological saline, and most preferably physiological saline; preferably, lipid composition: working solution = (0.05-20) g: 100 mL, more preferably (0.1-10) g: 100 mL, and most preferably (0.2-5) g: 100 mL.

[0292] In a specific embodiment of the present invention, a lipid drug composition preparation contains the aforementioned lipid drug composition and a pharmaceutically acceptable diluent or excipient, and the diluent or excipient is preferably any one of deionized water, ultrapure water, phosphate buffer solution and physiological saline, more preferably phosphate buffer solution or physiological saline, and most preferably physiological saline.

[0293] In the present invention, the preparation of the lipid drug composition preparation includes the following steps:

[0294] (1) Equilibrate the lipid composition in the diluent or excipient;

[0295] (2) Add the nucleic acid drug to the mixture of the equilibrated lipid composition and the diluent or excipient for complexation;

[0296] Among them, preferably, the equilibration time is 0.1-12 h, preferably 0.2-6 h, and more preferably 0.5-3 h; preferably, the complexation time is 0.1-12 h, preferably 0.2-5 h, and more preferably 0.5-2 h.

[0297] 5. Liposomes or lipid nanoparticles and their preparation

[0298] 5.1. Liposomes or lipid nanoparticles

[0299] In a specific embodiment of the present invention, a liposome or lipid nanoparticle contains any one of the aforementioned lipid drug compositions.

[0300] In a specific embodiment of the present invention, it is preferred that the aforementioned lipid nanoparticles are LNP-drug compositions, LPP-drug compositions or PNP-drug compositions; preferably LNP-drug compositions; more preferably LNP-nucleic acid drug compositions; more preferably LNP-mRNA drug compositions.

[0301] 5.2. Preparation of Liposomes or Lipid Nanoparticles

[0302] In a specific embodiment of the present invention, liposomes can be prepared by the following methods, including but not limited to thin-film dispersion method, ultrasonic dispersion method, reverse-phase evaporation method, freeze-drying method, freeze-thaw method, multiple emulsion method and injection method, preferably thin-film dispersion method, ultrasonic dispersion method and / or reverse-phase evaporation method.

[0303] In a specific embodiment of the present invention, lipid nanoparticles can be prepared by the following methods, including but not limited to microemulsion method, multiple emulsion method, high-shear homogenization ultrasonic method, thin-film hydration extrusion method, microfluidic method.

[0304] In a specific embodiment of the present invention, liposomes are prepared by the thin-film dispersion method, and the thin-film dispersion method includes the following steps:

[0305] (1) Weigh cationic lipid, steroid lipid, phospholipid and polyethylene glycolated lipid, dissolve them fully in an organic solvent, shake well, remove the organic solvent by rotary evaporation under reduced pressure to form an oil film, and dry it with a vacuum pump to remove the organic solvent;

[0306] (2) Add phosphate buffer solution dissolved with cryoprotectant, and perform ultrasonic bath to form a semi-transparent emulsion;

[0307] (3) Add the emulsion into a high-pressure homogenizer for overpressure, and then add the overpressurized emulsion into a liposome extruder for membrane filtration to form liposomes;

[0308] (4) Optionally, dry the liposomes in a freeze dryer to form liposome powder;

[0309] Among them, preferably, the organic solvent is dichloromethane, chloroform and / or methanol, more preferably chloroform and methanol; preferably, the rotation speed of the rotary evaporation under reduced pressure is 30-300 rpm, more preferably 50-200 rpm, most preferably 100-170 rpm; preferably, the temperature of the rotary evaporation under reduced pressure is 10-200 °C, more preferably 20-100 °C, most preferably 40-80 °C;

[0310] Preferably, the drying time with the vacuum pump is 1-72 h, more preferably 5-48 h, most preferably 15-36 h;

[0311] Preferably, the mass concentration of the cryoprotectant dissolved in the phosphate buffer solution is 0.1-80%, preferably 1-50%, more preferably 5-20%;

[0312] Preferably, the frequency of the water bath ultrasonic treatment is 10 - 300 kHz, more preferably 30 - 200 kHz, and most preferably 60 - 150 kHz;

[0313] Preferably, the time of the water bath ultrasonic treatment is 0.1 - 5 h, more preferably 0.2 - 2 h, and most preferably 0.25 - 1 h;

[0314] Preferably, the pressure of the high-pressure homogenizer is 50 - 240 MPa, more preferably 80 - 200 MPa, and most preferably 100 - 150 MPa;

[0315] Preferably, the number of overpressure times of the high-pressure homogenizer is any integer between 1 and 50, more preferably any integer between 3 and 20, and most preferably any integer between 5 and 10;

[0316] Preferably, the pressure of the liposome extruder is 50 - 300 MPa, more preferably 80 - 250 MPa, and most preferably 120 - 200 MPa;

[0317] Preferably, the number of membrane filtration times of the liposome extruder is any integer between 1 and 50, more preferably any integer between 3 and 30, and most preferably any integer between 5 and 20;

[0318] Preferably, the drying time of the freeze dryer is 1 - 120 h, more preferably 5 - 72 h, and most preferably 10 - 36 h.

[0319] In a specific embodiment of the present invention, in the method for preparing liposomes, the ratio of liposomes to phosphate buffer solution dissolved with cryoprotectant can be 1 mg:(0.1 - 100) mL, preferably 1 mg:(0.3 - 50) mL, and more preferably 1 mg:(0.5 - 5) mL.

[0320] In a specific embodiment of the present invention, preferably, lipid nanoparticles are prepared by a microfluidic method, and the steps are as follows:

[0321] (1) Dissolve each lipid component in an organic solvent to obtain a lipid composition dissolved in the organic phase; the organic phase is preferably ethanol;

[0322] (2) Add the nucleic acid drug to a buffer solution to obtain an aqueous solution; the aqueous phase is preferably citrate buffer salt or sodium acetate buffer solution;

[0323] (3) Mix the organic phase solution and the aqueous solution through a microfluidic device to form a lipid nanoparticle composition, and purify it by ultrafiltration or the like to remove the organic solvent and free nucleic acid molecules.

[0324] The preparation methods of cationic lipids, lipid compositions, and lipid pharmaceutical composition formulations, as well as the bioactivity tests of lipid pharmaceutical compositions, will be further described below in combination with some specific examples. The specific examples are for further detailed illustration of the present invention and do not limit the protection scope of the present invention. Among them, in the examples of preparing cationic lipids, the final products are characterized by nuclear magnetic resonance for their structures and confirmed for their molecular weights by mass spectrometry.

[0325] Example 1: Cationic Lipid (E1-1)

[0326]

[0327] The preparation process is as follows:

[0328] Step a: Under nitrogen protection, add dicyclohexylcarbodiimide (DCC, 3.63 g, 17.6 mmol) to a round-bottom flask containing 6-amino-1-hexanol (S1-1, 2.08 g, 9.6 mmol) with Boc-protected amino group, octanoic acid (S1-2, 1.15 g, 8.0 mmol), and 4-(dimethylamino)pyridine (DMAP, 0.24 g, 2.0 mmol) dissolved in dichloromethane (30 mL). React at room temperature for 16 h. After the reaction is completed, remove the precipitate by filtration, concentrate the filtrate, and purify the obtained crude product by column chromatography to obtain the esterified product S1-3 (2.55 g) containing Boc-protected amino group.

[0329] Step b: Remove the Boc protecting group. In a dry and clean round-bottom flask, prepare a solution of trifluoroacetic acid / dichloromethane (1:2, v / v). Slowly add a dichloromethane solution of S1-3 (1.86 g, 5.0 mmol) dropwise under ice bath conditions and react at room temperature for 2 hours. After the reaction is completed, add water to the reaction solution, stir evenly, extract, dry the organic phase with anhydrous magnesium sulfate, filter, concentrate the filtrate, and recrystallize to obtain the esterified product S1-4 (1.24 g, 91.4%) containing an exposed amino group.

[0330] Step c: Dissolve the above compound S1-4 (0.81 g, 3.0 mmol) and 1,2-epoxydecane (S1-5, 0.43 g, 3.0 mmol) in 15 mL of acetonitrile, then add calcium trifluoromethanesulfonate (Ca(OTf) 2 , 0.51 g, 1.5 mmol). Stir the reaction mixture at room temperature. After TLC shows that the reaction is complete, rotary evaporate to remove acetonitrile, add water (5 mL) and mix evenly, and extract with dichloromethane (5 mL * 3). Combine the organic layers, dry with anhydrous Na 2 SO 4 , filter, and concentrate. Purify the crude product by column chromatography to obtain the ring-opened product S1-6 (1.02 g) containing a hydroxyl group.

[0331] Step d: Under nitrogen protection, dissolve the above compound S1-6 (0.86 g, 2.0 mmol) in acetonitrile (15 mL). While stirring slowly, sequentially add S1-7 (1.05 g, 2.5 mmol, S1-7 is prepared by reacting 2-hexyldecanoic acid and 6-bromohexanol 1 H NMR (400 MHz, CDCl 3 3) δ: 4.03 (t, 4H), 3.57 - 3.54 (m, 1H), 3.10 - 2.83 (m, 6H), 2.31 - 2.24 (m, 3H), 1.86 - 1.75 (m, 4H), 1.65 - 1.20 (m, 64H), 0.88 (t, 12H). MS (ESI): m / z = 766.72 ([M + H] + +).

[0332]

[0333] Example 2: Cationic lipid (E2-1)

[0334]

[0335] The preparation process is as follows:

[0336] Step a: Under nitrogen protection, dissolve glycerol with a TBS-protected hydroxyl group (S2-1, 2.06 g, 10.0 mmol), potassium carbonate (K 2 2CO 3 3, 4.14 g, 30.0 mmol), and bromohexane (S2-2, 1.80 g, 11.0 mmol) in 30 mL of DMF. Stir the mixture at 110 °C for 16 hours. After TLC shows that the reaction is complete, pour the reaction solution into water (30 mL) for precipitation, filter, concentrate, and purify by column chromatography to obtain the glycerol ether S2-3 with a TBS-protected hydroxyl group (3.00 g, 88.7%).

[0337] Step b: Dissolve the above product S2-3 (1.88 g, 8.0 mmol) in THF (20 mL). Under nitrogen protection, add TBAF (20 mL, 1 M), and react overnight to remove the TBS protection. After the reaction is completed, concentrate the reaction solution to obtain the crude product of compound S2-4. Purify by column chromatography, concentrate, and dry with an oil pump to obtain the glycerol ether compound S2-4 containing a hydroxyl group (1.84 g, 88.4%).

[0338] Step d: Under nitrogen protection, add DCC (1.36 g, 6.6 mmol) to a round-bottom flask containing S2-4 (0.94 g, 3.6 mmol), 8-bromooctanoic acid (S2-5, 0.67 g, 3.0 mmol), and DMAP (91.50 mg, 0.8 mmol) dissolved in dichloromethane (20 mL). React at room temperature for 16 h. After the reaction is completed, remove the precipitate by filtration, concentrate the filtrate, and purify the obtained crude product by column chromatography to obtain the bromoester compound S2-6 (1.15 g).

[0339] Step c: Under nitrogen protection, dissolve compound S1-6 (0.64 g, 1.5 mmol) in acetonitrile (15 mL). Slowly stir and sequentially add S2-6 (0.87 g, 1.9 mmol) and DIPEA (0.14 g, 1.5 mmol), and stir and react at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous magnesium sulfate, filter, concentrate, and purify by column chromatography to obtain the cationic lipid E2,1 (1.03 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 5.13 - 5.06 (m, 1H), 4.05 - 3.99 (t, 2H), 3.57 - 3.49 (m, 5H), 3.46 - 3.36 (m, 4H), 2.53 - 2.43 (m, 2H), 2.38 - 2.17 (m, 8H), 1.60 - 1.20 (m, 62H), 0.88 (t, 12H). MS (ESI): m / z = 812.73 ([M+H] + ).

[0340]

[0341] Example 3: Cationic lipid (E3-1)

[0342]

[0343] The preparation process is as follows:

[0344] Step a: Dissolve 1,3-propanediol (S3-1, 9.50 g, 50 mmol) containing a TBS-protected hydroxyl group in 200 mL of dichloromethane solution, add pyridinium chlorochromate (PCC, 16.13 g, 75.0 mmol), stir at 15 °C for at least 2 hours, filter, concentrate under reduced pressure, and purify by column chromatography to obtain 3-hydroxypropionic acid S3-2 with a TBS-protected hydroxyl group (6.14 g).

[0345] Step b: Dissolve the above compound S3-2 (5.64 g, 30.0 mmol) and 1-octanol (S3-3, 9.75 g, 75.0 mmol) in 150 mL of dichloromethane solution, add p-toluenesulfonic acid monohydrate (TsOH·H 2 O, 1.14 g, 6.0 mmol) and anhydrous sodium sulfate (10.65 g, 75.0 mmol). Stir at 15 °C for at least 24 hours, filter, concentrate under reduced pressure, and purify the crude product by column chromatography to obtain acetal S3-4 containing a TBS-protected hydroxyl group (3.00 g).

[0346] Step c: Dissolve the above product S3-4 (2.16 g, 5.0 mmol) in THF (30 mL), under nitrogen protection, add TBAF (30 mL, 1 M), react overnight to remove the TBS protection. After the reaction is completed, concentrate the reaction solution to obtain the crude product of compound S3-5. Purify by column chromatography, concentrate, and dry with an oil pump to obtain acetal S3-5 with an exposed hydroxyl group (1.38 g, 87.1%).

[0347] Step d: Under nitrogen protection, add DCC (0.91 g, 4.4 mmol) to a round-bottom flask containing S3-5 (0.76 g, 2.4 mmol), S3-6 (0.39 g, 2.0 mmol), and DMAP (61.00 mg, 0.5 mmol) dissolved in dichloromethane (15 mL), and react at room temperature for 16 h. After the reaction is completed, remove the precipitate by filtration, concentrate the filtrate, and purify the obtained crude product by silica gel column chromatography to obtain bromoester S3-7 (0.81 g).

[0348] Step e: Under nitrogen protection, dissolve compound S1-6 (0.43 g, 1.0 mmol) in acetonitrile (10 mL), and sequentially add S3-7 (0.62 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) with slow stirring, and stir at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence, combine the organic phases, dry with anhydrous magnesium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E3-1 (0.71 g). 11H NMR (400 MHz, CDCl 3 ) δ: 4.65 (t, 1H), 4.07 (t, 4H), 3.57 - 3.54 (m, 1H), 3.52 - 3.36 (m, 4H), 2.53 - 2.43 (m, 2H), 2.38 - 2.19 (m, 8H), 1.71 - 1.22 (m, 68H), 0.87 (t, 12H). MS (ESI): m / z = 840.76 ([M+H] + ).

[0349]

[0350] Example 4: Cationic Lipid (E4-1)

[0351]

[0352] The preparation process is as follows:

[0353] Step a: Under nitrogen protection, to a round-bottom flask containing S2-4 (2.50 g, 9.6 mmol) dissolved in dichloromethane (30 mL), Boc-protected 8-aminooctanoic acid (S4-1, 2.07 g, 8.0 mmol), and DMAP (0.24 g, 2.0 mmol), add DCC (3.63 g, 17.6 mmol). React at room temperature for 16 h. After the reaction is completed, remove the precipitate by filtration, concentrate the filtrate, and purify the obtained crude product by column chromatography to obtain the esterified product S4-2 (3.45 g) containing Boc-protected amino group.

[0354] Step b: Remove the Boc protecting group. In a dry and clean round-bottom flask, prepare a solution of trifluoroacetic acid / dichloromethane (1:2, v / v). Slowly add a dichloromethane solution of S4-2 (2.51 g, 5.0 mmol) dropwise under ice bath conditions and react at room temperature for 2 h. After the reaction is completed, add water to the reaction solution, stir evenly, extract, dry the organic phase with anhydrous magnesium sulfate, filter, concentrate the filtrate, and recrystallize to obtain the esterified product S4-3 (1.84 g, 91.6%) containing an exposed amino group.

[0355] Step c: Dissolve the above compound S4-3 (1.21 g, 3.0 mmol) and S1-5 (0.43 g, 3.0 mmol) in 20 mL of acetonitrile, then add Ca(OTf) 2 (0.51 g, 1.5 mmol), stir the reaction mixture at room temperature. After TLC shows that the reaction is complete, evaporate the acetonitrile under reduced pressure, add water (10 mL), mix evenly, and extract with dichloromethane (10 mL * 3). Combine the organic phases, and use anhydrous Na 2 SO 4Dry, filter, and concentrate. The crude product was purified by column chromatography to obtain the ring-opened product S4-4 containing a hydroxyl group (1.33 g).

[0356] Step d: Under nitrogen protection, dissolve the above compound S4-4 (1.12 g, 2.0 mmol) in acetonitrile (20 mL). While stirring slowly, sequentially add S3-7 (1.24 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry over anhydrous magnesium sulfate, filter, concentrate, and purify by column chromatography to obtain the cationic lipid E4-1 (1.63 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 5.13 - 5.06 (m, 1H), 4.64 (t, 1H), 4.07 (t, 2H), 3.57 - 3.36 (m, 13H), 2.52 - 2.43 (m, 2H), 2.39 - 2.17 (m, 8H), 1.71 - 1.22 (m, 72H), 0.89 (t, 15H). MS (ESI): m / z = 970.86 ([M+H] + ).

[0357] Example 5: Cationic lipid (E5-1)

[0358]

[0359] The preparation process is as follows:

[0360] Step a: Under nitrogen protection, dissolve 6-bromohexyl 4-nitrophenyl carbonate (S5-1, 3.46 g, 10.0 mmol, S5-1 was prepared by reacting p-nitrophenyl chloroformate with 6-bromohexanol) in dichloromethane (100 mL). While stirring at room temperature, dropwise add pentadecane-7-ol (S5-2, 9.12 g, 40.0 mmol), and then slowly dropwise add pyridine (1.00 mL, 12.5 mmol) over 10 min. Then, add DMAP (0.24 g, 2.0 mmol) in one portion. Stir the reaction at room temperature for 16 h. After the reaction is completed, extract twice with dichloromethane, combine the organic phases, wash with brine, then dry over anhydrous magnesium sulfate, filter, and concentrate to obtain the crude product. The crude product was separated and purified by column chromatography to obtain the brominated carbonate (S5-3, 1.13 g).

[0361] Step b: Under nitrogen protection, dissolve the above compound S1-6 (0.64 g, 1.5 mmol) in acetonitrile (10 mL). Under slow stirring, sequentially add S5-3 (0.82 g, 1.9 mmol) and DIPEA (0.14 g, 1.5 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous magnesium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E5-1 (0.99 g). 1 H NMR(400MHz, CDCl 3 )δ: 4.71-4.63(m, 1H), 4.11(t, 2H), 4.05(t, 2H), 3.57-3.54(m, 1H), 2.53-2.43(m, 2H), 2.38-2.18(m, 6H), 1.71-1.22(m, 68H), 0.89(t, 12H). MS(ESI): m / z = 782.72([M+H] + ).

[0362]

[0363] Example 6: Cationic lipid (E6-1)

[0364]

[0365] The preparation process is as follows:

[0366] Under nitrogen protection, dissolve the above compound S1-6 (0.64 g, 1.5 mmol) in acetonitrile (10 mL). Under slow stirring, sequentially add S6-1 (0.87 g, 1.9 mmol, S6-1 is prepared by reacting 8-bromooctanoic acid and 9-heptadecanol and DIPEA (0.14 g, 1.5 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous magnesium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E6-1 (1.02 g). 1 H NMR(400MHz, CDCl 3)δ: 4.91 - 4.83 (m, 1H), 4.06 (t, 2H), 3.57 - 3.54 (m, 1H), 2.53 - 2.43 (m, 2H), 2.38 - 2.17 (m, 8H), 1.71 - 1.22 (m, 74H), 0.88 (t, 12H). MS(ESI): m / z = 808.77 ([M+H] + )。

[0367]

[0368] Example 7: Cationic Lipid (E7-1)

[0369]

[0370] The preparation process is as follows:

[0371] Step a: Dissolve compound S1-4 (0.81 g, 3.0 mmol) and 1,2-epoxy-9-decene (S7-1, 0.46 g, 3.0 mmol) in 15 mL of acetonitrile, then add Ca(OTf) 2 (0.51 g, 1.5 mmol). The reaction mixture is stirred at room temperature. After TLC shows the reaction is complete, remove acetonitrile by rotary evaporation, add water (5 mL) and mix evenly, and extract with dichloromethane (5 mL * 3). Combine the organic layers, dry with anhydrous Na 2 SO 4 Dry, filter, and concentrate. The crude product is purified by column chromatography to obtain the ring-opened product S7-2 containing a hydroxyl group (1.02 g).

[0372] Step b: Under nitrogen protection, dissolve the above compound S7-2 (0.85 g, 2.0 mmol) in acetonitrile (15 mL). Slowly stir and sequentially add S1-7 (1.05 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol), and stir at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, and extract with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. Combine the organic phases, dry with anhydrous magnesium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E7-1 (1.28 g). 1 H NMR (400 MHz, CDCl 3)δ: 5.87 - 5.75 (m, 1H), 5.05 - 4.82 (d, 2H), 4.07 (t, 4H), 3.57 - 3.54 (m, 1H), 2.53—2.43 (m, 2H), 2.38 - 2.17 (m, 7H), 2.11 - 1.94 (m, 2H), 1.71 - 1.22 (m, 64H), 0.88 (t, 9H). MS(ESI): m / z = 764.71 ([M + H] + ).

[0373]

[0374] Example 8: Cationic Lipid (E8 - 1)

[0375]

[0376] Replace the raw material S7 - 1 in Example 7 with the raw material glycidyl butyrate (S8 - 1, 0.43 g, 3.0 mmol), and prepare according to the same reaction steps to obtain E8 - 1 (1.26 g). 1 H NMR(400 MHz, CDCl 3 )δ: 4.12 - 4.00 (m, 6H), 3.86—3.78 (m, 1H), 2.53 - 2.43 (m, 2H), 2.32 - 2.19 (m, 9H), 1.72 - 1.22 (m, 56H), 0.90 (t, 12H). MS(ESI): m / z = 754.65 ([M + H] + ).

[0377]

[0378] Example 9: Cationic Lipid (E9 - 1)

[0379]

[0380] Replace the raw material S7 - 1 in Example 7 with the raw material allyl glycidyl ether (S9 - 1, 0.34 g, 3.0 mmol), and prepare according to the same reaction steps to obtain E9 - 1 (1.21 g). 1 H NMR(400 MHz, CDCl 3)δ: 5.85 - 5.76 (m, 1H), 5.16 - 5.07 (d, 2H), 4.07 (t, 4H), 3.96 - 3.92 (d, 2H), 3,76 - 3.69 (m, 1H), 3.36 - 3.32 (m, 2H), 2.53 - 2.43 (m, 2H), 2.38 - 2.17 (m, 7H), 1.71 - 1.22 (m, 54H), 0,88 (t, 9H). MS(ESI): m / z = 724.64 ([M + H] + )。

[0381]

[0382] Example 10: Cationic Lipid (E10 - 1)

[0383]

[0384] The preparation process is as follows:

[0385] Under nitrogen protection, DCC (0.91 g, 4.4 mmol) was added to a round-bottom flask containing E1 - 1 (1.84 g, 2.4 mmol), 3-(dimethylamino)propionic acid (S10 - 1, 0.23 g, 2.0 mmol), and DMAP (61.00 mg, 0.5 mmol) dissolved in dichloromethane (30 mL), and the reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated, and the crude product obtained was purified by column chromatography to obtain cationic lipid E10 - 1 (1.47 g). 1 H NMR (400 MHz, CDCl 3 )δ: 4.94 - 4.88 (m, 1H), 4.05 - 4.01 (m, 4H), 2.62 - 2.24 (m, 13H), 2.24 - 2.22 (s, 6H), 1.59 - 1.21 (m, 68H), 0.87 (t, 12H). MS(ESI): m / z = 865.79 ([M + H] + )。

[0386]

[0387] Example 11: Cationic Lipid (E11 - 1)

[0388]

[0389] The raw material E1 - 1 in Example 10 was replaced with raw material E2 - 1 (1.95 g, 2.4 mmol), and the preparation was carried out according to the same reaction steps to obtain E11 - 1 (1.52 g). 1 H NMR (400 MHz, CDCl 3)δ: 5.13 - 5.06 (m, 1H), 4.94 - 4.88 (m, 1H), 4.02 (t, 2H), 3.57 - 3.49 (m, 4H), 3.46 - 3.36 (m, 4H), 2.62 - 2.22 (m, 20H), 1.60 - 1.20 (m, 62H), 0.88 (t, 12H). MS(ESI): m / z = 911.79 ([M + H] + )。

[0390]

[0391] Example 12: Cationic Lipid (E12 - 1)

[0392]

[0393] Replace the raw material E1 - 1 in Example 10 with raw material E3 - 1 (2.02 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E12 - 1 (1.58 g). 1 H NMR (400 MHz, CDCl 3 )δ: 4.94 - 4.88 (m, 1H), 4.65 (t, 1H), 4.07 (t, 4H), 3.52 - 3.36 (m, 4H), 2.61 - 2.19 (m, 20H), 1.71 - 1.22 (m, 68H), 0.87 (t, 12H). MS(ESI): m / z = 939.83 ([M + H] + )。

[0394] Example 13: Cationic Lipid (E13 - 1)

[0395]

[0396] Replace the raw material E1 - 1 in Example 10 with raw material E4 - 1 (2.33 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E13 - 1 (1.79 g). 1 H NMR (400 MHz, CDCl 3 )δ: 5.13 - 5.06 (m, 1H), 4.94 - 4.86 (m, 1H), 4.65 (t, 1H), 4.07 (t, 2H), 3.57 - 3.36 (m, 12H), 2.62 - 2.17 (m, 20H), 1.71 - 1.22 (m, 72H), 0.89 (t, 15H). MS(ESI): m / z = 1069.93 ([M + H] + )。

[0397] Example 14: Cationic Lipid (E14 - 1)

[0398]

[0399] Replace the raw material E1-1 in Example 10 with raw material E5-1 (1.88 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E14-1 (1.46 g). 1 H NMR(400MHz, CDCl 3 ) δ: 4.98 - 4.87(m, 1H), 4.71 - 4.63(m, 1H), 4.10(t, 2H), 4.04(t, 2H), 2.64 - 2.57(m, 2H), 2.52 - 2.34(m, 8H), 2.31 - 2.18(m, 8H), 1.65 - 1.53(m, 10H), 1.39 - 1.23(m, 58H), 0.88(t, 12H). MS(ESI): m / z = 881.79([M+H] + )。

[0400] Example 15: Cationic lipid (E15-1)

[0401]

[0402] Replace the raw material E1-1 in Example 10 with raw material E6-1 (1.94 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E15-1 (1.52 g). 1 H NMR(400MHz, CDCl 3 ) δ: 4.94 - 4.83(m, 2H), 4.06(t, 2H), 2.63 - 2.17(m, 20H), 1.71 - 1.22(m, 74H), 0.88(t, 12H). MS(ESI): m / z = 907.85([M+H] + )。

[0403] Example 16: Cationic lipid (E16-1)

[0404]

[0405] Replace the raw material E1,1 in Example 10 with raw material E7-1 (1.83 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E16-1 (1.44 g). 1 H NMR(400MHz, CDCl 3)δ: 5.87 - 5.75 (m, 1H), 5.05 - 4.82 (m, 3H), 4.07 (t, 4H), 2.63 - 2.17 (m, 19H), 2.11 - 1.94 (m, 2H), 1.71 - 1.22 (m, 64H), 0.88 (t, 9H). MS(ESI): m / z = 863.77 ([M + H] + )。

[0406] Example 17: Cationic Lipid (E17 - 1)

[0407]

[0408] Replace the raw material E1 - 1 in Example 10 with raw material E8 - 1 (1.81 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E17 - 1 (1.44 g). 1 H NMR(400 MHz, CDCl 3 )δ: 4.94 - 4.88 (m, 1H), 4.12 - 4.00 (m, 6H), 2.63 - 2.19 (m, 21H), 1.72 - 1.22 (m, 56H), 0.90 (t, 12H). MS(ESI): m / z = 853.73 ([M + H] + )。

[0409] Example 18: Cationic Lipid (E18 - 1)

[0410]

[0411] Replace the raw material E1 - 1 in Example 10 with raw material E9 - 1 (1.74 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E18 - 1 (1.39 g). 1 H NMR(400 MHz, CDCl 3 )δ: 5.85 - 5.76 (m, 1H), 5.16 - 5.07 (d, 2H), 4.94 - 4.88 (m, 1H), 4.07 (t, 4H), 3.96 - 3.92 (d, 2H), 3.36 - 3.32 (m, 2H), 2.63 - 2.19 (m, 19H), 1.71 - 1.22 (m, 54H), 0.88 (t, 9H). MS(ESI): m / z = 823.71 ([M + H] + )。

[0412] Example 19: Cationic Lipid (E19 - 1)

[0413]

[0414] Replace the raw material S10-1 in Example 10 with the raw material 3-(4-phenyl-piperazin-1-yl)-propionic acid (S19-1, 0.34 g, 2.0 mmol), and prepare according to the same reaction steps to obtain E19-1 (1.56 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 4.93 - 4.87 (m, 1H), 4.04 - 4.00 (m, 4H), 2.68 - 2.24 (m, 24H), 1.60 - 1.21 (m, 68H), 0.86 (t, 12H). MS (ESI): m / z = 920.83 ([M+H] + ).

[0415]

[0416] Example 20: Cationic lipid (E20-1)

[0417]

[0418] Replace the raw material E1-1 in Example 19 with the raw material E2-1 (1.95 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E20-1 (1.62 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 5.13 - 5.06 (m, 1H), 4.93 - 4.87 (m, 1H), 4.05 - 3.99 (t, 2H), 3.57 - 3.49 (m, 4H), 3.46 - 3.36 (m, 4H), 2.68 - 2.24 (m, 25H), 1.60 - 1.20 (m, 62H), 0.88 (t, 12H). MS (ESI): m / z = 966.84 ([M+H] + ).

[0419] Example 21: Cationic lipid (E21-1)

[0420]

[0421] Replace the raw material E1-1 in Example 19 with the raw material E3-1 (2.02 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E21-1 (1.67 g). 1 H NMR (400 MHz, CDCl 3) δ: 4.93 - 4.87 (m, 1H), 4.65 (t, 1H), 4.07 (t, 4H), 3.52 - 3.36 (m, 4H), 2.63 - 2.23 (m, 25H), 1.71 - 1.22 (m, 68H), 0.87 (t, 12H). MS(ESI): m / z = 994.87 ([M + H] + )。

[0422] Example 22: Cationic Lipid (E22 - 1)

[0423]

[0424] Replace the raw material E1 - 1 in Example 19 with raw material E4 - 1 (2.33 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E22 - 1 (1.87 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 5.13 - 5.06 (m, 1H), 4.93 - 4.87 (m, 1H), 4.64 (t, 1H), 4.07 (t, 2H), 3.57 - 3.36 (m, 12H), 2.62 - 2.23 (m, 25H), 1.71 - 1.22 (m, 72H), 0.89 (t, 15H). MS(ESI): m / z = 1124.97 ([M + H] + )。

[0425] Example 23: Cationic Lipid (E23 - 1)

[0426]

[0427] Replace the raw material E1 - 1 in Example 19 with raw material E5 - 1 (1.88 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E23 - 1 (1.56 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 4.98 - 4.88 (m, 1H), 4.71 - 4.63 (m, 1H), 4.10 (t, 2H), 4.05 (t, 2H), 2.63 - 2.19 (m, 23H), 1.69 - 1.22 (m, 68H), 0.89 (t, 12H). MS(ESI): m / z = 936.83 ([M + H] + )。

[0428] Example 24: Cationic Lipid (E24 - 1)

[0429]

[0430] Replace the raw material E1-1 in Example 19 with raw material E6-1 (1.94 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E24-1 (1.61 g). 1 H NMR(400MHz, CDCl 3 ) δ: 4.93 - 4.83 (m, 2H), 4.06 (t, 2H), 2.68 - 2.24 (m, 25H), 1.71 - 1.22 (m, 74H), 0.88 (t, 1 2H). MS(ESI): m / z = 962.87 ([M+H] + )。

[0431] Example 25: Cationic lipid (E25-1)

[0432]

[0433] Replace the raw material E1-1 in Example 19 with raw material E7-1 (1.83 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E25-1 (1.55 g). 1 H NMR(400MHz, CDCl 3 ) δ: 5.87 - 5.75 (m, 1H), 5.05 - 4.82 (m, 3H), 4.07 (t, 4H), 2.67 - 2.23 (m, 24H), 2.11 - 1.94 (m, 2H), 1.71 - 1.22 (m, 64H), 0.88 (t, 9H). MS(ESI): m / z = 918.82 ([M+H] + )。

[0434] Example 26: Cationic lipid (E26-1)

[0435]

[0436] Replace the raw material E1-1 in Example 19 with raw material E8-1 (1.81 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E26-1 (1.53 g). 1 H NMR(400MHz, CDCl 3 ) δ: 4.93 - 4.87 (m, 1H), 4.12 - 4.00 (m, 6H), 2.66 - 2.23 (m, 26H), 1.72 - 1.22 (m, 56H), 0.90 (t, 12H). MS(ESI): m / z = 908.76 ([M+H] + )。

[0437] Example 27: Cationic lipid (E27-1)

[0438]

[0439] Replace the raw material E1-1 in Example 19 with the raw material E9-1 (1.74 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E27-1 (1.48 g). 1 H NMR(400MHz, CDCl 3 ) δ: 5.85 - 5.76(m, 1H), 5.16 - 5.07(d, 2H), 4.93 - 4.87(m, 1H), 4.07(t, 4H), 3.96 - 3.92(d, 2H), 3.36 - 3.32(m, 2H), 2.68 - 2.22(m, 24H), 1.71 - 1.22(m, 54H), 0.88(t, 9H). MS(ESI): m / z = 878.75([M + H] + )。

[0440] Example 28: Cationic lipid (E28-1)

[0441]

[0442] Replace the raw material S10-1 in Example 10 with the raw material 1-piperidinepropionic acid (S28-1, 0.31 g, 2.0 mmol), and prepare according to the same reaction steps to obtain E28-1 (1.54 g). 1 H NMR(400MHz, CDCl 3 ) δ: 4.92 - 4.87(m, 1H), 4.04 - 4.01(m, 4H), 2.68 - 2.24(m, 17H), 1.60 - 1.21(m, 74H), 0.86(t, 12H). MS(ESI): m / z = 905.82([M + H] + )。

[0443]

[0444] Example 29: Cationic lipid (E29-1)

[0445]

[0446] Replace the raw material E1-1 in Example 28 with the raw material E2-1 (1.95 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E29-1 (1.58 g). 1 H NMR(400MHz, CDCl 3)δ: 5.13 - 5.06 (m, 1H), 4.93 - 4.87 (m, 1H), 4.03 (t, 2H), 3.57 - 3.49 (m, 4H), 3.46 - 3.36 (m, 4H), 2.68 - 2.24 (m, 18H), 1.60 - 1.20 (m, 68H), 0.88 (t, 12H). MS(ESI): m / z = 951.83 ([M + H] + )。

[0447] Example 30: Cationic Lipid (E30 - 1)

[0448]

[0449] Replace the raw material E1 - 1 in Example 28 with raw material E3 - 1 (2.02 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E30 - 1 (1.64 g). 1 H NMR (400 MHz, CDCl 3 )δ: 4.93 - 4.87 (m, 1H), 4.65 (t, 1H), 4.03 (t, 4H), 3.52 - 3.36 (m, 4H), 2.63 - 2.23 (m, 18H), 1.71 - 1.22 (m, 74H), 0.87 (t, 12H). MS(ESI): m / z = 979.86 ([M + H] + )。

[0450] Example 31: Cationic Lipid (E31 - 1)

[0451]

[0452] Replace the raw material E1 - 1 in Example 28 with raw material E4 - 1 (2.33 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E31 - 1 (1.85 g). 1 H NMR (400 MHz, CDCl 3 )δ: 5.13 - 5.06 (m, 1H), 4.93 - 4.87 (m, 1H), 4.64 (t, 1H), 4.04 (t, 2H), 3.57 - 3.36 (m, 12H), 2.62 - 2.23 (m, 18H), 1.71 - 1.22 (m, 78H), 0.89 (t, 15H). MS(ESI): m / z = 1109.96 ([M + H] + )。

[0453] Example 32: Cationic Lipid (E32 - 1)

[0454]

[0455] Replace raw material E1-1 in Example 28 with raw material E5-1 (1.88 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E32-1 (1.56 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 4.97 - 4.87 (m, 1H), 4.71 - 4.62 (m, 1H), 4.12 (t, 2H), 4.04 (t, 2H), 2.64 - 2.20 (m, 16H), 1.68 - 1.22 (m, 74H), 0.89 (t, 12H). MS (ESI): m / z = 921.82 ([M+H] + )。

[0456] Example 33: Cationic Lipid (E33-1)

[0457]

[0458] Replace raw material E1-1 in Example 28 with raw material E6-1 (1.94 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E33-1 (1.58 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 4.94 - 4.88 (m, 1H), 4.87 - 4.81 (m, 1H), 4.03 (t, 2H), 2.65 - 2.23 (m, 18H), 1.60 - 1.22 (m, 80H), 0.85 (t, 12H). MS (ESI): m / z = 947.87 ([M+H] + )。

[0459] Example 34: Cationic Lipid (E34-1)

[0460]

[0461] Replace raw material E1-1 in Example 28 with raw material E7-1 (1.83 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E34-1 (1.52 g). 1 H NMR (400 MHz, CDCl 3 ) δ:. 5.88 - 5.75 (m, 1H), 5.04 - 4.82 (m, 3H), 4.07 (t, 4H), 2.67 - 2.23 (m, 17H), 2.11 - 1.94 (m, 2H), 1.71 - 1.22 (m, 70H), 0.88 (t, 9H). MS (ESI): m / z = 903.81 ([M+H] + )。

[0462] Example 35: Cationic Lipid (E35-1)

[0463]

[0464] Replace the raw material E1-1 in Example 28 with the raw material E8-1 (1.81 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E35-1 (1.51 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 4.93 - 4.87 (m, 1H), 4.12 - 4.00 (m, 6H), 2.66 - 2.23 (m, 19H), 1.72 - 1.22 (m, 62H), 0.90 (t, 12H). MS (ESI): m / z = 893.75 ([M+H] + )

[0465] Example 36: Cationic Lipid (E36-1)

[0466]

[0467] Replace the raw material E1-1 in Example 28 with the raw material E9-1 (1.74 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E36-1 (1.46 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 5.86 - 5.76 (m, 1H), 5.16 - 5.08 (d, 2H), 4.94 - 4.88 (m, 1H), 4.05 (t, 4H), 3.96 - 3.92 (d, 2H), 3.36 - 3.32 (m, 2H), 2.68 - 2.22 (m, 17H), 1.71 - 1.22 (m, 60H), 0.88 (t, 9H). MS (ESI): m / z = 863.74 ([M+H] + )

[0468] Example 37: Cationic Lipid (E37-1)

[0469]

[0470] Replace the raw material S10-1 in Example 10 with the raw material 3-(1-pyrrolidinyl)propionic acid (S37-1, 0.29 g, 2.0 mmol), and prepare according to the same reaction steps to obtain E37-1 (1.51 g). 1 H NMR (400 MHz, CDCl 3) δ: 4.94 - 4.87 (m, 1H), 4.04 - 4.00 (m, 4H), 2.68 - 2.24 (m, 17H), 1.75 - 1.21 (m, 72H), 0.86 (t, 12H). MS(ESI): m / z = 891.81 ([M + H] + )。

[0471]

[0472] Example 38: Cationic Lipid (E38-1)

[0473]

[0474] Replace the raw material E1-1 in Example 37 with raw material E2-1 (1.95 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E38-1 (1.56 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 5.14 - 5.06 (m, 1H), 4.93 - 4.87 (m, 1H), 4.05 - 3.99 (t, 2H), 3.57 - 3.49 (m, 4H), 3.46 - 3.36 (m, 4H), 2.68 - 2.24 (m, 18H), 1.75 - 1.20 (m, 66H), 0.88 (t, 12H). MS(ESI): m / z = 937.81 ([M + H] + )。

[0475] Example 39: Cationic Lipid (E39-1)

[0476]

[0477] Replace the raw material E1-1 in Example 37 with raw material E3-1 (2.02 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E39-1 (1.62 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 4.93 - 4.87 (m, 1H), 4.65 (t, 1H), 4.07 (t, 4H), 3.52 - 3.36 (m, 4H), 2.63 - 2.23 (m, 18H), 1.77 - 1.22 (m, 72H), 0.87 (t, 12H). MS(ESI): m / z = 965.84 ([M + H] + )。

[0478] Example 40: Cationic Lipid (E40-1)

[0479]

[0480] Replace the raw material E1-1 in Example 37 with the raw material E4-1 (2.33 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E40-1 (1.86 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 5.13 - 5.06 (m, 1H), 4.93 - 4.87 (m, 1H), 4.64 (t, 1H), 4.07 (t, 2H), 3.57 - 3.36 (m, 12H), 2.62 - 2.23 (m, 18H), 1.75 - 1.22 (m, 76H), 0.89 (t, 15H). MS (ESI): m / z = 1095.94 ([M+H] + )。

[0481] Example 41: Cationic lipid (E41-1)

[0482]

[0483] Replace the raw material E1-1 in Example 37 with the raw material E5-1 (1.88 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E41-1 (1.52 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 4.98 - 4.87 (m, 1H), 4.71 - 4.63 (m, 1H), 4.10 (t, 2H), 4.04 (t, 2H), 2.63 - 2.19 (m, 16H), 1.75 - 1.22 (m, 72H), 0.89 (t, 12H). MS (ESI): m / z = 907.80 ([M+H] + )。

[0484] Example 42: Cationic lipid (E42-1)

[0485]

[0486] Replace the raw material E1-1 in Example 37 with the raw material E6-1 (1.94 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E42-1 (1.56 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 4.93 - 4.83 (m, 2H), 4.06 (t, 2H), 2.68 - 2.24 (m, 18H), 1.76 - 1.22 (m, 78H), 0.88 (t, 12H). MS (ESI): m / z = 933.86 ([M+H] + )。

[0487] Example 43: Cationic Lipid (E43-1)

[0488]

[0489] Replace the raw material E1-1 in Example 37 with raw material E7-1 (1.83 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E43-1 (1.50 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 5.87 - 5.75 (m, 1H), 5.05 - 4.82 (m, 3H), 4.07 (t, 4H), 2.67 - 2.23 (m, 17H), 2.11 - 1.94 (m, 2H), 1.75 - 1.22 (m, 68H), 0.88 (t, 9H). MS (ESI): m / z = 889.79 ([M+H] + )

[0490] Example 44: Cationic Lipid (E44-1)

[0491]

[0492] Replace the raw material E1-1 in Example 37 with raw material E8-1 (1.81 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E44-1 (1.48 g). 1 H NMR (400 MHz, CDCl 3 ) δ: 4.93 - 4.87 (m, 1H), 4.12 - 4.00 (m, 6H), 2.66 - 2.23 (m, 19H), 1.75 - 1.22 (m, 60H), 0.90 (t, 12H). MS (ESI): m / z = 879.73 ([M+H] + )

[0493] Example 45: Cationic Lipid (E45-1)

[0494]

[0495] Replace the raw material E1-1 in Example 37 with raw material E9-1 (1.74 g, 2.4 mmol), and prepare according to the same reaction steps to obtain E45-1 (1.44 g). 1 H NMR (400 MHz, CDCl 3)δ: 5.85 - 5.76 (m, 1H), 5.16 - 5.07 (d, 2H), 4.93 - 4.87 (m, 1H), 4.07 (t, 4H), 3.96 - 3.92 (d, 2H), 3.36 - 3.32 (m, 2H), 2.68 - 2.22 (m, 17H), 1.76 - 1.22 (m, 58H), 0.88 (t, 9H). MS(ESI): m / z = 849.72 ([M+H] + ).

[0496] Example 46. Preparation of Polyethylene Glycolated Lipids E46-1 and E46-2

[0497] Example 46.1 Preparation of Polyethylene Glycolated Lipid E46-1

[0498]

[0499] The preparation process is as follows:

[0500] Step a: Compound S46-1 (20.00 g, 10.0 mmol, mPEG-OH, Mw about 2000, n 1 ≈45, PDI = 1.03), toluene (200 mL) were azeotropically dehydrated at 140 °C. After 60 mL of the solvent was distilled off, the reaction was cooled to room temperature. TEA (2.02 g, 20.0 mmol) and MsCl (2.05 g, 18.0 mmol) were added, and the reaction was stirred overnight at room temperature. After the reaction was completed, the reaction solution was poured into water (200 mL), extracted twice with EtOAc (100 mL * 2), the aqueous phase was retained, and the aqueous phase was further extracted twice with dichloromethane (100 mL * 2). The organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50 °C, and recrystallized in an ice bath, and filtered to obtain compound S46-2 (18.00 g, 90%).

[0501] Step b: The above compound S46-2 (18.00 g, 9.0 mmol) was added to 80 mL of water and stirred to dissolve at room temperature. Potassium carbonate (12.42 g, 90.0 mmol), compound S46-3 (9.58 g, 45.0 mmol) and tetra-n-butylammonium bromide (0.29 g, 0.9 mmol) were added, and the reaction solution was stirred at room temperature for 72 hours. After the reaction was completed, it was extracted twice with dichloromethane (100 mL * 2), the organic phases were combined, backwashed once with saturated sodium chloride aqueous solution (100 mL), the organic phase was retained, dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product of compound S46-4. It was purified by column chromatography, concentrated, and dried with an oil pump to obtain the target compound S46-4 (12.00 g).

[0502] Step c: The above compound S46-4 (12.00 g, 6.0 mmol) was dissolved in dry THF (120 mL), and NaH (60%, 2.40 g, 60.0 mmol) was slowly added thereto under an ice bath, and the reaction was carried out for 1 hour under the ice bath. Compound S46-5 (8.28 g, 30.0 mmol) was added, and after stirring and reacting for 1 hour under the ice bath, the reaction was slowly warmed to room temperature and reacted overnight. After the reaction was completed, the reaction was placed in an ice bath, and 2 mL of methanol was slowly added to quench the reaction. After stirring for 30 minutes, water (300 mL) was added and stirred. It was extracted twice with EtOAc (150 mL×2), and the aqueous phase was retained. Then it was extracted twice with dichloromethane (100 mL×2), and the organic phases were collected and combined, and washed once with saturated sodium chloride (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product of polyethylene glycolated lipid E46-1. It was purified by column chromatography, concentrated, and dried by an oil pump to obtain polyethylene glycolated lipid E46-1 (9.00 g). 1 H NMR(400MHz, CDCl 3 ) δ: 3.85 - 3.45 (m, 182H), 3.37 (s, 3H), 3.15 (t, 2H), 2.94 (t, 2H), 2.62 (t, 2H), 1.58 - 1.48 (m, 4H), 1.36 - 1.19 (m, 44H), 0.86 (t, 6H). Tested by MALDI-TOF-MS, the molecular weight of E46-1 was determined to be 2447 Da, and PDI = 1.03.

[0503]

[0504] Example 46.2 Preparation of Polyethylene Glycolated Lipid E46-2

[0505]

[0506] The preparation process is as follows:

[0507] The above compound S46-4 (11.26 g, 5.0 mmol), compound S46-6 (1.95 g, 6.0 mmol), and triethylamine (TEA, 0.76 g, 7.5 mmol) were dissolved in dichloromethane (100 mL), and the reaction was stirred overnight at room temperature. After the reaction solution was concentrated, it was dissolved in 100 mL of water, extracted twice with EtOAc (100 mL×2), and the aqueous phase was retained. Sodium chloride was added, and it was extracted twice with dichloromethane (100 mL×2), and the organic phases were combined and then washed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography, concentrated, and dried by an oil pump to obtain polyethylene glycolated lipid E46-2 (10.1 g, 84.8%). 1 HNMR(400MHz, CDCl3 ) δ: 3.84 - 3.45 (m, 182H), 3.37 (s, 3H), 3.35 (t, 2H), 3.18 (t, 2H), 2.27 (t, 2H), 1.56 - 1.40 (m, 4H), 1.36 - 1.18 (m, 46H), 0.87 (t, 6H). Determined by MALDI - TOF - MS, the molecular weight of E46 - 2 is 2461 Da and PDI = 1.03.

[0508]

[0509] Example 47: Preparation of LNP - mRNA Pharmaceutical Composition and Testing of Its Physicochemical Properties

[0510] Example 47.1: Preparation of LNP - mRNA Pharmaceutical Composition

[0511] In this example, multiple groups of LNP - mRNA pharmaceutical compositions containing Fluc - mRNA were prepared for comparison. The phospholipid in each composition is DSPC, and the sterol lipid is cholesterol. The differences lie in the two components of cationic lipid and polyethylene glycol - lipid. Among them, the control group L - 0: the cationic lipid is ALC - 0315 and contains polyethylene glycol - lipid PEG2k - DMG (abbreviated as DMG); experimental group series (L - 1 to L - 45): the cationic lipid is the cationic lipid prepared in the examples of this application, and the polyethylene glycol - lipid is PEG2k - DMG; experimental groups (L - 46 to L - 47): the cationic lipid is the cationic lipid prepared in the examples of this application, and the polyethylene glycol - lipid is the polyethylene glycol - lipid E46 - 1 or E46 - 2 prepared in this application; specifically as shown in Table 1.

[0512] The preparation method of the LNP - mRNA pharmaceutical composition is as follows:

[0513] Step a: Dissolve the cationic lipid, DSPC, cholesterol, and polyethylene glycol - lipid in ethanol according to a molar ratio of 48:9:42:1.5 to obtain an ethanol - phase solution;

[0514] Step b: Add Fluc - mRNA to 10 - 50 mM citrate buffer (pH = 4) to obtain an aqueous - phase solution;

[0515] Step c: Mix the ethanol - phase solution and the aqueous - phase solution (1:3 v / v) to prepare LNP - mRNA, and wash it by ultrafiltration with DPBS multiple times to remove ethanol and free molecules. Finally, filter it through a 0.2 - μm sterile filter to obtain the LNP - mRNA pharmaceutical composition.

[0516] Example 47.2: Testing of Physicochemical Properties of LNP - mRNA Pharmaceutical Composition

[0517] Determination of encapsulation efficiency: The encapsulation efficiency of the LNP-mRNA composition was determined using the Quant-it Ribogreen RNA Quantification Kit. The results showed that the lipid compositions (L-1 to L-47) of the present invention had a high encapsulation efficiency for nucleic acid drugs (mRNA), all within the range of 80%-98%, and most of the encapsulation efficiencies were within the range of 90%-98%. The results indicated that the lipid compositions prepared from most of the cationic lipids in each experimental group could well encapsulate mRNA, showing an encapsulation efficiency similar to or even superior to that of the existing cationic lipid ALC-0315. There were also differences in the encapsulation efficiencies of different cationic lipids.

[0518] Particle size determination: In this example, the particle size of LNP-mRNA was determined by dynamic light scattering (DLS), and the results are shown in Table 1 below. The measured LNP-mRNA had a high size uniformity, and its PDI was less than 0.3. The particle size of LNP-mRNA prepared from the lipid compositions of the present application was within the range of 90-120 nm.

[0519] Table 1: Summary table of the formulations of each lipid composition and the particle size and encapsulation efficiency of the LNP-mRNA prepared therefrom

[0520]

[0521] Example 48: Biological activity test of the LNP-mRNA pharmaceutical composition formulation

[0522] (1) Study on cytotoxicity (biocompatibility)

[0523] The cytotoxicity of the LNP-mRNA pharmaceutical composition formulation of the present invention was tested by the MTT staining method. The LNP-mRNA pharmaceutical composition formulation was dissolved in the culture medium to prepare the required concentration, and an appropriate amount of co-solvent could be added if necessary. 293T cells were used as the cell model, and the inoculation density was 1×10 4 cells / well. The cell suspension was inoculated into a 96-well plate at 100 μL / well. After inoculation, at 37 °C and 4% CO 2Incubate and culture in a cell incubator for 24 h, then aspirate and discard the old culture medium, and administer 100 μL of the culture medium containing the LNP-mRNA drug composition (prepared in Example 47) at a dose of 0.2 μg mRNA per well. Add 100 μL of fresh culture medium to the blank control group, with 6 replicates per group. After co-incubating the LNP-mRNA drug composition preparation with 293T cells for 24 h, add 20 μL of PBS buffer containing 5 mg / mL MTT to each well. After incubating MTT with 293T cells for 4 h, aspirate and discard the mixture of the culture medium and MTT buffer, and add 150 μL / well of DMSO to dissolve the purple crystal formazan of living cells. After sufficient oscillation, measure the absorbance with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate based on the measured absorbance values. The results show that compared with the blank control group, the cell survival rates of the LNP-mRNA drug composition preparations prepared in the control group and the experimental group of the present invention are both greater than 96%. The results are shown in Table 2, indicating that the LNP-mRNA drug composition preparation of the present invention has good biocompatibility.

[0524] Table 2: Cytotoxicity results

[0525] Group L-0 L-1 L-2 L-3 L-4 L-5 L-6 L-7 Cell survival rate 98.05％ 99.04％ 99.13％ 97.64％ 96.41％ 97.18％ 96.73％ 98.62％ Group L-8 L-9 L-10 L-11 L-12 L-13 L-14 L-15 Cell survival rate 97.75％ 98.77％ 99.26％ 97.42％ 98.29％ 98.16％ 99.06％ 99.01％ Group L-16 L-17 L-18 L-19 L-20 L-21 L-22 L-23 Cell survival rate 96.49％ 98.25％ 98.62％ 99.28％ 97.63％ 97.42％ 98.73％ 98.83％ Group L-24 L-25 L-26 L-27 L-28 L-29 L-30 L-31 Cell survival rate 98.56％ 97.33％ 96.58％ 98.81％ 98.50％ 97.46％ 97.51％ 97.27％ Group L-32 L-33 L-34 L-35 L-36 L-37 L-38 L-39 Cell survival rate 97.52％ 99.44％ 96.57％ 97.53％ 99.19％ 98.58％ 97.82％ 98.61％ Group L-40 L-41 L-42 L-43 L-44 L-45 L-46 L-47 Cell survival rate 97.63％ 98.86％ 97.82％ 96.69％ 97.58％ 98.82％ 98.84％ 97.97％

[0526] (2) Serum stability evaluation

[0527] Add the LNP-mRNA drug composition to the culture medium containing 10% fetal bovine serum (FBS), stir at 37 °C, and take samples at regular intervals to measure the particle size change of LNP-mRNA. Analyze the serum stability of the LNP-mRNA drug composition preparation by testing its particle size change. The experimental results show that within 7 days, the particle size changes of both the control group and the experimental group are less than 10%. In particular, the particle size changes of L-1, L-2, L-10, L-14, L-19, L-24, L-28, and L-33 in the experimental group are less than 5%, indicating that the LNP-mRNA drug composition preparation prepared with the cationic lipid of the present invention has good serum stability.

[0528] (3) Study on the mRNA transfection rate at the cell level

[0529] To investigate the mRNA transfection efficiency of each group of LNP-mRNA pharmaceutical compositions prepared in Example 47 of the present invention at the cellular level, Luciferase bioluminescence was used for testing. The LNP-mRNA pharmaceutical composition was dissolved in the culture medium to prepare the required dose. Using 293T cells as the cell model, at an inoculation density of 6,000 cells / well, the cell suspension was inoculated into a 96-well plate with a black-edged transparent bottom at 100 μL / well. After inoculation, it was incubated in a cell culture incubator for 24 h, and then administered at a dose of 0.2 μg mRNA per well. The blank control group was added with the corresponding dose of free Fluc-mRNA. Each concentration in each group had 6 replicates. After 24 hours of transfection, the old culture medium was removed and replaced with a new culture medium containing the substrate of sodium D-luciferin (1.5 mg / mL). After incubating for 5 minutes, the bioluminescence was detected using a microplate reader. The stronger the fluorescence, the more Fluc-mRNA was transported into the cytoplasm and translated into the corresponding fluorescent protein. The experimental results are shown in Table 3. Among them, the relative fluorescence intensity value is the ratio of the fluorescence intensity value of each group to the fluorescence intensity of the blank control group. The experimental results show that the LNP-mRNA pharmaceutical composition prepared by the present invention has excellent in vitro transfection effects, that is, the LNP in the experimental group are all effective nucleic acid delivery vectors, and basically all are superior to the L-0 group prepared by the cationic lipids of the prior art (except for the L-5, L-13, L-23, and L-32 groups). Among them, the relative fluorescence values of the experimental groups L-1, L-2, L-10, L-14, L-16, L-17, L-19, and L-26 are relatively high, probably because they contain two ionizable tertiary amine structures and / or multiple degradable ester bonds and / or unsaturated bonds. The partial positive charges ionized by the ionizable tertiary amine bind to the negatively charged nucleic acid. The degradable ester bonds promote the endosomal escape of LNP-mRNA and promote the release of mRNA into the cytoplasm to exert its efficacy. The unsaturated hydrocarbon tail chains are arranged more loosely. While maintaining the stability and fluidity of the membrane, it can enhance the membrane fusion ability and increase the uptake of the LNP-mRNA pharmaceutical composition by cells; the relative fluorescence values of the experimental groups L-4, L-13, L-22, and L-40 are relatively low. The hydrophobic tail chains of the cationic lipids used in them have more numbers and contain ether bonds. Their encapsulation efficiency is not very low, but the cell transfection activity is poor, probably due to less cellular uptake of LNP-mRNA or blocked endosomal escape.

[0530] Table 3: Results of cell transfection test

[0531] Serial number Relative fluorescence value Serial number Relative fluorescence value Serial number Relative fluorescence value Blank 1 L-16 11.33 L-33 15.16 L-0 10.08 L-17 12.19 L-34 14.71 L-1 14.45 L-18 10.72 L-35 11.40 L-2 13.93 L-19 14.94 L-36 12.23 L-3 12.63 L-20 12.45 L-37 13.54 L-4 13.62 L-21 10.92 L-38 12.52 L-5 13.91 L-22 13.20 L-39 14.88 L-6 12.38 L-23 9.33 L-40 9.55 L-7 9.64 L-24 10.28 L-41 13.22 L-8 13.90 L-25 12.63 L-42 10.59 L-9 10.78 L-26 11.37 L-43 12.92 L-10 15.03 L-27 8.99 L-44 9.63 L-11 14.52 L-28 8.73 L-45 13.26 L-12 14.68 L-29 13.25 L-46 13.91 L-13 12.88 L-30 7.89 L-47 14.51 L-14 15.21 L-31 12.43 L-15 9.80 L-32 13.72

[0532] (4) Study on mRNA transfection efficiency at the animal level

[0533] The LNP-mRNA pharmaceutical compositions (L-1, L-10, L-19) were administered to 6-8-week-old female Babl / c mice at a dose of 10 μg mRNA / mouse by tail vein injection, and small animal in vivo fluorescence imaging was performed at 6 hours, 12 hours, and 24 hours after administration. After imaging at the last time point, the mice were euthanized, and the main organs (heart, liver, spleen, lung, kidney) of the mice and the muscle at the injection site were imaged. 0.2 mL of D fluorescein sodium (15 mg / mL) was intraperitoneally injected 10-15 min before imaging. The results showed that the LNP-mRNA pharmaceutical compositions prepared with the cationic lipids of the present invention also had excellent transfection efficiency in vivo. The in vivo transfection activity of L-10 is shown in Figure 1 .

[0534] The above are only the embodiments of the present invention, and thus do not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, shall be similarly included in the patent protection scope of the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although the present invention gives specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use, or improvement of the present invention, including those that depart from the scope disclosed in this application and are made with conventional techniques known in the art.

Claims

1. A cationic lipid, characterized in that its structure is as shown in the general formula (2): wherein, L 1 is - L c R, L c is a connecting key, and R is any one of linear pentyl, linear hexyl, linear heptyl, linear octyl, linear nonyl, and linear decyl; L 2 is -(CH 2 ) m -, m is an integer from 1 to 4; L 5 -R 3 selected from any one of the following structures: L 3 and L 4 each independently represents -OC(=O)- or -C(=O)O-; B 1 and B 2 each independently is C 2-10 an alkylene group; R 1 and R 2 are each independently a C 1-30 aliphatic hydrocarbon group or the C 1-30 aliphatic hydrocarbon group is a C 1-30 linear aliphatic hydrocarbon group or a C 1-30 branched aliphatic hydrocarbon group; the R 1 and R 2 are any one of the following cases: Case (1): R 1 , R 2 One of them is a C 1-30 linear aliphatic hydrocarbon group, and the other is a C 1-30 branched aliphatic hydrocarbon group Case (2): R 1 and R 2 are each independently a C 1-30 branched-chain aliphatic hydrocarbon group Case (3): R 1 and R 2 are each independently Case (4): R 1 , R 2 One of them is a C 1-30 linear aliphatic hydrocarbon group or a C 1-30 branched aliphatic hydrocarbon group The other is The said C 1-30 The straight-chain aliphatic hydrocarbon group is selected from any one of; The said selected from any one of; The selected from any one of them; wherein, t is an integer from 0 to 12; or a pharmaceutically acceptable salt thereof.

2. The cationic lipid according to claim 1, characterized in that the R is a straight-chain octyl group.

3. The cationic lipid according to claim 1, characterized in that The said L 3 and L 4 are both -OC(=O)- or both -C(=O)O- at the same time.

4. The cationic lipid according to claim 1, characterized in that the structure of the cationic lipid satisfies any one of the following general formulas:

5. The cationic lipid according to claim 1, characterized in that the structure of the cationic lipid is selected from any one of the following structures:

6. A lipid composition, characterized in that it contains the cationic lipid according to any one of claims 1-5.

7. The lipid composition according to claim 6, characterized in that it further contains one or more of phospholipids, steroid lipids and polyethylene glycolated lipids; selected from any one of the following situations: Situation (1): It further contains phospholipids; Situation (2): It further contains steroid lipids; Situation (3): It further contains polyethylene glycolated lipids; Situation (4): It further contains phospholipids and steroid lipids; Situation (5): It further contains phospholipids and polyethylene glycolated lipids; Situation (6): It further contains steroid lipids and polyethylene glycolated lipids; Situation (7): It further contains phospholipids, steroid lipids and polyethylene glycolated lipids.

8. The lipid composition according to claim 6, characterized in that it also contains three lipids, namely phospholipids, steroid lipids and polyethylene glycolated lipids, at the same time.

9. The lipid composition according to any one of claims 7-8, characterized in that The phospholipids are selected from any one of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dielaidoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dioleoyl phosphatidylserine, dipalmitoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine and their combinations; or the steroid lipids are selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and their combinations; Or the polyethylene glycolated lipid is selected from polyethylene glycol-1,2-dimyristoyl glycerol, polyethylene glycol-distearoyl phosphatidylethanolamine, PEG-cholesterol, polyethylene glycol-diacylglycerol, polyethylene glycol-dialkoxypropyl, specifically including any one of polyethylene glycol 500-dipalmitoyl phosphatidylcholine, polyethylene glycol 2000-dipalmitoyl phosphatidylcholine, polyethylene glycol-500-stearoyl phosphatidylethanolamine, polyethylene glycol 2000-distearoyl phosphatidylethanolamine, polyethylene glycol 500-1,2-dioleoyl phosphatidylethanolamine, polyethylene glycol 2000-1,2-dioleoyl phosphatidylethanolamine, and polyethylene glycol 2000-2,3-dimyristoyl glycerol and their combinations.

10. The lipid composition according to any one of claims 7-8, wherein, it contains 20-80% of cationic lipid, 5-15% of phospholipid, 25-55% of steroid lipid, and 0.5-10% of polyethylene glycolated lipid, and the percentages are the molar percentages of each lipid in the total lipid in the solution containing the solvent.

11. The lipid composition according to claim 10, wherein, the molar percentage of the cationic lipid in the total lipid in the solution containing the solvent is 30-65%.

12. The lipid composition according to claim 11, wherein, the molar percentage of the cationic lipid in the total lipid in the solution containing the solvent is any one of 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 55%.

13. The lipid composition according to claim 10, wherein, the molar percentage of the phospholipid in the total lipid in the solution containing the solvent is 7.5-13%.

14. The lipid composition according to claim 13, wherein, the molar percentage of the phospholipid in the total lipid in the solution containing the solvent is any one of 8%, 9%, 10%, 11%, 12%.

15. The lipid composition according to claim 10, wherein, the molar percentage of the steroid lipid in the total lipid in the solution containing the solvent is 35-50%.

16. The lipid composition according to claim 15, wherein, the molar percentage of the steroid lipid in the total lipid in the solution containing the solvent is any one of 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.

17. The lipid composition according to claim 10, wherein, the molar percentage of the polyethylene glycolated lipid in the total lipid in the solution containing the solvent is 0.5-5%.

18. The lipid composition according to claim 17, wherein, the molar percentage of the polyethylene glycolated lipid in the total lipid in the solution containing the solvent is 1-3%.

19. The lipid composition according to claim 17, wherein, the molar percentage of the polyethylene glycolated lipid in the total lipid in the solution containing the solvent is any one of 1.5%, 1.6%, 1.7%, 1.8%, 1.9%.

20. A lipid drug composition, characterized in that, it contains the lipid composition described in any one of claims 6 - 19 and a drug, and the drug is selected from nucleic acid drugs.

21. The lipid drug composition according to claim 20, characterized in that, the nucleic acid drug is selected from any one of RNA, DNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir and ribozyme, and the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA and siRNA.

22. The lipid drug composition according to claim 20, characterized in that, the nucleic acid drug is any one of DNA, mRNA, miRNA and siRNA.

23. A lipid drug composition preparation, characterized in that, it contains the lipid drug composition described in any one of claims 20 - 22 and a pharmaceutically acceptable diluent or excipient.

24. The lipid drug composition preparation according to claim 23, characterized in that, the diluent or excipient is any one of deionized water, ultrapure water, phosphate buffer solution and physiological saline.

25. The lipid drug composition preparation according to claim 23, characterized in that, the diluent or excipient is phosphate buffer solution or physiological saline.

26. The lipid drug composition preparation according to claim 24, characterized in that, the diluent or excipient is physiological saline.

27. A liposome or lipid nanoparticle, characterized in that, it contains the lipid composition described in any one of claims 6 - 19.

28. The liposome or lipid nanoparticle according to claim 27, characterized in that, the lipid nanoparticle is an LNP - nucleic acid drug composition.

29. The liposome or lipid nanoparticle according to claim 28, characterized in that, the lipid nanoparticle is an LNP - mRNA drug composition.

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