An amino acid cationic lipid containing an unsaturated bond

Abstract

The present invention provides a novel amino acid cationic lipid containing an unsaturated bond, and the structure is shown in the general formula (1), where the definitions of each symbol are consistent with those described herein. The amino acid or amino acid derivative raw materials used in the preparation process are simple and easily available, and have the advantages of simplicity, safety and cost savings in production. The amino acid cationic lipid containing an unsaturated bond of the present invention contains an unsaturated hydrocarbon group tail chain and a tertiary amine structure derived from an amino acid residue. The lipid nanoparticle LNP prepared therefrom has higher membrane fluidity, which enhances the membrane fusion ability, is conducive to improving cell uptake, and thus improves the delivery efficiency of the amino acid cationic lipid containing an unsaturated bond as a delivery material.

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, in particular to an amino acid cationic lipid containing an unsaturated bond, and a lipid composition, a lipid drug composition, its preparation and application comprising the amino acid cationic lipid. Background Art

[0002] Therapeutic methods based on messenger RNA (mRNA) have been proven and have made certain progress in various applications, including mRNA vaccines, protein replacement therapy, cancer immunotherapy, and gene editing. Despite the great progress, mRNA drugs still face significant challenges in effective delivery. Due to its physicochemical properties, mRNA is easily degraded by ribozymes, cannot pass through the cell membrane by itself, and is usually unstable in the blood circulation. Therefore, it is urgent to develop safe and efficient delivery vectors to improve its gene transfection efficiency. Common delivery vectors include two major categories: viral vectors and non-viral vectors. Although viral vectors have high transfection efficiency, their potential safety risks such as immunogenicity and carcinogenicity, as well as disadvantages such as small loading capacity, limit their clinical applications. Non-viral vectors have advantages such as good safety, large loading capacity, low cost, and can be prepared in large quantities. For example, lipid nanoparticles (LNP) have received extensive attention because they are used in COVID-19 mRNA vaccines. LNP provides many benefits for mRNA delivery, including simple formulation, modularity, good biocompatibility, and a large mRNA payload capacity. LNP contains cationic lipids, phospholipid lipids, sterol lipids, and polyethylene glycolated lipids. Among them, cationic lipids interact with drug molecules (such as negatively charged nucleic acids) through electrostatic forces, and phospholipid lipids play a role in preventing lipid oxidation or connecting ligands to the surface of lipid nanoparticles; sterol lipids have strong membrane fusion properties, promoting the intracellular uptake and cytoplasmic entry of drug molecules; polyethylene glycolated lipids are located on the surface of lipid nanoparticles, improving their hydrophilicity, avoiding rapid clearance by the immune system, preventing particle aggregation, and increasing stability.

[0003] Most of the LNP prepared from cationic lipids in the prior art are liver-targeted, and the problem of effective delivery to organs other than the liver (such as the spleen, lung, and kidney) needs to be solved urgently. Moreover, most cationic lipids contain saturated aliphatic hydrocarbon tail chains, and the cellular uptake is relatively poor. In this application, both the head and tail chains of amino acid lipids are modified to obtain an amino acid cationic lipid containing an unsaturated bond with a higher cellular uptake rate, a higher delivery efficiency, and certain targeting properties to other organs. Summary of the Invention

[0004] The present invention provides a novel amino acid cationic lipid containing unsaturated bonds, a preparation method thereof, a lipid composition containing the amino acid cationic lipid, a lipid pharmaceutical composition and its preparation containing the lipid composition, a liposome or lipid nanoparticle containing the lipid composition, especially an LNP-nucleic acid pharmaceutical composition and its preparation containing the lipid composition, which has the advantages of high delivery efficiency, low safety and toxicity, and high biocompatibility, and can improve the therapeutic and / or preventive effect of drugs.

[0005] The above object of the present invention is achieved by the following technical solutions:

[0006] An embodiment of the present invention provides an amino acid cationic lipid:

[0007]

[0008] Wherein, AA is a residue of an amino acid or an amino acid derivative;

[0009] Each occurrence of B2 is independently a linking bond or C 1-20 alkylene;

[0010] Each occurrence of L2 and L3 is independently a linking bond or a divalent linking group;

[0011] R1 is a C 10-40 aliphatic hydrocarbon group containing 1, 2 or more unsaturated bonds, and each occurrence of R2 is independently a C 1-40 aliphatic hydrocarbon group or Wherein, t is an integer from 0 to 12; R e 、R f are independently C 1-20 alkyl, C 2-20 alkenyl and C 2-20 alkynyl;

[0012] Each occurrence of R3 is independently a hydrogen atom, an alkyl group, an alkoxy group, a C 3-6 carbocyclic group, a nitrogen-containing heterocyclic group, -NR d R d 、-SR d 、-C(=O)R d 、-C(=O)OR d 、-OC(=O)R d 、or a functional group R 01 that can react with a biologically related substance; wherein, R d each occurrence is independently C 1-12 alkyl;

[0013] b and c are each independently 1 or 2; when the fragment -B2-L2-R2 and / or -L3-R3 is led out from the amino terminus of an amino acid and its derivatives, b and c are each independently 1 or 2; when the fragment -B2-L2-R2 and / or -L3-R3 is led out from the carboxyl terminus of an amino acid and its derivatives, b and c are each independently 1; when b is 2, the two -B2-L2-R2 fragments are the same or different; when c is 2, the two -L3-R3 fragments are independently the same or different from each other;

[0014] or a salt, tautomer, stereoisomer or solvate thereof.

[0015] The present invention also provides a lipid composition, and the embodiments are as follows:

[0016] A lipid composition containing an amino acid cationic lipid having the structure shown in formula (1).

[0017] The present invention also provides a lipid pharmaceutical composition, and the embodiments are as follows:

[0018] A lipid pharmaceutical composition containing a lipid composition and a drug, and the lipid composition contains an amino acid 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.

[0019] The present invention also provides a preparation of a lipid pharmaceutical composition, and the embodiments are as follows:

[0020] A preparation of a lipid pharmaceutical composition containing the aforementioned lipid pharmaceutical composition and a pharmaceutically acceptable diluent or excipient.

[0021] The present invention also provides a liposome or lipid nanoparticle, and the embodiments are as follows:

[0022] A liposome or lipid nanoparticle containing a lipid composition, and the lipid composition contains an amino acid cationic lipid having the structure shown in formula (1).

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] Aiming at the problems of poor uptake and low transfection efficiency of the LNP-mRNA pharmaceutical composition, the present invention designed and synthesized a series of amino acid cationic lipids containing unsaturated bonds, and the unsaturated hydrocarbon tail chains contained therein are arranged more loosely, which plays a key role in maintaining the stability and fluidity of the membrane, can enhance the membrane fusion ability, increase the uptake of the LNP-mRNA pharmaceutical composition by cells, and thus improve the delivery efficiency of nucleic acid drugs.

[0025] The LNP-mRNA pharmaceutical composition prepared from the novel amino acid cationic lipid containing unsaturated bonds of the present invention is mainly distributed in the liver and spleen, providing some ideas for further studying the targeting problems of other organs.

[0026] For the novel amino acid cationic lipid containing unsaturated bonds of the present invention, amino acids or amino acid derivatives are selected as the hydrophilic head groups. Through electrostatic interaction, their positive charges are easily complexed with negatively charged drugs or mRNA, thereby increasing their stability in blood circulation. In addition, the present invention uses amino acids or amino acid derivatives with good biocompatibility as the backbone, further improving the biocompatibility of the cationic lipid. Moreover, the raw materials of amino acids or amino acid derivatives are simple and easy to obtain, can be obtained naturally or synthesized simply, and have the advantages of simplicity, safety, and cost savings in production.

[0027] The linking arm of the novel amino acid cationic lipid containing unsaturated bonds of the present invention selects a degradable group. The presence of the degradable group enables the LNP-drug composition prepared therefrom to be degraded in a timely manner in the body and have low cytotoxicity, solving the problem that the LNP-drug composition prepared from lipids that cannot be degraded in the prior art will accumulate in endosomes and acidify the endosomal environment, resulting in hindered endosomal escape of drugs (such as mRNA) and the drugs delivered into cells being unable to fully play their roles. Description of the Drawings

[0028] Figure 1 It is the animal fluorescence imaging diagram and in vivo organ distribution diagram of the LNP-mRNA composition (L-2).

[0029] Embodiments

[0030] Term Explanation

[0031] In the present invention, unless otherwise described, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. The disclosures of all patents and other publications cited herein are incorporated herein by reference in their entirety. In the case of any conflict between the description and interpretation of any term herein and any document incorporated herein by reference, the following description and interpretation of the term shall prevail.

[0032] In the present invention, when the structures involved have isomers, without special designation, any one of the isomers may be used. For example, for structures with cis-trans isomers, it may be either the cis structure or the trans structure; for structures 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 right-handed form.

[0033] In the present invention, the interpretation of a numerical range includes not only a numerical range marked with a short dash (such as 0 - 12), but also a numerical range marked with a wavy line (such as (0~12)), and also a numerical range marked with "to" (such as 0 to 12, 1 to 12). In the present invention, unless otherwise specified, an integer range marked in interval form can represent a set composed of all integers within the range of the interval, and the range includes both endpoints. For example, the integer range 0 - 12 represents a set composed of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. The numerical ranges in the present invention include, but are not limited to, numerical ranges represented by integers, non-integers, percentages, and fractions. Unless otherwise specified, both endpoints are included.

[0034] In the present invention, the terms "about" and "around" in relation to a numerical value generally refer to a numerical range of ±10%, and in some cases, it can be enlarged to ±15%, but not exceeding ±20%. Based on a preset numerical value. For example, if the molar percentage of steroid lipid in the total lipid in a solution containing a solvent is about 40%, it can generally be considered to include the case where the molar percentage of steroid lipid is 30% - 50%.

[0035] In the present invention, unless otherwise specified, "any" includes any one, any two, and any two or more.

[0036] In the present invention, unless otherwise specified, the terms "comprise", "include", "contain" and similar expressions should be interpreted in an open and inclusive sense as "including but not limited to" in this specification and the claims.

[0037] In the present invention, when two or more objects are "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, or one can be the maximum range and the other can be any one of the preferred cases, or they can be selected from the same level of preference.

[0038] In the present invention, "each independently at each occurrence" not only means that in different groups, they can each independently be any one of the definitions, but also means that when they appear at different positions in the same group, they can also each independently be any one of the definitions. For example, "each independently is a linking bond, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, -NH-, -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- wherein each occurrence of R c is independently a hydrogen atom or C 1-12 alkyl", in the group "-NR c C(=O)NR c -", the two Rs c are independently a hydrogen atom or C 1-12 alkyl, i.e., the two Rs c can be the same or different.

[0039] For the divalent linking group in the present invention, such as an alkylene group, an alkylidene group, an arylene group, an 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 GroupA and GroupB, it can be GroupA-C(=O)NH-GroupB or GroupB-NHC(=O)-GroupA.

[0040] In the structural formula of the present invention, when the end group of the linking group is likely to be confused with the substituent 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 above two structural formulas respectively represent -CH(CH2CH2CH3)2- and -CH2CH2CH(CH3)2-CH2CH2-.

[0041] In the present invention, the range of the number of carbon atoms in the group is marked in the subscript position of C in the form of a subscript, 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 a compound obtained by substituting a hydrogen atom of C 1-12 alkyl. "C 1-12 substituted alkyl" refers to a compound having 1 - 12 carbon atoms obtained after substituting a hydrogen atom of the alkyl. For another example, when a group can be selected from C 1-12 alkylene group, it can be an alkylene group with any number of carbon atoms within the range shown by the subscript, that is, it can be selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12Any one of the alkylene groups. In the present invention, unless otherwise specified, subscripts marked in the form of an interval represent any integer that can be selected from within the range, and this range includes both endpoints.

[0042] The heteroatoms in the present invention are not particularly limited and include, but are not limited to, O, S, N, P, Si, F, Cl, Br, I, B, etc.

[0043] 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".

[0044] In the present invention, "substituted" means that at least one hydrogen atom of any of the above groups (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 a C 1-12 alkyl group); a carboxyl group (-COOH); an amine group (-NR c R c , and two Rs c are each independently H, C 1-12 alkyl group); a C 1-12 alkyl group and a cycloalkyl group. In some embodiments, the substituent is a C 1-12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. 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.

[0045] In the present invention, "optional" or "optionally" (for example, optionally substituted) means that the subsequent described situation or event may or may not occur, and this description includes examples where the event or situation occurs and examples 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 this description includes substituted hydrocarbon groups and unsubstituted hydrocarbon groups.

[0046] In the present invention, a "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 referred to as a "main chain heteroatom", such as A-S-CH2-B, A-O-CH2-B, (The atomic interval is denoted as 4) is regarded as containing a heteroatom in the main chain. The carbon chain linking group can be divided into a hydrocarbon group and a carbon chain linking group with a heteroatom in the side chain; the carbon chain linking group with a heteroatom in the side chain includes but is not limited to oxo (=O), thio (=S), amino (linked to the main chain carbon through a carbon-nitrogen double bond), an oxyhydrocarbon group in the form of an ether bond, a thiohydrocarbon group in the form of a thioether bond, an azahydrocarbon group in the form of a tertiary amino group, etc. The "carbon chain linking group" has a main chain composed entirely of carbon atoms, and the side chain of the carbon chain is allowed to contain heteroatoms. That is, it is formed by connecting methylene or substituted methylene. The substituted methylene can be substituted by a monovalent substituent, two monovalent substituents or a divalent substituent (such as divalent oxygen, such as jointly forming a three-membered ring with divalent methylene ) substitution. The substituted methylene can be that one hydrogen atom is substituted (such as -CH(CH3)-), or two hydrogen atoms are respectively substituted (such as -(CH3)C(OCH3)-), or two hydrogen atoms are simultaneously substituted (such as a carbonyl group, a thiocarbonyl group, -C(=NH)-, -C(=N + H2)-), or a cyclic side group (such as The atomic interval is denoted as 1).

[0047] In the present invention, for a compound or a group, it can be simultaneously substituted and hybridized. For example, a nitro phenyl group substitutes a hydrogen atom, or -CH2-CH2-CH2- is replaced by -CH2-S-CH(CH3)-.

[0048] In the present invention, the "linking bond" 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.

[0049] The "group" in the present invention contains at least 1 atom and refers to a radical formed by a compound losing one or more atoms. Relative to a compound, the group formed after losing some groups is also called a residue. The valence state of the group is not particularly limited. By way of example, it can be divided into a monovalent group, a divalent group, a trivalent group, a tetravalent group,..., a hundred-valent group, etc. Among them, groups with a valence state greater than or equal to 2 are collectively called linking groups. The linking group can also contain only one atom, such as an oxy group, a thio group.

[0050] In the present invention, "hydrocarbon" refers to a hydrocarbon compound composed of carbon atoms and hydrogen atoms.

[0051] In the present invention, according to the hydrocarbon group category, hydrocarbons are divided into two types: aliphatic hydrocarbons and aromatic hydrocarbons. Hydrocarbons that do not contain a benzene ring or any structure in a benzene ring substituted by a hydrocarbon group are defined as aliphatic hydrocarbons. Hydrocarbons that contain at least one benzene ring or a benzene ring substituted by a hydrocarbon group are defined as aromatic hydrocarbons. And aromatic hydrocarbons can contain an aliphatic hydrocarbon group structure, such as toluene, diphenylmethane, 2,3-dihydroindene, etc.

[0052] In the present invention, hydrocarbons are classified into saturated hydrocarbons and unsaturated hydrocarbons according to the saturation situation. 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, but not limited to, alkenes (containing double bonds), alkynes (containing triple bonds), dienes (containing conjugated double bonds), etc. When the aliphatic hydrocarbon part in the aromatic hydrocarbon is a saturated structure, it is also called an arylalkane, such as toluene.

[0053] In the present invention, there is no particular limitation on the structure of hydrocarbons, and they can be in the form of a straight-chain structure without side chains, a branched-chain structure with side chains, a cyclic structure, a dendritic structure, a comb-like structure, a hyperbranched structure, etc. In the absence of a special definition, a straight-chain structure without side chains, a branched-chain structure with side chains, and a cyclic structure are preferably selected, corresponding to straight-chain hydrocarbons, branched-chain hydrocarbons, and cycloalkanes, respectively. Among them, hydrocarbons without cyclic structures are collectively called open-chain hydrocarbons, including but not limited to a straight-chain structure without side chains and a branched-chain structure with side chains. Open-chain hydrocarbons belong to aliphatic hydrocarbons. Therefore, straight-chain hydrocarbons can also be called straight-chain aliphatic hydrocarbons. Branched-chain hydrocarbons can also be called branched-chain aliphatic hydrocarbons.

[0054] In the present invention, "hydrocarbyl" refers to the 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 (losing one hydrogen atom), divalent hydrocarbyl (losing two hydrogen atoms, also called a hydrocarbonylene), trivalent hydrocarbyl (losing three hydrogen atoms), and so on. By analogy, when n hydrogen atoms are lost, the valence state of the formed hydrocarbyl is n. In the absence of a special designation, the hydrocarbyl in the present invention specifically refers to a monovalent hydrocarbyl. Unless otherwise expressly stated in this specification, the hydrocarbyl is optionally substituted.

[0055] In the present invention, the source of the hydrocarbyl is not particularly limited. For example, it can be derived from aliphatic hydrocarbons or aromatic hydrocarbons, or from saturated hydrocarbons or unsaturated hydrocarbons, or from straight-chain hydrocarbons, branched-chain hydrocarbons or cycloalkanes, or from hydrocarbons or heterohydrocarbons, etc. From the perspective of saturation, for example, it can be derived from alkanes, alkenes, alkynes, dienes, etc.; for cycloalkanes, for example, it can be derived from cycloaliphatic hydrocarbons or aromatic hydrocarbons, monocyclic hydrocarbons or polycyclic hydrocarbons; for heterocyclic hydrocarbons, for example, it can be derived from aliphatic heterocyclic hydrocarbons or aromatic heterocyclic hydrocarbons.

[0056] In the present invention, "aliphatic hydrocarbyl" refers to the residue formed after an aliphatic hydrocarbon loses at least one hydrogen atom. In the absence of a special designation, the aliphatic hydrocarbyl in the present invention specifically refers to a monovalent aliphatic hydrocarbyl. Aliphatic hydrocarbyl includes saturated aliphatic hydrocarbyl and unsaturated aliphatic hydrocarbyl. Unless otherwise expressly stated in this specification, the aliphatic hydrocarbyl is optionally substituted.

[0057] In the present invention, "alkyl" refers to a hydrocarbyl group formed from an alkane. Without specific designation, it refers to a hydrocarbyl group formed by removing a hydrogen atom at any position, which can be straight-chain or branched-chain, and can 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 explicitly stated in this specification, the alkyl group is optionally substituted.

[0058] In the present invention, "unsaturated hydrocarbyl" refers to a hydrocarbyl group formed by removing a hydrogen atom from an unsaturated hydrocarbon. The hydrocarbyl group formed by removing a hydrogen atom from an unsaturated carbon of an unsaturated hydrocarbon can be classified into alkenyl, alkynyl, diene group, etc. For example, propenyl and propynyl. The hydrocarbyl group formed by removing a hydrogen atom from a saturated carbon of an unsaturated hydrocarbon is called an olefin group, alkyne, diene group, etc. according to the different unsaturated bonds. Specifically, for example, allyl and propargyl.

[0059] In the present invention, "alkenyl" or "alkenyl group" means a substituted or unsubstituted straight-chain or branched-chain alkenyl group including two or more carbon atoms (such as 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.

[0060] In the present invention, "alkynyl" or "alkynyl group" means an optionally substituted straight-chain or branched-chain hydrocarbon including two or more carbon atoms (such as 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.

[0061] In the present invention, "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, alkylene (C 1-24 alkylene) with 1 to 24 carbon atoms, alkylene (C 1-12 alkylene) with 1 to 12 carbon atoms. Specifically, for example, methylene, ethylene, propylene, n-butylene, vinyl, propenyl, n-butenyl, propynyl, n-butynyl, etc. Unless otherwise explicitly stated in this specification, the alkylene is optionally substituted.

[0062] In the present invention, "alkylene" which is also divalent alkyl includes open-chain alkylene and divalent cycloalkyl. Open-chain alkylene refers to a divalent alkyl without a cyclic structure, and divalent cycloalkyl refers to a divalent alkyl with a cyclic structure. Unless otherwise explicitly stated in this specification, the alkylene is optionally substituted.

[0063] In the present invention, "molecular weight" characterizes the mass of a compound molecule, and "average molecular weight" characterizes the mass of the components of a general formula compound in a macroscopic substance. When there is no special regulation, "average molecular weight" generally refers to "number-average molecular weight" M n . For the number-average molecular weight, it can be the molecular weight of a polydisperse block or substance, or the molecular weight of a monodisperse block or substance. When not specifically stated, the measurement unit of "molecular weight" and "average molecular weight" is Dalton, Da. The "degree of polymerization" can also be used to characterize the molecular weight of the polyethylene glycol chain, specifically referring to the number of repeating units (ethylene oxide units, EO units) in a compound molecule. Correspondingly, "average degree of polymerization", "number-average degree of polymerization" or "number of EO units" are used to characterize the average value and number average value of the number of repeating units.

[0064] In the present invention, for the percentage, "about" generally means ±0.5%.

[0065] In the present invention, "stable existence" and "degradability" of a group are a pair of relative concepts. For detailed examples of groups that can stably exist and groups that can degrade, see paragraphs

[0134] -

[0145] in CN113402405A.

[0066] In the present invention, the "hydroxy protecting group" includes all groups that can be used as the protecting group for a normal hydroxy group. The hydroxy protecting group is preferably an alkanoyl group (such as acetyl, tert - butanoyl), an aralkanoyl group (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. The most commonly used are ammonolysis with NH3 / 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 metallic sodium 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 / CH3OH, etc.). It can be removed with fluoride ions (such as Bu4N + F - ) in a tetrahydrofuran solution, or can also be removed with aqueous acetic acid at room temperature.

[0067] In the present invention, the "carboxy protecting group" refers to a protecting group that can be converted into a carboxy group through hydrolysis or a de - protection reaction of the carboxy protecting group. The carboxy protecting group is preferably an alkyl group (such as methyl, ethyl, tert - butyl) or an aralkyl group (such as benzyl), more preferably tert - butyl (tBu), methyl (Me) or ethyl (Et). In the present invention, the "protected carboxy" refers to a group formed after the carboxy group is protected by a suitable carboxy protecting group, preferably methoxycarbonyl, ethoxycarbonyl, tert - butoxycarbonyl, benzyloxycarbonyl. The said carboxy 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, tert - butyl can be removed under mild acidic conditions, and benzyl can be removed by hydrogenolysis. The reagents for removing the carboxy protecting group are selected from TFA, H2O, LiOH, NaOH, KOH, MeOH, EtOH and their combinations, preferably the combination of TFA and H2O, the combination of LiOH and MeOH, or the combination of LiOH and EtOH. The de - protection of the protected carboxy generates the corresponding free acid, and the said de - protection is carried out in the presence of a base, and the base and the free acid formed by the said de - protection form a pharmaceutically acceptable salt.

[0068] In the present invention, the "amino protecting group" includes all groups that can be used as the protecting group 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 1-6Alkylsulfonyl, 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, H2O, LiOH, MeOH, EtOH, and combinations thereof, preferably the combination of TFA and H2O, 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 N,N-dimethylformamide (DMF) solution containing 20% piperidine.

[0069] In the present invention, "cation" means that the corresponding structure permanently or non-permanently bears a positive charge in response to certain conditions (such as pH). Therefore, cations include both permanent cations and cationizable ones. A permanent cation means that the corresponding compound or group or atom bears a positive charge at any pH value or hydrogen ion activity in its environment. Typically, a 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 or group or atom bears a positive charge at a lower pH and does not bear a charge 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 bears a positive charge at a high hydrogen ion concentration and does not bear a charge 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 pH or hydrogen ion concentration it bears a charge or not. 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 bearing a positive charge, which is well known to those skilled in the art. For example, in some embodiments, if a compound or moiety is cationizable, preferably, it bears a positive charge 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.

[0070] In the present invention, the "cationic component / compound" typically refers to a charged molecule that carries a positive charge (cation) at a pH value typically of about 1 to 9. In some embodiments, the cationic component / compound preferably carries a charge at a pH value equal to or lower than 9 (such as 5 to 9), equal to or lower than 8 (such as 5 to 8), equal to or lower than 7 (such as 5 to 7), and most preferably at physiological pH (such as about 7.3 to 7.4). Thus, the cationic peptides, proteins, polysaccharides, lipids or polymers according to an embodiment of the present invention carry a positive charge under physiological conditions, especially under the physiological conditions of cells in vivo.

[0071] In the present invention, liposome nanoparticles, cationic peptides, proteins, polysaccharides, lipids or polymers are uncharged, have a neutral charge or are electrically neutral respectively under physiological conditions, especially under the physiological conditions of cells in vivo. The cationic peptides or proteins preferably contain a relatively large amount of cationic amino acids, such as Arg, His, Lys or Orn in a larger number than other amino acid residues (especially more cationic amino acids than anionic amino acid residues such as Asp or Glu) or contain a moiety mainly formed by cationic amino acid residues. The term "cationic" can also refer to a "poly-cationic" component / compound. The cationic component / compound can also refer to a cationic lipid capable of carrying a positive charge. For example, the cationic lipid contains one or more amine groups carrying a positive charge, and the preferred cationic lipids are ionizable so that they can exist in a positively charged form or a neutral form according to the pH. The ionization of the cationic lipid affects the surface charge of lipid nanoparticles (LNPs) under different pH conditions. This charge state can affect plasma protein uptake, blood clearance and tissue distribution, as well as the ability to form non-bilayer structures crucial for intracellular delivery of nucleic acids.

[0072] In the present invention, the "PEGylated lipid" refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety.

[0073] In the present invention, the "neutral lipid" refers to any of a number of lipid substances that exist in an uncharged or neutral zwitterionic form at a selected pH, preferably a phospholipid, which can be of synthetic or natural origin.

[0074] In the present invention, the "steroid lipid" is a steroid or a steroid analogue.

[0075] In the present invention, an "amino acid residue" includes an amino acid in which a hydrogen atom is removed from an amino group and / or a hydroxyl group is removed from a carboxyl group and / or a hydrogen atom is removed from a mercapto group and / or the amino group is protected and / or the carboxyl group is protected and / or the mercapto group is protected. Loosely speaking, an amino acid residue can be referred to as an amino acid. The source of the amino acid in the present invention is not particularly limited without specific indication, and can be either a natural source, a non-natural source, or a mixture of both. The structural type of the amino acid in the present invention is not particularly limited without specific indication, and can refer to either the L-type, the D-type, or a mixture of both. In one embodiment of the present invention, the amino acid is a hydrophobic amino acid selected from any one of tryptophan (Trp), phenylalanine (Phe), valine (Val), isoleucine (Ile), leucine (Leu), and tyrosine (Tyr). In another embodiment of the present invention, the amino acid is a hydrophilic amino acid selected from any one of glutamic acid (Glu), aspartic acid (Asp), histidine (His), glutamine (Gln), asparagine (Asn), serine (Ser), threonine (Thr), proline (Pro), glycine (Gly), lysine (Lys), and arginine (Arg), preferably glycine or lysine, and more preferably lysine.

[0076] The variant form in the present invention refers to a structural form that can be transformed into a target reactive group through any one of chemical change processes such as oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and dissociation, ionization, protonation, deprotonation, substitution, deprotection, and change of leaving group.

[0077] In the present invention, the "variant form of a reactive group" refers to a form that remains active (is still a reactive group) after at least one chemical change process such as oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and dissociation, ionization, protonation, deprotonation, substitution, deprotection, and change of leaving group for a reactive group, or a non-active form after being protected.

[0078] The "micro-modification" in the present invention 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 dissociation, ionization, protonation, deprotonation, and transformation of leaving group.

[0079] The "micro-variant form" corresponds to the "micro-modification", and refers to a structural form that can form a target reactive group after experiencing simple chemical reaction processes such as deprotection, salt complexation and dissociation, ionization, protonation, deprotonation, and transformation of leaving group. The transformation of the leaving group, such as the transformation from an ester form to an acyl chloride form.

[0080] In the present invention, "any suitable linking group", "any suitable reactive group", etc., the "any suitable" means a structure that conforms to the basic principles of chemical structure and enables the smooth implementation of the preparation method of the present invention. The chemical structures described in this way can be regarded as having a clear and definite scope.

[0081] When at least two types of structural types are listed, the "any combination" of the listed structural types refers to the combination of any two or more than two structures among the aforementioned related structural types; and there is no limitation on the number of structural units. The number of any kind of structural unit can be zero, one, or more than one. When the number of the same type of structural units is more than 1, they can be structural units with the same or different chemical structures, and the total number of constituent units is at least 2. For example, examples of any combination of alkylene, divalent cycloalkyl, divalent cycloalkenyl, divalent cycloalkynyl, divalent cyclo-dienyl, arylene, carbon-carbon double bond, carbon-carbon triple bond, conjugated carbon-carbon double bond, divalent heteroatom-containing aliphatic ring linking group, divalent heteroatom-containing aromatic ring linking group, and carbon chain linking group with a heteroatom in the side chain include -Ph-CH2-Ph- (arylene-alkylene-arylene), -CH2-Ph-CH2CH2- (alkylene-arylene-alkylene, where the number of alkylene is 2 and has different chemical structures), or the benzene ring in the aforementioned examples is replaced with a cyclohexane ring, a diazahexane ring, or the structure of 1-(2-pyridyl)hexahydro-1H-1,4-diazepine. Another example is that cycloalkenyl hydrocarbon group = cycloalkenyl + alkylene group = cycloalkenyl as a substituent of the hydrocarbon group, and cyclo-dienyl hydrocarbon group = cyclo-dienyl as a substituent of the hydrocarbon group.

[0082] In the present invention, the "N / P ratio" refers to the molar ratio of nitrogen atoms in the cationic lipid to phosphoric acid in the nucleic acid.

[0083] In the present invention, "nucleic acid" refers to DNA or RNA or a modified form thereof, which contains purine or pyrimidine bases present in DNA (adenine "A", cytosine "C", guanine "G", thymine "T") or purine or pyrimidine bases present in RNA (adenine "A", cytosine "C", guanine "G", uracil "U").

[0084] 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, a chain-terminating nucleoside, a stem-loop, a polyadenylation sequence, and / or a polyadenylation signal. 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, for example, 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.

[0085] In the present invention, an antisense oligonucleotide or small interfering RNA (siRNA) can inhibit the expression of a target gene and a target protein in vitro or in vivo.

[0086] In the present invention, FLuc mRNA can express luciferase protein, which emits bioluminescence in the presence of a luciferin substrate, so FLuc is commonly used in mammalian cell culture to measure gene expression and cell viability.

[0087] In the present invention, "inhibiting the expression of a target gene" refers to the ability of a nucleic acid to silence, reduce, or inhibit the expression of a target gene. To test the degree of gene silencing, a test sample (e.g., a cell sample in a culture medium expressing the target gene) is contacted with a nucleic acid that inhibits the expression of the target gene. The expression of the target gene in the test sample or test animal is compared with the expression of the target gene in a control sample (e.g., a cell sample in a culture medium expressing the target gene) that has not been contacted or administered the nucleic acid. The expression of the target gene in the control sample can be assigned a value of 100%. In certain embodiments, inhibition of the expression of the target gene is achieved when the level of expression of the target gene in the test sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the level of expression of the target gene in the control sample or control mammal.

[0088] In the present invention, methods for determining the level of expression of a target gene include, but are not limited to, dot blot, northern blot, in situ hybridization, ELISA, immunoprecipitation, enzymatic action, and phenotypic assays.

[0089] In the present invention, "transfection" refers to the introduction of a species (such as RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo or in vivo.

[0090] In the present invention, an "antigen" typically 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 comprise 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). Peptides and protein fragments, variants and derivatives that contain at least one epitope are also understood as antigens in this context.

[0091] In the present invention, "delivery" refers to the provision of an entity to a target. For example, delivering a drug and / or therapeutic agent and / or prophylactic agent to a subject, which is a tissue and / or cell of a human and / or other animal.

[0092] 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 gel, 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, sodium saccharin, 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, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, 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.

[0093] In the present invention, a vaccine is a prophylactic or therapeutic material that provides at least one antigen or antigen function. The antigen or antigen function can stimulate the adaptive immune system of the body to provide an adaptive immune response.

[0094] 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.

[0095] 1. Amino acid cationic lipid

[0096] One embodiment of the present invention:

[0097] An amino acid cationic lipid, characterized in that its structure is shown in the general formula (1):

[0098]

[0099] Wherein, AA is a residue of an amino acid or an amino acid derivative;

[0100] Each occurrence of B2 is independently a linking bond or C 1-20 alkylene;

[0101] Each occurrence of L2 and L3 is independently a linking bond or a divalent linking group;

[0102] R1 is a C 10-40 aliphatic hydrocarbon group containing one, two or more unsaturated bonds, and each occurrence of R2 is independently a C 1-40 aliphatic hydrocarbon group or Wherein, t is an integer from 0 to 12; R e , R f are each independently a C 1-20 alkyl group, a C 2-20 alkenyl group and a C 2-20 alkynyl group;

[0103] Each occurrence of R3 is independently a hydrogen atom, an alkyl group, an alkoxy group, a C 3-6 carbocyclic group, a nitrogen-containing heterocyclic group, -NR d R d , -SR d , -C(=O)R d , -C(=O)OR d , -OC(=O)R d , or a functional group R 01 ; capable of reacting with a biologically relevant substance; wherein, R d is each independently a C 1-12 alkyl group;

[0104] b and c are each independently 1 or 2; when the fragment -B2-L2-R2 and / or -L3-R3 is led out from the amino terminus of the amino acid and its derivatives, b and c are each independently 1 or 2; when the fragment -B2-L2-R2 and / or -L3-R3 is led out from the carboxyl terminus of the amino acid and its derivatives, b and c are each independently 1; when b is 2, the two -B2-L2-R2 fragments are the same or different; when c is 2, the two -L3-R3 fragments are the same or different;

[0105] or a salt, tautomer, stereoisomer or solvate thereof.

[0106] 1.1. Amino acid or amino acid derivative residue AA

[0107] In the present invention, AA is a residue of an amino acid or an amino acid derivative.

[0108] In a specific embodiment of the present invention, the aforementioned amino acid or amino acid derivative is preferably any one of arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, histidine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine and amino acid derivatives of any one of the aforementioned amino acids; more preferably any one of lysine, glutamic acid and histidine; more preferably any one of the following structures and forms in which 1-3 terminal groups of any one of them are protected:

[0109]

[0110] In a specific embodiment of the present invention, it is preferred that AA is or wherein, R a each independently represents a linking bond, H, methyl, ethyl, propyl or isopropyl each time it appears; or AA is a case where 1-2 carbonyl groups in any one of the aforementioned structures are each independently capped with an oxygen atom or a secondary amine atom:

[0111] More preferably, AA is any one of the following structures:

[0112]

[0113]

[0114] 1.2. B2

[0115] In the present invention, B2 each independently represents a linking bond or C 1-20 alkylene

[0116] In a specific embodiment of the present invention, it is preferred that B2 each independently represents a linking bond or C1-20 Alkylene group, the C 1-20 The alkylene group is any one of methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene and eicosylene; more preferably, each occurrence of B2 is independently a linking bond or C 2-10 Alkylene group; the C 1-20 Alkylene group and C 2-10 The alkylene group is optionally substituted with 0-5 identical or different C 1-12 Alkyl group, cycloalkyl group, heterocyclic group or hydroxyl group, wherein, C 1-12 The alkyl group is preferably methyl, ethyl or propyl.

[0117] 1.3.R1, R2, R3

[0118] 1.3.1.R1

[0119] In the present invention, R1 is a C 10-40 aliphatic hydrocarbon group containing 1, 2 or more than 2 unsaturated bonds.

[0120] In a specific embodiment of the present invention, R1 is a C 10-40 linear aliphatic hydrocarbon group containing 1, 2 or 3 unsaturated bonds; preferably a linear C 10-20 aliphatic hydrocarbon group containing 1 or 2 unsaturated bonds; more preferably, R1 is selected from any one of the following structures:

[0121]

[0122]

[0123] In a specific embodiment of the present invention, preferably R1 is a branched aliphatic hydrocarbon group containing 2 or more than 2 unsaturated bonds; preferably a branched C 15-40 aliphatic hydrocarbon group containing 2 or more than 2 unsaturated bonds; preferably, R1 is selected from any one of the following structures:

[0124]

[0125]

[0126] 1.3.2.R2

[0127] In the present invention, each occurrence of R2 is independently a linear alkyl group, a branched alkyl group, a linear alkenyl group, a branched alkenyl group, a linear alkynyl group, a branched alkynyl group or

[0128] In a specific embodiment of the present invention, the linear alkyl group is preferably C 1-25 linear alkyl group; more preferably each independently is C 1-17 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.

[0129] In a specific embodiment of the present invention, the branched alkenyl group or branched alkynyl group is represented as wherein, R e , R f each independently is any one of C 1-20 alkyl group, C 2-20 alkenyl group and C 2-20 alkynyl group, and t is an integer from 0 to 12; more preferably R e , R f each independently is selected from any one of pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, octadeca-6,9-dienyl; more preferably the branched alkyl group, branched alkenyl group or branched alkynyl group is selected from any one of the following structures:

[0130]

[0131] In a specific embodiment of the present invention, is selected from any one of the following structures:

[0132]

[0133] 1.3.3.R3

[0134] In the present invention, R3 is a hydrogen atom, an alkyl group, an alkoxy group, C 3-6 carbocyclic group, a nitrogen-containing heterocycle, -NR d R d , -SR d , -C(=O)R d , -C(=O)OR d , -OC(=O)R d , or a functional group R 01 that can react with a biologically relevant substance; wherein, R d is each independently C 1-12 alkyl group each time it appears.

[0135] In a specific embodiment of the present invention, it is preferred that R3 is each independently a hydrogen atom, -R d , -OR d , -NR d R d , -C(=O)Rd -1. -C(=O)OR d . -OC(=O)R d . -OC(=O)OR d and R 01 any one of; R3 is more preferably each independently any one of the following cases:

[0136] Case (1): a hydrogen atom, an alkyl group, an alkoxy group, -C(=O)OR d . -OC(=O)R d . -OC(=O)OR d . epoxy group, hydroxyl group, protected 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;

[0137] Case (2): a functional group with therapeutic targeting; preferably a residue of any one of folic acid and N-acetylgalactosamine or a residue of a functional derivative of any one of them; more preferably any one of the following structures:

[0138]

[0139] 1.4.1.L2

[0140] In the present invention, each occurrence of L2 is independently a linking bond or a divalent linking group.

[0141] In a specific embodiment of the present invention, it is preferred that each occurrence of L2 is independently a linking bond, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, -NH-, -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 any one of C(=O)S-, wherein, R c is independently a hydrogen atom or C 1-12 alkyl each time it appears, s is 2, 3 or 4; more preferably, L2 is a linking bond, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, -O(CH2) 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-; more preferably, L2 is independently a linking bond, -C(=O)-, -O-, -NH-, -OC(=O)-, -C(=O)O-, -NHC(=O)- and -C(=O)NH- each time it appears.

[0142] 1.4.2. L3

[0143] In the present invention, L3 is independently a linking bond or a divalent linking group each time it appears.

[0144] In a specific embodiment of the present invention, preferably, L3 is independently a linking bond, -O-, -NH-, -C(=O)-, -(CH2) t -, -(CH2) t O-, -O(CH2) t -, -(CH2) t NH-, -(CH2) t C(=O)-, -(CH2) t O(CH2) t -, -C(=O)O(CH2) t -, -(CH2) t C(=O)O- and -(CH2) t OC(=O)- any one of them.

[0145] 1.5. Fragment -L3-R3, -B2-L2-R2

[0146] 1.5.1. -L3-R3

[0147] In a specific embodiment of the present invention, preferably, the -L3-R3 fragment is selected from any one of the following structures:

[0148]

[0149] 1.5.2. -B2-L2-R2

[0150] In a specific embodiment of the present invention, preferably, each occurrence of -B2-L2-R2 is independently selected from any one of the following structures:

[0151]

[0152] 1.6. Examples of structural general formulas

[0153] In a specific embodiment of the present invention, preferably, the structure of the amino acid cationic lipid satisfies any one of the following general formulas:

[0154] Wherein, B2, L2, and L3 are not linker bonds; more preferably, each occurrence of L2 is independently any one of -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH-, -NHC(=O)O-; even more preferably, each occurrence of L2 is independently any one of -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-.

[0155] 1.7. Examples of specific structures

[0156]

[0157]

[0158]

[0159]

[0160] 2. Preparation of amino acid cationic lipid

[0161] 2.1. Method 1

[0162] In the present invention, the preparation of any of the aforementioned amino acid cationic lipids can be carried out by methods including but not limited to the following:

[0163] Step 1: React one molecule of A-1 (AA1) with two molecules of the same or different A-2 (R3'-F1) to generate a small molecule intermediate A-3 containing a divalent linker L3 Among them, small molecule A-1 is an amino acid or amino acid derivative containing 1, 2 or 3 identical or different amino acid end groups, and the amino acid end groups are protected or unprotected, including but not limited to amino group, carboxyl group, hydroxyl group, mercapto group, protected amino group, protected carboxyl group, protected hydroxyl group, protected mercapto group; small molecule A-2 contains a reactive group F1, which can react with the amino acid end group of AA1 to form a divalent linker L3, preferably -OH, -COOH, -NH2, -Br, etc., and the R3' end contains a reactive group R 01 or contains R 01 of the micro-variation form; the micro-variation form refers to the structural form that can form the target reactive group after simple chemical reaction processes such as deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group; AA” is an amino acid derivative residue containing 2 identical or different amino acid end groups, and the amino acid end groups are protected or unprotected, including but not limited to amino group, carboxyl group, hydroxyl group, mercapto group, protected amino group, protected carboxyl group, protected hydroxyl group, protected mercapto group; c is 1 or 2, depending on the type of the end group of AA1 reacting with F1 and the feeding situation of R3'-F1. When the end group of AA1 reacting with F1 is carboxyl or hydroxyl group, c is 1, and when it is amino group, c is 1 or 2; and when c is 2, the two -L3-R3' fragments can be the same or different, that is, when F1 reacts with the amino end group of AA1, A-1 can react with different A-2 successively to obtain A-3 with c being 2 and the two -L3-R3' fragments being different, or 1 molecule of A-1 can react with 2 molecules of the same A-2 in one step or stepwise to obtain A-3 with c being 2 and the two -L3-R3' fragments being the same;

[0164] Step 2: React small molecule A-4 (R2-F2) with small molecule A-5 (F3-B2-F N ) to generate a small molecule intermediate A-6 (R2-L2-B2-F N ) containing a divalent linker L2, with a reactive group F N at one end and R2 at the other end; among them, small molecule A-4 contains a reactive group F2, small molecule A-5 contains a pair of hetero-functional groups F3 and F N , F2 can react with F3 to form a divalent linker L2, and F N is a reactive group capable of reacting with an amino group, preferably -F, -Cl, -Br, etc.;

[0165] Step 3: React one molecule or two molecules of the same or different small molecule intermediate A-6 (R2-L2-B2-F N ) with small molecule intermediate A-3 to obtain amino acid derivative A-7 Among them, AA’ is an amino acid derivative residue, containing 1 amino acid terminal group, and the amino acid terminal group is protected or unprotected, including but not limited to amino group, carboxyl group, hydroxyl group, mercapto group, protected amino group, protected carboxyl group, protected hydroxyl group, protected mercapto group; b is 1 or 2, depending on the terminal group type of AA” reacting with F N and the feeding situation of R2-L2-B2-F N When reacting with F N if the terminal group of AA” is carboxyl group or hydroxyl group, b is 1, and if it is amino group, b is 1 or 2; and when b is 2, the two -L6-B2-L2-R2 segments can be the same or different, that is, when F N reacts with the amino terminal group of AA”, A-3 can react with different A-6 successively to obtain A-7 with b being 2 and the two -L6-B2-L2-R2 segments being different, or 1 molecule of A-3 can react with 2 molecules of the same A-6 stepwise or in multiple steps to obtain A-7 with b being 2 and the two -L6-B2-L2-R2 segments being the same;

[0166] Step 4. The amino acid derivative A-7 reacts with the small molecule intermediate A-8 (a small molecule alcohol derivative obtained by reacting R1-OH, a bromide R1-Br with metallic magnesium under anhydrous and anaerobic conditions, or can also be obtained by purchase) to generate the amino acid cationic lipid A-9’ AA is an amino acid or amino acid derivative residue;

[0167] When R3’ is equal to R3, the obtained structure A-9’ is the structure corresponding to the general formula (1);

[0168] When R3’ is not equal to R3, A-9’ is subjected to terminal micro - modification to obtain A-9 which is the structure corresponding to the general formula (1); the terminal micro - modification is selected from the following chemical reactions: de - protection, salt complexation and de - complexation, ionization, protonation, de - protonation, changing the leaving group;

[0169] Among them, the definitions of L2, L3, B2, R3, R1, R2, b, and c are the same as those described in the general formula (1), and will not be elaborated here.

[0170] The foregoing various small molecule raw materials A-1, A-2, A-3, A-4, A-5, A-8, etc. can be obtained by purchase or can be synthesized independently. For example, in Example 4, the small molecule A-3 is which can be synthesized independently using N - methyl lysine and TBS - protected bromoethane as raw materials; in Example 1, the small molecule A-3 is which can be obtained by purchase and then carrying out Boc protection of the amino group, thus omitting Step 1.

[0171] Step 1

[0172]

[0173] Step 2

[0174]

[0175] Step 3

[0176]

[0177] Step 4

[0178]

[0179] In this method, A-3 in Step 3 The two amino acid end groups contained in AA” can also react with A-8 (R1-OH) simultaneously to obtain A-10'; at this time, both L2 and B2 are linking bonds, R1 and R2 are the same, and the definitions of L2, L3, B2, R3, R1, R2, b, and c are the same as those described in general formula (1), which will not be elaborated here;

[0180] When R3' is equal to R3, the obtained structure A-10' corresponds to the structure shown in general formula (1);

[0181] When R3' is not equal to R3, A-10' is subjected to terminal micro-modification to obtain A-10, which corresponds to the structure shown in general formula (1); the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group.

[0182] For the foregoing preparation steps, the reaction with multiple molecules can be carried out in one step or in multiple steps.

[0183]

[0184] In the reaction raw material R2-F2 in the foregoing preparation method, R2 can be an etherified aliphatic hydrocarbon derivative residue wherein, each t independently represents an integer from 0 to 12 each time it appears; R e and R f each independently represents C 1-15 alkyl, C 2-15 alkenyl, and C 2-15 alkynyl, any one of them. More specifically, R1-F1 can be It can be obtained by purchase or synthesized independently. When synthesized independently, aldol addition can be used. For example, one molecule reacts with two molecules of R e -OH to undergo addition to obtain At this time, Re and R f are the same; R1-F1 can also be obtained by purchase or synthesized independently. When synthesized independently, it can be obtained by reacting with a relevant alkylating reagent. The alkylating reagent is preferably a halide, such as It can be obtained by reacting one molecule of glycerol with a hydroxyl group protected by TBS and two molecules of bromohexane followed by deprotection. It can be obtained by reacting one molecule of glycerol with a hydroxyl group protected by TBS and two molecules of bromohexane followed by deprotection.

[0185] 2.1.1. Specific method

[0186] 2.1.1.1. Specific method 1:

[0187] Step 1: React one molecule of A-1 with one or two molecules of the same or different A-2 (R3’-F1) to generate a small molecule intermediate A-3 containing a divalent linker L3 Among them, the small molecule A-1 is an amino acid or amino acid derivative containing 2 or 3 identical or different amino acid end groups, and the amino acid end groups are protected or unprotected, including but not limited to amino group, carboxyl group, hydroxyl group, mercapto group, protected amino group, protected carboxyl group, protected hydroxyl group, protected mercapto group; L AA ’ is an amino acid derivative residue containing 1 amino acid end group, and the amino acid end group is protected or unprotected, including but not limited to amino group, carboxyl group, hydroxyl group, mercapto group, protected amino group, protected carboxyl group, protected hydroxyl group, protected mercapto group; the small molecule A-2 contains a reactive group F1, which can react with the amino acid end group of L AA ' to generate a divalent linker L3, preferably -OH, -COOH, -NH2, -Br, etc., and the R3’ end contains a reactive group R 01 or contains R 01 or a micro-variation form containing R 01 ; The micro-variation form refers to a group that can be transformed into R AA through any one of the chemical processes of deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group; L m is -NH(CH2) t5 -, -OC(=O)(CH2) t5 -, or -NHC(=O)(CH2) AA -, the left side of L etc., whose amino terminus and carboxyl terminus can be protected or unprotected, such as S1-3 in Example 1, S4-1 in Example 4, S14-1 in Example 14, etc.; c is 1 or 2, depending on L reacting with F1 AA 's end group type and the feeding situation of R3'-F1. When the end group of L AA ' reacting with F1 is a carboxyl group or a hydroxyl group, c is 1; when it is an amino group, c is 1 or 2; and when c is 2, the two -L3-R3' fragments can be the same or different. That is, when F1 reacts with the AA amino terminal group of L', A-1 can react with different A-2 successively to obtain A-3 with c being 2 and the two -L3-R3' fragments being different, or 1 molecule of A-1 can react with 2 molecules of the same A-2 in one step or in multiple steps to obtain A-3 with c being 2 and the two -L3-R3' fragments being the same;

[0188] Step 2: The amino acid derivative A-3 undergoes an esterification reaction with one molecule of the alcohol derivative A-4 (R1-OH) to form the intermediate A-5 wherein A-4 can be generated by the action of bromide R1-Br on metallic magnesium or can be obtained by purchase, such as S1-2 in Example 1, S14-4 in Example 14, S15-3 in Example 15, etc.;

[0189] Step 3: One molecule or two molecules of the same or different bromides A-6( x is an integer from 0 to 7, such as S1-6 in Example 1, S7-1 and S1-6 in Example 7, S2-3 in Example 8, etc.) or succinimide derivatives A-7( such as S13-3 in Example 13) react with A-5 to obtain the amino acid lipid A-8' or A-9' wherein, b is 2, and the two -(CH2) x -L2-R2 fragments can be the same or different, depending on the feeding situation and type when A-6 reacts with A-5; that is, when A-5 reacts with different A-6 successively to obtain A-8' with the two -(CH2) x -L2-R2 fragments being different, for example, when A-5 reacts with 2 molecules of the same A-6 in one step or in multiple steps to obtain A-9' with the two -(CH2) x -L2-R2 fragments being the same;

[0190] In the reaction raw material A-6 in the foregoing preparation method, R2 in the molecule can be an etherified aliphatic hydrocarbon derivative residue wherein, t is independently an integer from 0 to 12 each time it appears; R e 、R f are independently C1-C15 Any one of alkyl, C2-C 15 alkenyl, and C2-C 15 alkynyl. More specifically, R1-F1 can be obtained by purchase or synthesized independently. When synthesized independently, 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. For example, in Example 9, S9-5 is obtained by reacting one molecule of 3-hydroxypropanal with TBS-protected hydroxyl group and two molecules of 1-octanol, followed by deprotection; R1-F1 can also be obtained by purchase or synthesized independently. When synthesized independently, it can be obtained by reacting with a related alkylating agent. The alkylating agent is preferably a halide. For example, in Example 10, S10-4 is obtained by reacting one molecule of glycerol with TBS-protected hydroxyl group and two molecules of bromohexane, followed by deprotection.

[0191] When R3’ is equal to R3, the obtained structure A-8’ or A-9’ corresponds to the structure shown in the general formula (1), where corresponds to the AA nucleus in the general formula (1);

[0192] When R3’ is not equal to R3, A-8’ or A-9’ is subjected to terminal micro-modification to obtain A-8 or A-9 corresponding to the structure shown in the general formula (1); the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group;

[0193] Among them, the definitions of L3, R3, R1, R2, b, and c are the same as those described in the general formula (1), and will not be elaborated here. The foregoing small molecule raw materials A-1, A-2, A-3, A-4, A-6, etc. can be obtained by purchase or synthesized independently. For example, in Example 4, the small molecule A-3 is which can be synthesized independently using N-methyllysine and bromoethane with TBS-protected hydroxyl group as raw materials; in Example 1, the small molecule A-3 is which can be obtained by purchase and then performing Boc protection on the amino group, thus omitting Step 1.

[0194] Step 1

[0195]

[0196] Step 2

[0197]

[0198] Step Three

[0199]

[0200] 2.1.1.2. Specific Method 2:

[0201] Step 1: React one molecule of B-1 with one or two molecules of the same or different B-2 (R3'-F1) to form a small molecule intermediate B-3 containing a divalent linker L3 wherein the small molecule B-1 is an amino acid or amino acid derivative containing 2 or 3 identical or different amino acid end groups, and the amino acid end groups are protected or unprotected, including but not limited to amino group, carboxyl group, hydroxyl group, protected amino group, protected carboxyl group, protected hydroxyl group; L AA ' is an amino acid derivative residue containing 1 amino acid end group, and the amino acid end group is protected or unprotected, including but not limited to amino group, carboxyl group, hydroxyl group, protected amino group, protected carboxyl group, protected hydroxyl group; the small molecule B-2 contains a reactive group F1, which can react with the amino acid end group of L AA ' to form a divalent linker L3, preferably -OH, -COOH, -NH2, -Br, etc., and the R3' end contains a reactive group R 01 or contains a micro-variant form of R 01 ; the micro-variant form refers to a group that can be transformed into R 01 through any one of the chemical processes of deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group; L AA is -NH-, and the left side of L AA is connected to L3; B-3 can be whose carboxyl terminus can be protected or unprotected, such as S3-1 in Example 3, S6-1 in Example 6, etc.; c is 1 or 2, depending on the end group type of L AA ' reacting with F1 and the feeding situation of R3'-F1. When the secondary amino group of L AA ' reacts with F1, c is 1; when it is the primary amino group, c is 1 or 2; and when c is 2, the two -L3-R3' fragments can be the same or different, that is, when F1 reacts with the primary amino group of L AA ', B-1 can react with different B-2 successively to obtain B-3 with c being 2 and the two -L3-R3' fragments being different, or one molecule of B-1 can react with two molecules of the same B-2 in one step or stepwise to obtain B-3 with c being 2 and the two -L3-R3' fragments being the same;

[0202] Step 2. Amino acid derivative B-3 ( such as S1-3 in Example 3, S6-1 in Example 6, etc.) reacts with two molecules of alcohol derivative B-4 (R1-OH) to form amino acid lipid B-5’ wherein B-4 can be formed by the action of bromide R1-Br on metallic magnesium or can be obtained by purchase, such as S1-2 in Example 3;

[0203] When R3’ is equal to R3, the obtained structure B-5’ is the structure corresponding to the general formula (1), wherein, corresponding to the AA core in the general formula (1), R1 and R2 are the same, and b is 1;

[0204] When R3’ is not equal to R3, B-5’ is subjected to terminal micro-modification to obtain the structure B-5 corresponding to the general formula (1); the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, changing the leaving group;

[0205] wherein, the definitions of L3, R3, R1, R2, and c are the same as those described in the general formula (1), and will not be elaborated here. The aforementioned small molecule raw materials B-1, B-2, B-3, B-5, etc. can be obtained by purchase or can be synthesized independently. For example, the small molecule B-3 in Example 6 is which can be synthesized independently using N-methylglutamic acid and bromoethane protected by TBS hydroxyl as raw materials; the small molecule B-3 in Example 3 is which can be obtained by purchase, and thus Step 1 can be omitted.

[0206] Step 1

[0207]

[0208] Step 2

[0209]

[0210] 2.2. Instructions on relevant raw materials and / or steps during the preparation process

[0211] 2.2.1. Condensing agent, oxidizing agent, reducing agent

[0212] In the present invention, the condensing agent used in the reaction is not limited, but N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) are preferred, and DCC is most preferred. Generally, the amount of the condensing agent is 1 to 20 times the molar equivalent of the carboxylic acid, preferably 5 - 10 times. An appropriate catalyst (such as 4-dimethylaminopyridine) can be added to this reaction.

[0213] In the present invention, the oxidizing agent used in the reaction is not particularly limited, as long as it is a compound or a combination of multiple compounds that can increase the valence of the substrate. Preferred are phenyl iodide bis(trifluoroacetate), 1,4-benzoquinone, benzyltrimethylammonium tribromide, pyridinium dichromate, potassium dichromate, ozone, oxygen, hypofluorous acid, sodium hypochlorite, cobalt(III) acetate, cobalt(II) acetate, manganese(II) acetate, palladium(II) acetate, copper(II) acetate, monoperoxyphthalic acid, iodine, N-iodosuccinimide, iodobenzene dichloride, 2-iodoxybenzoic acid, dimethyldioxirane, dimethyl sulfoxide - oxalyl chloride, dimethyl sulfoxide - acetic anhydride, DDQ, dichloro(tris(triphenylphosphine))ruthenium, manganese dioxide, diacetoxyiodobenzene, periodic acid, sodium periodate, sodium periodate - osmium tetroxide, potassium permanganate, sodium metaborate, peroxybenzoic acid, benzoyl peroxide, nickel peroxide, hydrogen peroxide, cumene hydroperoxide, tert-butyl hydroperoxide, peracetic acid, m-chloroperoxybenzoic acid, N-chlorosuccinimide, pyridinium chlorochromate, palladium(II) chloride - copper(II) chloride, urea hydrogen peroxide complex, triphenylmethyl tetrafluoroborate, tributyltin oxide, cobalt(III) fluoride, vanadyl trifluoride, chromium(III) oxide, manganese(III) acetate, TEMPO, ammonium cerium(IV) nitrate, bromine, N-oxidopyridine, silver oxide, O-ethyl peroxycarbonate, manganese(III) acetylacetonate, vanadyl acetylacetonate, aluminum isopropoxide, potassium peroxymonosulfate, dichloroiodobenzene, etc., or a combination of one or more of them. More preferably, it is a combination of one or more of oxygen, sodium hypochlorite, hydrogen peroxide, dichloroiodobenzene, potassium peroxymonosulfate, etc. The amount of the oxidizing agent is 1 to 50 times the molar equivalent of the hydroxyl group in the intermediate compound, preferably 1 to 20 times, and more preferably 5 to 10 times.

[0214] In the present invention, the reducing agent used in the reaction is not particularly limited, as long as it can reduce the Schiff base formed by ammonia and aldehyde or ketone to an amino group; preferred are sodium borohydride, sodium cyanoborohydride, lithium aluminum hydride, borane, diborane, diisobutylaluminum hydride, diisopinocampheylborane, lithium borohydride, zinc borohydride, borane - pyridine, borane - methyl sulfide, borane - tetrahydrofuran, etc., or a combination of one or more of them; more preferably sodium cyanoborohydride. The equivalent of the reducing agent is 1 to 50 times the molar equivalent of the amino group to be modified, preferably 1 to 20 times, and more preferably 5 to 10 times.

[0215] In the present invention, the reaction temperature is from 0 to 200 °C, preferably from 0 to 100 °C, more preferably from 0 to 25 °C, and the reaction time is preferably from 10 minutes to 48 hours, more preferably from 30 minutes to 24 hours. The obtained product can be purified by purification methods such as extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, thin film dialysis or supercritical extraction.

[0216] In the present invention, the reaction solvent can be solvent-free or an aprotic solvent. The aprotic solvents include toluene, benzene, xylene, acetonitrile, ethyl acetate, ether, methyl tert-butyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, preferably tetrahydrofuran, dichloromethane, dimethyl sulfoxide, dimethylformamide.

[0217] In the present invention, the base used in the reaction is an inorganic base or an organic base, preferably an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine); preferably triethylamine, pyridine. The amount of the base is 1 to 50 times the molar equivalent of the carboxylic acid, preferably 1 to 10 times, more preferably 2 to 3 times.

[0218] 2.2.2. "Protection" and "deprotection" of related groups involved in the reaction process

[0219] 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. And when there are two or more functional groups, only the target functional group is selectively made to react, 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 needed. Therefore, in organic synthesis, it is important to deprotect only the protecting group bonded to the designated functional group under appropriate conditions.

[0220] 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.

[0221] In the present invention, the hydroxyl group protected by the hydroxyl protecting group is not particularly limited, and can be, for example, 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, and can come from, for example, 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.

[0222] 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 benzyl, silyl ether, acetal, or tert-butyl as an example, the corresponding deprotection methods are as follows:

[0223] A: Deprotection of the benzyl protecting group

[0224] Benzyl deprotection 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.

[0225] There is no limitation on the hydrogenation reduction catalyst, and palladium and nickel are preferred. However, the carrier is not limited, but alumina or carbon is preferred, and carbon is more preferred. The dosage of palladium is 1 to 100 wt% of the protected hydroxy compound, preferably 1 to 20 wt% of the protected hydroxy compound.

[0226] There is no particular limitation on the reaction solvent as long as both the raw materials and products can be dissolved in the solvent. However, methanol, ethanol, ethyl acetate, tetrahydrofuran, and acetic acid are preferred; methanol is more preferred. There is no particular limitation on the hydrogen donor, but hydrogen, cyclohexene, 2-propanol, ammonium formate, etc. are preferred. The reaction temperature is preferably 25 to 40 °C. There is no particular limitation on the reaction time, and the reaction time is negatively correlated with the dosage of the catalyst, preferably 1 to 5 hours.

[0227] B: Deprotection of silyl ether protecting groups

[0228] Compounds used for such hydroxy protection include trimethylsilyl ether, triethylsilyl ether, dimethyl tert-butylsilyl ether, tert-butyldiphenylsilyl ether, etc. The deprotection of such silyl ethers is carried out by a fluoride ion-containing compound, preferably tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid, potassium fluoride, and more preferably tetrabutylammonium fluoride and potassium fluoride. The dosage of the fluorine-containing reagent is 5 to 20 times the molar equivalent of the protected hydroxy group, preferably 8 to 15 times the initiator. If the dosage of fluorine is less than 5 times the molar equivalent of the protected hydroxy group, incomplete deprotection will occur; when the dosage of the deprotection reagent is greater than 20 times the molar equivalent of the protected hydroxy group, the excess reagent or compound will cause trouble in purification and may be mixed into subsequent steps, thus causing side reactions. There is no particular limitation on the reaction solvent as long as it can dissolve the reactants and products. Aprotic solvents are 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.

[0229] C: Deprotection of tert-butyl protecting groups

[0230] The deprotection of tert-butyl is carried out under acidic conditions, and the solution pH is preferably 0 to 4. There is no particular limitation on the acid, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and nitric acid are preferred, and hydrochloric acid is more preferred. There is no particular limitation on the reaction solvent as long as it can dissolve the reactants and products. Water is preferred. The reaction temperature is preferably 0 to 30 °C.

[0231] 2.2.3. Alkylation reaction

[0232] 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 sequence. Examples are as follows:

[0233] 2.2.3.1. Alkylation of substrate alcohols with sulfonates and halides

[0234] In the presence of a base, an amine 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 brings trouble to purification and may be mixed into the subsequent steps, resulting in an increase in side reactions in the next step and an increase in purification difficulty.

[0235] 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, the anion exchange resin is not particularly limited 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. The solvents for osmosis and ultrafiltration are not limited. Generally, water or organic solvents can be used. The organic solvents are not particularly limited as long as the product can be dissolved in them. Dichloromethane, chloroform, etc. are preferred.

[0236] The reaction solvent is not restricted. 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.

[0237] The base includes organic bases (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine) or inorganic bases (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.

[0238] 2.2.3.2. Alkylation of substrate amines with sulfonates and halides

[0239] A. Alkylation of substrate amines with sulfonates and halides

[0240] 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 and may be mixed into subsequent steps, resulting in an increase in side reactions in the next step and an increase in the difficulty of purification.

[0241] The obtained product is a mixture of an amine intermediate and excess sulfonate and halide, which can be purified by column chromatography, 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. Preferably, it is an ion exchange resin of tertiary amine or quaternary ammonium salt with dextran, agarose, polyacrylate, polystyrene, polydiphenylethylene, etc. as the backbone. The solvent for osmosis and ultrafiltration is not limited. Generally, it can be water or an organic solvent. There is no particular limitation on the organic solvent as long as the product can be dissolved in it. Preferably, it is dichloromethane, chloroform, etc.

[0242] The reaction solvent is not restricted. Preferably, it is an aprotic solvent, such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide. More preferably, it is dimethylformamide, dichloromethane, dimethyl sulfoxide or tetrahydrofuran.

[0243] 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). Preferably, it is an organic base, and more preferably triethylamine or pyridine. 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.

[0244] 2.2.3.3. Alkylation reaction of substrate amine with aldehyde derivatives

[0245] After obtaining an imine intermediate from the reaction of a substrate amine with an aldehyde derivative, an 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, 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 causes trouble in purification, may be mixed into subsequent steps, and increases 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 such as cation exchange resin, osmosis, ultrafiltration, etc. to obtain the intermediate. The cation exchange resin has no particular limitation as long as it can exchange with quaternary ammonium cations to achieve a separation effect. The solvents for osmosis and ultrafiltration are not limited. Generally, water or organic solvents can be used. The organic solvents have no particular limitation as long as the product can be dissolved in them. Preferred are dichloromethane, chloroform, etc.

[0246] The reaction solvent is not limited. Preferred are organic solvents such as methanol, ethanol, water, toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, etc.; more preferred are water and methanol.

[0247] The reducing agent has no particular limitation as long as it can reduce the imine to an amine. Preferred are sodium borohydride, lithium aluminum hydride, sodium cyanoborohydride, Zn / AcOH, etc., and more preferred is sodium cyanoborohydride. Generally, the amount of the reducing agent used is 0.5 to 50 times the amount of substance of the aldehyde derivative, and more preferably 1 - 10 times.

[0248] 2.2.4. Terminal linear functionalization

[0249] The method of terminal linear functionalization has no particular limitation and is related to the type of the final functional group or its protected form. It mainly includes the functionalization of terminal hydroxyl groups and the transformation of reactive groups into the target functional group or its protected form.

[0250] For the preparation method of the functionalization of terminal hydroxyl groups, starting from the terminal hydroxyl group of A, functional groups of types A to J or their protected forms are obtained through functionalization. The specific preparation method is as described in paragraphs

[0960] to

[1205] of document CN104530417A. The reaction general formula is as follows:

[0251]

[0252] Among them, q and q1 are each independently 0 or 1; Z1 and Z2 are each independently divalent linking groups; R 01 is a functional group capable of reacting with biologically relevant substances.

[0253] Based on the transformation of reactive groups into target functional groups or their protected forms, it can be achieved by any of the following methods:

[0254] Method 1: Direct modification. Based on the direct modification of reactive groups, the target functional group or its protected form is obtained. As an example, the transformation of a carboxyl group into an acyl halide, acyl hydrazide, ester, thioester, dithioester, etc., and the transformation of hydroxyl, mercapto, alkynyl, amino, carboxyl groups, etc. into the corresponding protected structures, etc. Another example is the modification of hydroxyl, amino, etc. by acid anhydrides.

[0255] Method 2: Coupling reaction between two reactive groups. Using a hetero-functionalized reagent containing one type of reactive group and the target functional group or its protected form as a raw material, the target functional group or its protected form is introduced through the reaction between one of the reactive groups and the reactive group at the A end. There are no particular restrictions on the reaction mode and method between the two reactive groups. Its reaction conditions are related to the type of divalent linker formed by the reaction and can adopt existing publicly available technologies. Such as alkylation, alkenyl addition reaction, alkynyl addition reaction, Schiff base reaction combined with reduction reaction, condensation reaction, etc. Among them, the alkylation reaction is preferably an alkylation reaction based on mercapto or amino, corresponding to the formation of thioether bond, secondary amino or tertiary amino in sequence. Among them, the condensation reaction includes but is not limited to condensation reactions for generating ester groups, thioester groups, amide groups, imine bonds, hydrazone bonds, carbamate groups, etc. Another example is using a hetero-functionalized reagent containing groups such as azide, alkynyl, alkenyl, trithioester group, mercapto, dienyl, furyl, 1,2,4,5-tetrazinyl, cyanate, etc. and the target functional group or its protected form as a raw material, and introducing the target functional group or its protected form through click reaction. The reaction between the two reactive groups is accompanied by the formation of a new bond. Typical representatives of the newly formed divalent linker are amide bond, urethane bond, ester group, secondary amine bond, thioether bond, triazole group, etc.

[0256] Method 3: Obtaining the target functional group or its protected form through a combination of direct modification and coupling reaction.

[0257] In the present invention, the raw materials used in each preparation method can be obtained by purchase or self-synthesis.

[0258] In the present invention, the intermediates and final 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 final product, characterization methods including but not limited to nuclear magnetic resonance, electrophoresis, ultraviolet-visible spectrophotometer, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism spectrometry, mass spectrometry, etc. can be used.

[0259] 3.1. Lipid composition

[0260] In the present invention, a lipid composition contains any one of the amino acid cationic lipids having the structure shown in the general formula (1) as described above.

[0261] In a specific embodiment of the present invention, preferably, in addition to the cationic lipid having the structure 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:

[0262] Situation (1): It further contains phospholipids;

[0263] Situation (2): It further contains steroid lipids;

[0264] Situation (3): It further contains polyethylene glycolated lipids;

[0265] Situation (4): It further contains phospholipids and steroid lipids;

[0266] Situation (5): It further contains phospholipids and polyethylene glycolated lipids;

[0267] Situation (6): It further contains steroid lipids and polyethylene glycolated lipids;

[0268] Situation (7): It further contains phospholipids, steroid lipids, and polyethylene glycolated lipids;

[0269] More preferably, it further contains three lipids, namely neutral lipids, steroid lipids, and polyethylene glycolated lipids, simultaneously.

[0270] In a specific embodiment of the present invention, the phospholipid in the lipid composition is preferably any one of 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-bisdocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 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-bisdocosahexaenoyl-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) and their combinations.

[0271] 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.

[0272] In a specific embodiment of the present invention, the polyethylene glycolylated 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 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-oleoyl phosphatidylethanolamine, polyethylene glycol 2000-1,2-oleoyl phosphatidylethanolamine, and polyethylene glycol 2000-2,3-dimyristoyl glycerol (PEG-DMG) and its composition.

[0273] In a specific embodiment of the present invention, the polyethylene glycolylated lipid in the lipid composition is preferably any one of the following structures and its composition:

[0274] Wherein, n1 is an integer from 25 to 300, and more preferably n1 is any one of 44, 45, 46, 47, 48.

[0275] In a specific embodiment of the present invention, it is preferred that any one of the foregoing lipid compositions contains 20-80% of the amino acid cationic lipid shown in formula (1), 5-15% of phospholipids, 25-55% of steroid lipids, and 0.5-10% of polyethylene glycolylated lipids, and the percentages are the molar percentages of each lipid in the total lipids in the solution containing the solvent.

[0276] In a specific embodiment of the present invention, it is preferred that in any one of the foregoing lipid compositions, the molar percentage of the cationic lipid in the total lipids 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%.

[0277] In a specific embodiment of the present invention, it is preferred that in any one of the foregoing lipid compositions, the molar percentage of the phospholipid 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%.

[0278] In a specific embodiment of the present invention, preferably in any of the aforementioned lipid compositions, the molar percentage of steroid lipid in the total lipid in the solution containing the solvent is 35 - 50%, more preferably any one of about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.

[0279] In a specific embodiment of the present invention, preferably in any of the aforementioned lipid compositions, the molar percentage of polyethylene glycolated lipid in the total lipid in the solution containing the solvent is 0.5 - 5%; preferably 1 - 3%; more preferably any one of about 1.5%, 1.6%, 1.7%, 1.8%, 1.9%.

[0280] 3.2. Preparation of lipid composition

[0281] 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.

[0282] 4. Lipid drug composition and its preparation

[0283] 4.1. Lipid drug composition

[0284] In an embodiment of the present invention, a lipid drug composition contains any of the aforementioned lipid compositions and a drug, wherein the lipid composition contains any of the aforementioned amino acid cationic lipids having a structure as shown in general 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.

[0285] In a specific embodiment of the present invention, in the lipid drug composition, 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; preferably the nucleic acid drug is any one of DNA, mRNA, miRNA, and siRNA.

[0286] In a specific embodiment of the present invention, preferably the lipid drug composition is preferably used as a drug and is selected from any one of the following drugs: anti-tumor agent, antiviral agent, anti-fungal agent, and vaccine.

[0287] In a specific embodiment of the present invention, preferably, the drug includes but is not limited to doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracycline, nitrogen mustard, thiotepa, chlorambucil, lomustine, melphalan, carmustine, romustine, busulfan, dibromomannitol, mitomycin C, cis-dichlorodiammineplatinum(II), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, dibucaine, chlorpromazine, propranolol, timolol, labetalol, clonidine, hydralazine, imipramine, amitriptyline, doxepin, phenytoin, diphenhydramine, chlorpheniramine, promethazine, gentamicin, ciprofloxacin, cefoxitin, miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, naftifine, amphotericin B, antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma drugs, vitamins, sedatives, and imaging agents, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, colchicine, daunorubicin, mitoxantrone, mithramycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, puromycin, maytansinoids.

[0288] In a specific embodiment of the present invention, preferably, 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;

[0289] 4.2. Lipid Drug Composition Preparation

[0290] 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, the 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.

[0291] In 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.

[0292] In the present invention, the preparation of the lipid drug composition preparation includes the following steps:

[0293] (1) Equilibrate the lipid composition in the diluent or excipient;

[0294] (2) Add the nucleic acid drug to the mixture of the equilibrated lipid composition and the diluent or excipient for complexation;

[0295] Wherein, preferably, the equilibration time is 0.1 - 12 h, preferably 0.2 - 6 h, more preferably 0.5 - 3 h; preferably, the complexation time is 0.1 - 12 h, preferably 0.2 - 5 h, more preferably 0.5 - 2 h.

[0296] 5. Liposomes or lipid nanoparticles and their preparation

[0297] 5.1. Liposomes or lipid nanoparticles

[0298] In a specific embodiment of the present invention, a liposome or lipid nanoparticle contains any one of the lipid drug compositions described above.

[0299] In a specific embodiment of the present invention, preferably, the aforementioned lipid nanoparticle is an LNP - drug composition, an LPP - drug composition or a PNP drug composition; preferably an LNP - drug composition; more preferably an LNP - nucleic acid drug composition; more preferably an LNP - mRNA drug composition.

[0300] 5.2. Preparation of liposomes or lipid nanoparticles

[0301] 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 the thin - film dispersion method, ultrasonic dispersion method and / or reverse - phase evaporation method.

[0302] 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.

[0303] 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:

[0304] (1) Weigh the cationic lipid, steroid lipid, neutral lipid 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;

[0305] (2) Add a phosphate buffer solution containing a cryoprotectant, and perform ultrasonic bath to form a semi - transparent emulsion;

[0306] (3) Add the emulsion to a high-pressure homogenizer for overpressure, and then add the overpressurized emulsion to a liposome extruder for membrane filtration to form liposomes;

[0307] (4) Optionally, dry the liposomes in a freeze dryer to form liposome powder;

[0308] 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, and most preferably 100-170 rpm; preferably, the temperature of the rotary evaporation under reduced pressure is 10-200 °C, more preferably 20-100 °C, and most preferably 40-80 °C;

[0309] Preferably, the drying time by the vacuum pump is 1-72 h, more preferably 5-48 h, and most preferably 15-36 h;

[0310] Preferably, the mass concentration of the cryoprotectant dissolved in the phosphate buffer solution is 0.1-80%, preferably 1-50%, and more preferably 5-20%;

[0311] Preferably, the frequency of the water bath ultrasonic treatment is 10-300 kHz, more preferably 30-200 kHz, and most preferably 60-150 kHz;

[0312] 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;

[0313] Preferably, the pressure of the high-pressure homogenizer is 50-240 MPa, more preferably 80-200 MPa, and most preferably 100-150 MPa;

[0314] 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;

[0315] Preferably, the pressure of the liposome extruder is 50-300 MPa, more preferably 80-250 MPa, and most preferably 120-200 MPa;

[0316] 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;

[0317] Preferably, the drying time of the freeze dryer is 1-120 h, more preferably 5-72 h, and most preferably 10-36 h.

[0318] 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.

[0319] In a specific embodiment of the present invention, it is preferred that lipid nanoparticles are prepared by a microfluidic method, and the steps are as follows:

[0320] (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.

[0321] (2) Add the nucleic acid drug to a buffer solution to obtain an aqueous solution; the aqueous phase is preferably citrate buffer or sodium acetate buffer solution.

[0322] (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.

[0323] The following further describes the preparation methods of amino cationic lipids, lipid compositions, and lipid drug composition preparations and the bioactivity tests of lipid drug compositions in combination with some specific examples. The specific examples are for further detailed description of the present invention and do not limit the protection scope of the present invention. Among them, in the examples for preparing cationic lipids, the final product is characterized by nuclear magnetic resonance for its structure and confirmed by mass spectrometry for its molecular weight.

[0324] Example 1: Cationic Lipid (E1-1)

[0325]

[0326] The preparation process is as follows:

[0327] Step a: Add freshly activated magnesium turnings (0.40 g, 16.6 mmol) to a clean round-bottom flask equipped with a magnetic stir bar, addition funnel, and reflux condenser. Degas and flush the apparatus with argon, and add 2 mL of anhydrous diethyl ether to the flask via syringe. Dissolve bromide 18-bromo-6,9-octadecadiene (S1-1, 4.37 g, 13.3 mmol) in anhydrous diethyl ether (50 mL), and add it to the dropping funnel. With vigorous stirring, add approximately 1 mL of this ether solution to the magnesium turnings. Note the exothermic reaction (to confirm / accelerate the formation of the Grignard reagent, add 1 mg of iodine, and immediate decolorization is observed, confirming the formation of the Grignard reagent), and the diethyl ether begins to reflux. Add the remaining bromide solution dropwise while maintaining the reaction under gentle reflux by cooling the flask in water. After the addition is complete, maintain the reaction mixture at 35 °C for 1 hour, then cool it in an ice bath. Dissolve ethyl formate (0.44 g, 6.0 mmol) in anhydrous diethyl ether (8 mL), then transfer it to the addition funnel and add it dropwise to the reaction mixture with stirring. Observe the exothermic reaction, and the reaction mixture begins to reflux. After the reaction is initiated, rapidly add the remaining ether solution of the formate in a stream, and stir the reaction mixture at room temperature for an additional 1 hour. Quench the reaction by adding 5 mL of acetone dropwise, then add ice water (10 mL). Treat the reaction mixture with aqueous H2SO4 (10% v / v, 50 mL) until the solution becomes homogeneous, and allow it to stand and separate. Extract the aqueous phase with diethyl ether (20 mL * 2). Dry the combined diethyl ether layers over anhydrous magnesium sulfate, concentrate it to obtain the crude product, and purify it by column chromatography to obtain the colorless oil compound S1-2 (2.46 g).

[0328] Step b: Under a nitrogen atmosphere, add N,N'-dicyclohexylcarbodiimide (DCC, 1.00 g, 4.8 mmol) to a round-bottom flask containing S1-2 (1.39 g, 2.6 mmol), Boc-protected N,N-dimethyllysine (S1-3, 0.60 g, 2.2 mmol), and 4-dimethylaminopyridine (DMAP, 67.10 mg, 0.55 mmol) dissolved in dichloromethane (20 mL), and react at room temperature for 16 h. After the reaction is complete, remove the precipitate by filtration, concentrate the filtrate, and purify the resulting residue by column chromatography to obtain compound S1-4 (1.48 g).

[0329] Step c: Removal of the Boc protecting group. In a dry and clean round-bottom flask, a solution of trifluoroacetic acid / dichloromethane (1:2, v / v) was prepared. The dichloromethane solution of S1-4 (1.18 g, 1.5 mmol) was slowly added dropwise under an ice bath condition, and the reaction was carried out at room temperature for 2 hours. After the reaction was completed, the reaction solution was concentrated, diluted with purified water, extracted with dichloromethane, the organic phase was dried over anhydrous magnesium sulfate, filtered, the filtrate was concentrated, and recrystallized to obtain compound S1-5 (0.92 g, 90.1%).

[0330] Step d: Under nitrogen protection, compound S1-5 (0.69 g, 1.0 mmol) was dissolved in acetonitrile (20 mL). Under slow stirring, 6-bromohexyl 2-hexyldecanoate (S1-6, 1.05 g, 2.5 mmol, S1-6 was prepared by the reaction of 2-hexyldecanoic acid and 6-bromohexanol ) and N,N-diisopropylethylamine (DIPEA, 0.18 g, 2.0 mmol) were successively added, and the reaction was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated and then dissolved in dichloromethane, and successively extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The combined organic phases were dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E1-1 (1.11 g). 1 HNMR(400MHz,CDCl3)δ:5.35-5.24(m,8H),4.92-4.80(m,1H),4.02(t,4H),3.05(t,1H),2.76-2.68(m,4H),2.36-2.26(m,14H),2.03-1.97(m,8H),1.69-1.14(m,110H),0.89(t,18H). MS(ESI):m / z=1362.30([M+H] + )。

[0331]

[0332] Example 2: Cationic lipid (E2-1)

[0333]

[0334] The preparation process is as follows:

[0335] Step a: Under a nitrogen atmosphere, DCC (1.81 g, 8.8 mmol) was added to a round-bottom flask containing 8-bromononanoic acid (S2-1, 0.89 g, 4.0 mmol), 9-heptadecanol (S2-2, 1.23 g, 4.8 mmol) and DMAP (0.12 g, 1.0 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 residue obtained was purified by column chromatography to obtain 2-octyl octyl 8-bromooctanoate (S2-3, 1.52 g).

[0336] Step b: Under nitrogen protection, compound S1-5 (0.55 g, 0.8 mmol) was dissolved in acetonitrile (20 mL), and S2-3 (0.92 g, 2.0 mmol) and DIPEA (0.14 g, 1.6 mmol) were successively added under slow stirring, and the reaction was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated and dissolved in dichloromethane, and was successively extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E2-1 (0.95 g). 1 H NMR (400 MHz, CDCl3) δ: 5.42 - 5.25 (m, 8H), 4.93 - 4.81 (m, 3H), 3.08 (t, 1H), 2.83 - 2.68 (m, 4H), 2.49 - 2.30 (m, 12H), 2.26 (t, 4H), 2.07 - 2.00 (m, 8H), 1.50 - 1.19 (m, 122H), 0.88 (t, 18H). MS (ESI): m / z = 1446.39 ([M + H] + )

[0337]

[0338] Example 3: Cationic lipid (E3-1)

[0339]

[0340] The preparation process is as follows:

[0341] Dissolve S1-2 (1.98 g, 3.8 mmol) and N,N-dimethylglutamic acid (S3-1, 0.26 g, 1.5 mmol) separately in anhydrous dichloromethane. After stirring evenly, combine the two-component solutions. To the mixed solution, sequentially add DMAP (18.30 mg, 0.15 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 0.72 g, 3.8 mmol), and DIPEA (0.68 g, 7.5 mmol), and stir the reaction at room temperature for 9 h. After the reaction is completed, dilute the reaction solution with dichloromethane (20 mL), then wash it twice with saturated sodium carbonate solution (10 mL * 2), wash it twice with 10 mL aqueous solution, and wash it once with saturated brine. The organic phase is dried with anhydrous sodium sulfate, and the solvent is concentrated to obtain the crude product. The crude product is separated and purified by column chromatography, collect the target eluate, and concentrate to obtain cationic lipid E3-1 (0.95 g). 1 HNMR(400MHz,CDCl3)δ:5.45-5.26(m,16H),4.92-4.82(m,2H),3.06(t,1H),2.83-2.68(m,8H),2.43(s,6H),2.26(t,2H),2.03-1.97(m,16H),1.71-1.23(m,82H),0.89(t,12H). MS(ESI):m / z=1197.11([M+H] + )。

[0342]

[0343] Example 4: Cationic Lipid (E4-2)

[0344]

[0345] The preparation process is as follows:

[0346] Step a: Under nitrogen protection, dissolve TBS-N-hydroxyethyl-N-methyllysine (S4-1, 0.64 g, 2.0 mmol, S4-1 is N-methyllysine protected by amino Boc reacted with 2-bromoethanol whose hydroxyl group is protected by TBS and obtained after de-Boc) in acetonitrile (30 mL). Under slow stirring, sequentially add S1-6 (2.10 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 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 S4-2 (1.63 g).

[0347] Step b: Under a nitrogen atmosphere, DCC (0.59 g, 2.9 mmol) was added to a round-bottom flask containing S4-2 (1.29 g, 1.3 mmol), S1-2 (0.82 g, 1.6 mmol) and DMAP (39.65 mg, 0.3 mmol) dissolved in dichloromethane (20 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 residue obtained was purified by column chromatography to obtain E4-1 (1.61 g).

[0348] Step c: The above compound E4-1 (1.20 g, 0.8 mmol) was dissolved in THF (20 mL), placed in a nitrogen-protected flask, tetrabutylammonium fluoride solution (TBAF, 20 mL, 1 M) was added, and the reaction was carried out overnight to remove the TBS protection. After the reaction was completed, the mixture was concentrated, extracted, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography to obtain cationic lipid E4-2 (0.98 g, 88.4%). 1 H NMR (400 MHz, CDCl3) δ: 1 HNMR (400 MHz, CDCl3) δ: 5.40 - 5.32 (m, 8H), 4.90 (t, 1H), 4.05 (t, 4H), 3.60 - 3.55 (m, 2H), 3.22 (t, 1H), 2.85 - 2.67 (m, 6H), 2.43 - 2.28 (m, 11H), 2.08 - 2.03 (m, 8H), 1.44 - 1.22 (m, 110H), 0.87 (t, 18H). MS (ESI): m / z = 1414.29 ([M+Na] + )。

[0349]

[0350] Example 5: Cationic lipid (E5-2)

[0351]

[0352] The preparation process is as follows:

[0353] Step a: Under nitrogen protection, compound S4-1 (0.64 g, 2.0 mmol) was dissolved in acetonitrile (30 mL), and S2-3 (2.30 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were successively added under slow stirring, and the reaction was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated and then dissolved in dichloromethane, and extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered and concentrated, and purified by column chromatography to obtain S5-1 (1.77 g).

[0354] Step b: Under a nitrogen atmosphere, DCC (0.59 g, 2.9 mmol) was added to a round-bottom flask containing S5-1 (1.40 g, 1.3 mmol), S1-2 (0.82 g, 1.6 mmol), and DMAP (39.65 mg, 0.3 mmol) dissolved in dichloromethane (30 mL). 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 residue obtained was purified by column chromatography to obtain E5-1 (1.70 g).

[0355] Step c: The above compound E5-1 (1.27 g, 0.8 mmol) was dissolved in THF (20 mL) and placed in a nitrogen-protected flask. TBAF solution (20 mL, 1 M) was added, and the reaction was carried out overnight to remove the TBS protection. After the reaction was completed, the mixture was concentrated, extracted, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography gave the cationic lipid E5-2 (1.11 g, 88.1%). 1 H NMR (400 MHz, CDCl3) δ: 1 H NMR (400 MHz, CDCl3) δ: 5.42 - 5.29 (m, 8H), 4.93 - 4.83 (m, 3H), 3.57 (t, 2H), 3.22 (t, 1H), 2.81 - 2.70 (m, 6H), 2.47 - 2.17 (m, 13H), 2.08 - 1.99 (m, 8H), 1.69 - 1.08 (m, 122H), 0.88 (t, 18H). MS (ESI): m / z = 1498.39 ([M+Na] + )。

[0356]

[0357] Example 6: Cationic lipid (E6-2)

[0358]

[0359] The preparation process is as follows:

[0360] Step a: Dissolve S1-2 (2.64 g, 5.0 mmol) and TBS-N-hydroxyethyl-N-methylglutamic acid (S6-1, 0.64 g, 2.0 mmol) separately in anhydrous dichloromethane. After stirring evenly, combine the two-component solutions. To the mixed solution, sequentially add DMAP (24.40 mg, 0.2 mmol), EDCI (0.96 g, 5.0 mmol), and DIPEA (0.90 g, 10.0 mmol), and stir the reaction at room temperature for 9 h. After the reaction is completed, dilute the reaction solution with dichloromethane (20 mL), then wash it twice with saturated sodium carbonate solution (10 mL * 2), wash it twice with 10 mL aqueous solution, and then wash it once with saturated brine. The organic phase is dried over anhydrous sodium sulfate, and the solvent is concentrated to obtain a crude product. The crude product is separated and purified by column chromatography, collect the target eluate, and concentrate to obtain product E6-1 (1.43 g).

[0361] Step b: Dissolve the above compound E6-1 (1.07 g, 0.8 mmol) in THF (20 mL), place it in a flask under nitrogen protection, add TBAF solution (20 mL, 1 M), react overnight to remove the TBS protection. After the reaction is completed, concentrate, extract, and combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and purify by column chromatography to obtain cationic lipid E6-2 (0.87 g, 88.4%). 1 1H NMR (400 MHz, CDCl3) δ: 5.45 - 5.26 (m, 16H), 4.92 - 4.81 (m, 2H), 3.58 (t, 2H), 3.06 (t, 1H), 2.85 - 2.69 (m, 10H), 2.43 (s, 3H), 2.26 (t, 2H), 2.03 - 1.97 (m, 16H), 1.71 - 1.22 (m, 82H), 0.88 (t, 12H). MS (ESI): m / z = 1227.13 ([M + H] + )。

[0362]

[0363] Example 7: Cationic Lipid (E7-1)

[0364]

[0365] The preparation process is as follows:

[0366] Step a: Dissolve 1,2-epoxytetradecane (S7-1, 0.32 g, 1.5 mmol) and S1-5 (1.03 g, 1.5 mmol) in 30 mL of acetonitrile, then add calcium trifluoromethanesulfonate (Ca(OTf)2, 0.25 g, 0.8 mmol). The reaction mixture is stirred at room temperature. After TLC shows that the reaction is complete, evaporate the acetonitrile, add water (30 mL), and extract with dichloromethane (10 mL * 3). Combine the organic layers, dry over anhydrous Na2SO4, filter, and evaporate. The crude product is separated and purified by column chromatography. Collect the target eluate and concentrate to obtain compound S7-2 (1.27 g, 94.7%).

[0367] Step b: Under nitrogen protection, dissolve the above compound S7-2 (0.90 g, 1.0 mmol) in acetonitrile (20 mL). While stirring slowly, sequentially add S1-6 (0.52 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 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 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 cationic lipid E7-1 (1.02 g). 1 H NMR (400 MHz, CDCl3) δ: 5.35 - 5.24 (m, 8H), 4.92 - 4.80 (m, 1H), 4.02 (t, 2H), 3.55 - 3.53 (m, 1H), 3.05 (t, 1H), 2.76 - 2.68 (m, 4H), 2.40 (s, 6H), 2.36 - 2.26 (m, 7H), 2.03 - 1.97 (m, 8H), 1.71 - 1.22 (m, 102H), 0.88 (t, 15H). MS (ESI): m / z = 1250.20 ([M + H] + )。

[0368]

[0369] Example 8: Cationic Lipid (E8-1)

[0370]

[0371] The preparation process is as follows:[[]]END]]

[0372] Under nitrogen protection, dissolve the above-mentioned compound S7-2 (0.90 g, 1.0 mmol) in acetonitrile (20 mL). While stirring slowly, successively add S2-3 (0.58 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 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 E8-1 (1.06 g). 1 H NMR (400 MHz, CDCl3) δ: 5.36 - 5.24 (m, 8H), 4.92 - 4.82 (m, 2H), 3.55 - 3.52 (m, 1H), 3.06 (t, 1H), 2.76 - 2.68 (m, 4H), 2.40 (s, 6H), 2.36 - 2.26 (m, 8H), 2.03 - 1.98 (m, 8H), 1.71 - 1.22 (m, 108H), 0.88 (t, 15H). MS (ESI): m / z = 1292.25 ([M+H] + )。

[0373]

[0374] Example 9: Cationic Lipid (E9-1)

[0375]

[0376] The preparation process is as follows:

[0377] Step a: Dissolve 1,3-propanediol (S9-1, 9.50 g, 50 mmol) containing one TBS-protected hydroxyl group in 300 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 silica gel column chromatography to obtain S9-2 (6.02 g).

[0378] Step b: Dissolve the above-mentioned compound S9-2 (5.64 g, 30.0 mmol) and 1-octanol (S9-3, 9.75 g, 75.0 mmol) in 200 mL of dichloromethane solution, add p-toluenesulfonic acid monohydrate (TsOH·H2O, 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 the crude product under reduced pressure, and purify by column chromatography to obtain compound S9-4 (2.84 g).

[0379] Step c: Dissolve the above product S9-4 (2.16 g, 5.0 mmol) in THF (20 mL), place it in a flask under nitrogen protection, add TBAF (20 mL, 1 M), and react overnight to remove the TBS protection. After the reaction, concentrate, extract, and combine the organic phases. Dry over anhydrous sodium sulfate, filter, and concentrate. Purify by column chromatography to obtain compound S9-5 (1.40 g, 88.6%).

[0380] Step d: Under a nitrogen atmosphere, add DCC (0.91 g, 4.4 mmol) to a round-bottom flask containing S9-5 (0.76 g, 2.4 mmol), S9-6 (0.39 g, 2.0 mmol), and DMAP (61.00 mg, 0.5 mmol) dissolved in dichloromethane (20 mL). React at room temperature for 16 h. After the reaction, remove the precipitate by filtration, concentrate the filtrate, and purify the resulting residue by silica gel column chromatography to obtain S9-7 (0.81 g).

[0381] Step e: Under nitrogen protection, dissolve compound S1-5 (0.34 g, 0.5 mmol) in acetonitrile (20 mL). Slowly stir and sequentially add S9-7 (0.62 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol). Stir and react at room temperature for about 20 h. After the reaction, 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 over anhydrous magnesium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E9-1 (0.60 g). 1 H NMR (400 MHz, CDCl3) δ: 5.45 - 5.29 (m, 8H), 4.93 - 4.83 (m, 1H), 4.64 (t, 2H), 4.20 (t, 4H), 3.52 - 3.36 (m, 8H), 3.06 (t, 1H), 2.83 - 2.68 (m, 4H), 2.49 - 2.30 (m, 12H), 2.26 (t, 4H), 2.07 - 1.98 (m, 12H), 1.71 - 1.21 (m, 106H), 0.88 (t, 18H). MS (ESI): m / z = 1510.36 ([M + H] + )。

[0382]

[0383] Example 10.1: Cationic Lipid (E10-1)

[0384]

[0385] The preparation process is as follows:

[0386] Step a: Under a nitrogen atmosphere, glycerol (S10-1, 2.06 g, 10.0 mmol) containing one TBS-protected hydroxyl group, potassium carbonate (K2CO3, 4.14 g, 30.0 mmol), and bromohexane (S10-2, 1.80 g, 11.0 mmol) were dissolved in 80 mL of DMF. The mixture was stirred at 110 °C for 16 hours. After confirming the completion of the reaction by thin-layer chromatography, the reaction solution was poured into water (50 mL) for precipitation, filtered, and further separated and purified by column chromatography to obtain compound S10-3 (3.35 g, 89.3%).

[0387] Step b: The above product S10-3 (1.88 g, 5.0 mmol) was dissolved in THF (20 mL), placed in a nitrogen-protected flask, and TBAF (20 mL, 1 M) was added. The reaction was carried out overnight to remove the TBS protection. After the reaction, the solution was concentrated, extracted, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography gave compound S10-4 (1.14 g, 87.9%).

[0388] Step d: Under a nitrogen atmosphere, DCC (0.91 g, 4.4 mmol) was added to a round-bottom flask containing S10-4 (0.62 g, 2.4 mmol), 8-bromooctanoic acid (S10-5, 0.45 g, 2.0 mmol), and DMAP (61.00 mg, 0.5 mmol) dissolved in dichloromethane (20 mL). The reaction was carried out at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The residue obtained was purified by silica gel column chromatography to obtain S10-6 (0.76 g).

[0389] Step c: Under nitrogen protection, compound S1-5 (0.34 g, 0.5 mmol) was dissolved in acetonitrile (20 mL). S10-6 (0.58 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) were successively added with slow stirring, and the reaction was stirred at room temperature for about 20 h. After the reaction, the reaction solution was concentrated and then dissolved in dichloromethane. It was extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E10-1 (0.59 g). 11H NMR (400 MHz, CDCl3) δ: 5.41 - 5.25 (m, 8H), 5.14 - 5.08 (m, 2H), 4.93 - 4.85 (m, 1H), 3.59 - 3.49 (m, 8H), 3.48 - 3.36 (m, 8H), 3.08 (t, 1H), 2.84 - 2.66 (m, 4H), 2.46 - 2.26 (m, 16H), 2.06 - 1.99 (m, 8H), 1.63 - 1.18 (m, 98H), 0.87 (t, 18H). MS (ESI): m / z = 1454.30 ([M + H] + )。

[0390]

[0391] Example 10.2: Cationic Lipid (E10 - 2)

[0392]

[0393] Replace the raw material S10 - 2 in Example 10.1 with raw material 1 - bromoheptane ( 1.97 g, 11.0 mmol), and prepare according to the same reaction steps to obtain cationic lipid E10 - 2 (0.62 g). 1 1H NMR (400 MHz, CDCl3) δ: 5.43 - 5.24 (m, 8H), 5.14 - 5.08 (m, 2H), 4.92 - 4.85 (m, 1H), 3.59 - 3.48 (m, 8H), 3.48 - 3.36 (m, 8H), 3.06 (t, 1H), 2.85 - 2.66 (m, 4H), 2.46 - 2.26 (m, 16H), 2.08 - 1.98 (m, 8H), 1.63 - 1.18 (m, 106H), 0.89 (t, 18H). MS (ESI): m / z = 1510.37 ([M + H] + )。

[0394] Example 11: Cationic Lipid (E11 - 2)

[0395]

[0396] The preparation process is as follows:

[0397] Step a: Under nitrogen protection, dissolve compound S4-1 (0.96 g, 3.0 mmol) in acetonitrile (50 mL). While stirring slowly, add S9-7 (3.71 g, 7.5 mmol) and DIPEA (0.54 g, 6.0 mmol) successively, 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 over anhydrous magnesium sulfate, filter, concentrate, and purify by column chromatography to obtain compound S11-1 (2.74 g).

[0398] Step b: Under a nitrogen atmosphere, add DCC (0.91 g, 4.4 mmol) to a round-bottom flask containing S1-2 (1.27 g, 2.4 mmol), S11-1 (2.18 g, 2.0 mmol) and DMAP (61.00 mg, 0.5 mmol) dissolved in dichloromethane (50 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 residue by silica gel column chromatography to obtain E11-1 (2.67 g).

[0399] Step c: Dissolve the above product E11-1 (1.66 g, 1.0 mmol) in THF (20 mL), place it in a flask protected by nitrogen, add TBAF (20 mL, 1 M), react overnight to remove the TBS protection. After the reaction is completed, concentrate, extract, and combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and purify by column chromatography to obtain cationic lipid E11-2 (1.35 g, 87.6%). 1 H NMR (400 MHz, CDCl3) δ: 5.44 - 5.29 (m, 8H), 4.91 - 4.83 (m, 1H), 4.63 (t, 2H), 4.20 (t, 4H), 3.60 - 3.55 (m, 2H), 3.52 - 3.36 (m, 8H), 3.06 (t, 1H), 2.83 - 2.68 (m, 6H), 2.48 - 2.30 (m, 9H), 2.25 (t, 4H), 2.07 - 1.99 (m, 12H), 1.71 - 1.21 (m, 106H), 0.88 (t, 18H). MS (ESI): m / z = 1540.37 ([M + H] + )。

[0400]

[0401] Example 12: Cationic Lipid (E12-2)

[0402]

[0403] The preparation process is as follows:

[0404] Step a: Under nitrogen protection, dissolve compound S4-1 (0.96 g, 3.0 mmol) in acetonitrile (50 mL). With slow stirring, successively add S10-6 (3.50 g, 7.5 mmol) and DIPEA (0.54 g, 6.0 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 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 compound S12-1 (2.61 g).

[0405] Step b: Under a nitrogen atmosphere, add DCC (0.91 g, 4.4 mmol) to a round-bottom flask containing S1-2 (1.27 g, 2.4 mmol), S12-1 (2.17 g, 2.0 mmol) and DMAP (61.00 mg, 0.5 mmol) dissolved in dichloromethane (30 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 residue by silica gel column chromatography to obtain E12-1 (2.58 g).

[0406] Step c: Dissolve the above product E12-1 (1.60 g, 1.0 mmol) in THF (20 mL), place it in a flask under nitrogen protection, add TBAF (20 mL, 1 M), and react overnight to remove the TBS protection. After the reaction is completed, concentrate, extract, and combine the organic phases, dry with anhydrous sodium sulfate, filter and concentrate, and purify by column chromatography to obtain cationic lipid E12-2 (1.29 g, 86.9%). 1 H NMR (400 MHz, CDCl3) δ: 5.43 - 5.25 (m, 8H), 5.14 - 5.08 (m, 2H), 4.93 - 4.85 (m, 1H), 3.60 - 3.49 (m, 10H), 3.48 - 3.36 (m, 8H), 3.08 (t, 1H), 2.84 - 2.67 (m, 6H), 2.45 - 2.26 (m, 13H), 2.06 - 1.98 (m, 8H), 1.68 - 1.18 (m, 98H), 0.87 (t, 18H). MS (ESI): m / z = 1484.31 ([M + H] + )。

[0407]

[0408] Example 13: Cationic Lipid (E13-2)

[0409]

[0410] The preparation process is as follows:

[0411] Step a: 1,2-Di-O-tetradecyl-5H-glycerol ester (S13-1, 2.43 g, 5.0 mmol, S13-1 is glycerol with the hydroxyl group protected by TBS reacted with tetradecyl bromide and then the TBS protecting group was removed. For the specific operation method, refer to Step a of Example 10) and N,N'-succinimidyl carbonate (DSC, S13-2, 1.92 g, 7.5 mmol) were placed in dichloromethane (50 mL), and stirred in an ice bath. Triethylamine (TEA, 2.1 mL, 15.0 mmol) was added to the stirred solution, and then the reaction mixture was stirred overnight at room temperature. The progress of the reaction was detected by TLC. After the reaction was completed, the reaction mixture was diluted with dichloromethane, and the organic layer was washed successively with water (50 mL) and aqueous sodium bicarbonate solution (50 mL), concentrated, further purified by column chromatography, concentrated, and dried under vacuum to obtain compound S13-3 (2.72 g).

[0412] Step b: S4-1 (1.15 g, 3.6 mmol) was dissolved in dichloromethane (30 mL), and S13-3 (1.88 g, 3.0 mmol) and TEA (0.75 mL, 5.4 mmol) were added successively, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated to obtain a crude product. It was purified by column chromatography, concentrated, and dried with an oil pump to obtain compound S13-4 (1.96 g).

[0413] Step c: Under a nitrogen atmosphere, DCC (0.91 g, 4.4 mmol) was added to a round-bottom flask containing S1-2 (1.27 g, 2.4 mmol), S13-4 (1.66 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 residue obtained was purified by silica gel column chromatography to obtain E13-1 (2.17 g).

[0414] Step d: The above product E13-1 (1.34 g, 1.0 mmol) was dissolved in THF (20 mL), placed in a nitrogen-protected flask, and TBAF (20 mL, 1 M) was added, and the reaction was carried out overnight to remove the TBS protection. After the reaction was completed, it was concentrated, extracted, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography to obtain cationic lipid E13-2 (1.05 g, 85.7%). 11H NMR (400 MHz, CDCl3) δ: 5.35 - 5.24 (m, 8H), 4.92 - 4.80 (m, 1H), 4.13 - 4.01 (m, 2H), 3.59 - 3.33 (m, 9H), 3.17 - 3.00 (m, 3H), 2.76 - 2.68 (m, 6H), 2.43 - 2.28 (m, 3H), 2.03 - 1.97 (m, 8H), 1.71 - 1.21 (m, 94H), 0.88 (t, 12H). MS (ESI): m / z = 1226.13 ([M+H] + ).

[0415]

[0416] Example 14: Cationic Lipid (E14-2)

[0417]

[0418] The preparation process is as follows:

[0419] Step a: Under nitrogen protection, TBS-N-hydroxybutyl-N-methyl lysine (S14-1, 1.04 g, 3.0 mmol, S14-1 is N-methyl lysine protected by amino Boc reacted with 4-bromobutanol with a hydroxyl group protected by TBS and obtained after de-Boc) was dissolved in acetonitrile (50 mL). Under slow stirring, 6-bromohexyl 2-hexyldecanoate (S14-2, 3.14 g, 7.5 mmol, S14-2 is prepared by reacting 2-hexyldecanoic acid and 6-bromo-1-hexanol) and DIPEA (0.54 g, 6.0 mmol) were added and stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, and extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain S14-3 (2.51 g).

[0420] Step b: Under a nitrogen atmosphere, DCC (0.91 g, 4.4 mmol) was added to a round-bottom flask containing compound S14-3 (2.05 g, 2.0 mmol), 10-undecen-1-ol (S14-4, 0.41 g, 2.4 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 residue obtained was purified by column chromatography to obtain compound E14-1 (1.97 g).

[0421] Step c: Dissolve the above compound E14-1 (1.76 g, 1.5 mmol) in THF (20 mL), place it in a flask under nitrogen protection, add TBAF solution (20 mL, 1 M), react overnight to remove the TBS protection. After the reaction, concentrate, extract, combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate, and purify by column chromatography to obtain cationic lipid E14-2 (1.39 g, 87.3%). 1 H NMR (400 MHz, CDCl3) δ: 5.86 - 5.75 (m, 1H), 5.05 - 4.81 (m, 2H), 4.05 (t, 6H), 3.60 - 3.55 (m, 2H), 3.18 - 2.99 (m, 3H), 2.76 - 2.68 (m, 2H), 2.43 - 2.26 (m, 11H), 1.74 - 1.22 (m, 88H), 0.87 (t, 12H). MS (ESI): m / z = 1061.97 ([M + H] + )。

[0422]

[0423] Example 15: Cationic Lipid (E15-1)

[0424]

[0425] The preparation process is as follows:

[0426] Step a: Under nitrogen protection, dissolve 1-methylhistidine (S15-1, 0.51 g, 3.0 mmol) in acetonitrile (50 mL), and successively add S14-2 (3.14 g, 7.5 mmol) and DIPEA (0.54 g, 6.0 mmol) with slow stirring, and stir at room temperature for about 20 h. After the reaction, concentrate the reaction solution, dissolve it in dichloromethane, extract 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 S15-2 (2.08 g).

[0427] Step b: Under a nitrogen atmosphere, add DCC (0.91 g, 4.4 mmol) to a round-bottom flask containing compound S15-2 (1.69 g, 2.0 mmol), S15-3 (0.67 g, 2.4 mmol) and DMAP (61.00 mg, 0.5 mmol) dissolved in dichloromethane (30 mL), and react at room temperature for 16 h. After the reaction, remove the precipitate by filtration, concentrate the filtrate, and purify the obtained residue by column chromatography to obtain cationic lipid E15-1 (1.79 g). 11H NMR (400 MHz, CDCl3) δ: 7.60 (s, 1H), 6.89 (s, 1H), 5.86 - 5.75 (m, 2H), 5.05 - 4.81 (m, 5H), 4.64 (t, 1H), 4.02 (t, 4H), 3.71 (s, 3H), 3.22 - 3.09 (m, 2H), 2.43 - 2.28 (m, 6H), 2.11 - 1.93 (m, 4H), 1.70 - 1.25 (m, 88H), 0.89 (t, 12H). MS (ESI): m / z = 1108.99 ([M+H] + )。

[0428]

[0429] Example 16: Preparation of LNP - mRNA Pharmaceutical Composition and Testing of Its Physicochemical Properties

[0430] Example 16.1: Preparation of LNP - mRNA Pharmaceutical Composition

[0431] In this example, multiple groups of LNP - mRNA pharmaceutical compositions containing Fluc - mRNA were prepared for comparison. The phospholipid in each group was DSPC, the sterol lipid was cholesterol, the polyethylene glycol - lipid in experimental groups L - 1 to L - 15 was PEG2k - DMG (abbreviated as DMG), and the polyethylene glycol - lipid in experimental group L - 16 was PL - 3( prepared by referring to the method disclosed in CN115515924A); among them, the cationic lipid of control group L - 0 was an amino acid cationic lipid of the prior art (abbreviated as CL - 1, prepared by referring to the method disclosed in CN104168887A, and the structure is ). The cationic lipids of the experimental group series (L - 1 to L - 16) were the amino acid cationic lipids containing unsaturated bonds prepared in the examples of this application, specifically as shown in Table 1.

[0432] The preparation method of the LNP - mRNA pharmaceutical composition is as follows:

[0433] 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;

[0434] Step b: Add Fluc - mRNA to 10 - 50 mM citrate buffer (pH = 4) to obtain an aqueous - phase solution;

[0435] 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.

[0436] Example 16.2: Physicochemical property test of LNP-mRNA pharmaceutical composition

[0437] Determination of encapsulation efficiency: Use the Quant-it Ribogreen RNA quantification kit to determine the encapsulation efficiency of the LNP-mRNA composition. The results show that the lipid compositions (L-1 to L-15) of the present invention have a high nucleic acid encapsulation efficiency for nucleic acid drugs, all in the range of 88%-95%, and most of the encapsulation efficiencies are in the range of 90%-95%. The results are shown in Table 1 below. The results indicate that the lipid compositions prepared from the amino acid cationic lipids in each experimental group can well encapsulate mRNA, showing an encapsulation efficiency superior to that of existing amino acid cationic lipids, and there are also differences in the encapsulation efficiencies of different amino acid cationic lipids.

[0438] Particle size determination: In this example, the particle size of LNP-mRNA was determined by dynamic light scattering (DLS). The results are shown in Table 1 below. The measured LNP-mRNA has a high size uniformity, and its PDI is less than 0.3. The particle size of LNP-mRNA prepared from the lipid composition of the present application is in the range of 90-110 nm.

[0439] Table 1: Summary table of the formulations of each lipid composition and the particle size and encapsulation efficiency of the LNP-mRNA prepared therefrom

[0440]

[0441]

[0442] Example 17: Biological activity test of LNP-mRNA pharmaceutical composition formulation

[0443] (1) Study on cytotoxicity (biocompatibility)

[0444] The MTT staining method was used to test the cytotoxicity of the LNP-mRNA pharmaceutical composition formulation of the present invention. The LNP-mRNA pharmaceutical composition formulation was dissolved in the culture medium to prepare the required concentration. If necessary, an appropriate amount of cosolvent could be added. Hela cells were used as the cell model, and the seeding density was 1×10 4Cells / well, and 100 μL of the cell suspension was inoculated into a 96-well plate per well. After inoculation, it was incubated in a cell culture incubator at 37 °C and 4% CO2 for 24 h. Then, the old culture medium was aspirated and discarded, and the drug was administered at a dose of 0.1 - 0.3 μg mRNA dissolved in 100 μL of culture medium per well (the LNP-mRNA drug composition was prepared in Example 16). The blank control group was added with 100 μL of fresh culture medium. There were 6 replicates for each concentration (0.1 μg, 0.15 μg, 0.20 μg, 0.25 μg, and 0.3 μg) in each group. After the LNP-mRNA drug composition preparation was co-incubated with Hela cells for 24 h, 20 μL of PBS buffer containing 5 mg / mL of MTT was added to each well. After MTT was incubated with cancer cells for 4 h, the mixed solution of the culture medium and the MTT buffer was aspirated and discarded, and 150 μL of DMSO was added to each well to dissolve the purple crystal formazan of living cells. After sufficient oscillation, the absorbance was measured with an enzyme-linked immunosorbent assay (ELISA) reader. Calculations were made based on the measured absorbance values. The results showed that compared with the blank control group, the cell survival rate of the LNP-mRNA drug composition preparation prepared in the present invention was greater than 95%, indicating that the LNP-mRNA drug composition preparation of the present invention had good biocompatibility.

[0445] (2) Serum stability evaluation

[0446] The LNP-mRNA drug composition was added to the culture medium containing 10% fetal bovine serum (FBS), stirred at 37 °C, and samples were taken at regular intervals to measure the particle size change of LNP-mRNA. The serum stability of the LNP-mRNA drug composition preparation was analyzed by testing its particle size change. The experimental results showed that within 7 days, the particle size changes of both the control group and the experimental group were less than 10%. In particular, the particle size changes of the experimental groups L-1, L-2, L-4, L-5, L-10, and L-15 were less than 5%, indicating that the LNP-mRNA drug composition preparation prepared with the cationic lipid of the present invention had good serum stability.

[0447] (3) Study on cell transfection activity

[0448] To investigate the mRNA transfection efficiency of each group of LNP-mRNA drug compositions prepared in Example 16 of the present invention at the cellular level, Luciferase bioluminescence was used for testing. The LNP-mRNA drug composition was dissolved in the culture medium to prepare the required dose. Hela cells were used as the cell model, and the cell suspension was inoculated into a 96-well plate with a black edge and transparent bottom at a density of 6000 cells / well at a volume of 100 μL / well. After inoculation, the cells were 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. After 24 h of transfection, the old culture medium was removed and replaced with a new culture medium containing the substrate D-luciferin sodium (1.5 mg / mL), and after incubation 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 2. Among them, the relative fluorescence intensity is the ratio of the fluorescence intensity value of each group to the fluorescence intensity of the blank control group. The results show that the LNP-mRNA drug compositions prepared by the present invention all have excellent in vitro transfection effects, that is, the LNP in the experimental group are all effective nucleic acid delivery vectors and are superior to the L-0 group prepared by the amino acid cationic lipid of the prior art. Among them, the relative fluorescence values of the experimental groups L-1, L-2, L-9, L-10, and L-15 are relatively high, probably because they contain both an ionizable tertiary amine structure, multiple degradable ester bonds, and unsaturated aliphatic hydrocarbon chains. 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 the mRNA is released into the cytoplasm to exert its efficacy. The unsaturated hydrocarbon chains can increase the membrane fluidity and improve cell uptake. Compared with other experimental groups, although the encapsulation efficiency of the experimental groups L-2 and L-6 (only containing unsaturated aliphatic hydrocarbon chains) is not very low, their relative fluorescence intensity values are relatively low, indicating that the more unsaturated aliphatic chains are not necessarily better, and the transfection efficiency of the amino acid cationic lipid containing both unsaturated aliphatic hydrocarbon chains and degradable saturated aliphatic hydrocarbon chains is higher.

[0449] Table 2: Results of cell transfection test

[0450] Serial number Relative fluorescence value Serial number Relative fluorescence value Serial number Relative fluorescence value Blank 1 L-6 5.8 L-13 7.3 L-0 3.3 L-7 7.5 L-14 7.7 L-1 9.5 L-8 8.6 L-15 9.2 L-2 10.2 L-9 9.6 L-16 9.4 L-3 5.3 L-10 9.3 L-4 8.2 L-11 7.4 L-5 7.9 L-12 8.1

[0451] (4) Study on animal transfection activity

[0452] The LNP-mRNA pharmaceutical compositions (L-2, L-9, L-16) 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 from the amino cationic lipids of the present invention also had excellent transfection efficiency in vivo, and were mainly distributed in the liver and spleen in vivo. The small animal fluorescence imaging and organ distribution of L-2 (heart, liver, spleen, lung, kidney from left to right) are shown in Figure 1 .

[0453] The above are only the embodiments of the present invention, and 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.

[0454] 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 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 include any changes, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application and are made by using conventional techniques known in the art.

Claims

1. An amino acid cationic lipid, characterized in that, The structure is as shown in formula (1-3): Each occurrence of B2 is independently any one of pentamethylene, hexamethylene, heptamethylene, and octamethylene; Each occurrence of L2 is independently -OC(=O)- or -C(=O)O-; R1 is a linear C with one, two or more double bonds 10-40 aliphatic hydrocarbon group or branched C 10-40 aliphatic hydrocarbon group, and each occurrence of R2 is independently C 1-25 linear alkyl group, wherein, t is an integer from 0 to 12, R e , R f are each independently any one of pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, octadec-6,9-dienyl, t1 and t2 are each independently integers from 0 to 5, and t3 and t4 are 1; the linear C 10-40 aliphatic hydrocarbon group or branched C 10-40 aliphatic hydrocarbon group is selected from any one of the following structures: -L3-R3, each independently, when it appears, is any one of ; or a salt, tautomer, or stereoisomer thereof.

2. The amino acid cationic lipid according to claim 1, wherein The R1 is selected from any one of the following structures:

3. The amino acid cationic lipid according to claim 1, wherein The R1 containing one or two double bonds is selected from any one of the following structures:

4. The amino acid cationic lipid according to claim 1, wherein The B2 is hexamethylene or heptamethylene.

5. The amino acid cationic lipid according to claim 4, wherein The B2 is optionally substituted with 0-5 hydroxyl groups.

6. The amino acid cationic lipid according to claim 1, wherein The said C 1-25 The linear alkyl group is any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl and heptadecyl.

7. The amino acid cationic lipid according to claim 1, wherein The said Selected from any one of the following structures:

8. The amino acid cationic lipid according to claim 1, wherein The said Selected from any one of the following structures:

9. The amino acid cationic lipid according to claim 1, wherein Each occurrence of -B2-L2-R2 is independently selected from any one of the following structures:

10. The amino acid cationic lipid according to any one of claims 1-9, characterized in that, The structure of the amino acid cationic lipid is selected from any one of the following structures:

11. An amino acid cationic lipid, characterized in that, The structure is as follows:

12. A lipid composition, characterized in that, Containing the amino acid cationic lipid described in any one of claims 1-11.

13. The lipid composition according to claim 12, characterized in that, It also contains one or more of phospholipids, steroid lipids, and polyethylene glycolated lipids, selected from any one of the following situations: Situation (1): It also contains phospholipids; Situation (2): It also contains steroid lipids; Situation (3): It also contains polyethylene glycolated lipids; Situation (4): It also contains phospholipids and steroid lipids; Situation (5): It also contains phospholipids and polyethylene glycolated lipids; Situation (6): It also contains steroid lipids and polyethylene glycolated lipids; Situation (7): It also contains phospholipids, steroid lipids, and polyethylene glycolated lipids.

14. The lipid composition according to claim 12, characterized in that, It also simultaneously contains three lipids: phospholipids, steroid lipids, and polyethylene glycolated lipids.

15. The lipid composition according to any one of claims 13 - 14, 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-doundecanoyl-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 its compositions; Or the structure of the polyethylene glycolated lipid is selected from any one of the following structural formulas and its compositions: Wherein, n1 is an integer from 25 to 300.

16. The lipid composition according to any one of claims 13-14, characterized in that, Containing 20-80% of amino acid 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.

17. The lipid composition according to claim 16, wherein The molar percentage of the amino acid cationic lipid in the total lipid in the solution containing the solvent is 30-65%.

18. The lipid composition according to claim 16, wherein, The molar percentage of the amino acid 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%.

19. The lipid composition according to claim 16, wherein The molar percentage of the phospholipid in the total lipid in the solution containing the solvent is 7.5-13%.

20. The lipid composition according to claim 16, 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%.

21. The lipid composition according to claim 16, characterized in that, The molar percentage of the steroid lipid in the total lipid in the solution containing the solvent is 35-50%.

22. The lipid composition according to claim 16, 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%.

23. The lipid composition according to claim 16, wherein, The molar percentage of the polyethylene glycolated lipid in the total lipid in the solution containing the solvent is 0.5-5%.

24. The lipid composition according to claim 16, wherein The molar percentage of the polyethylene glycolated lipid in the total lipid in the solution containing the solvent is 1-3%.

25. The lipid composition according to claim 16, wherein The molar percentage of the polyethylene glycolated lipid in the total lipid in the solution containing the solvent is any one of about 1.5%, 1.6%, 1.7%, 1.8%, 1.9%.

26. A lipid-drug composition, characterized in that, Containing the lipid composition according to any one of claims 12-25 and a drug, 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.

27. The lipid-drug composition according to claim 26, 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.

28. The lipid-drug composition according to claim 26, wherein The nucleic acid drug is any one of DNA, mRNA, miRNA, and siRNA.

29. The lipid-drug composition according to claim 26, wherein The pharmaceutical composition is used as a medicine and is selected from any one of the following medicines: an anti-tumor agent, an antiviral agent, an anti-fungal agent, and a vaccine.

30. An LNP-drug composition preparation, characterized in that, Containing the lipid-drug composition according to claim 29 and a pharmaceutically acceptable diluent or excipient.

31. The LNP-drug composition preparation according to claim 30, characterized in that, The diluent or excipient is any one of deionized water, ultrapure water, phosphate buffer solution, and physiological saline.

32. The LNP-drug composition preparation according to claim 30, characterized in that, The diluent or excipient is phosphate buffer solution or physiological saline.

33. A LNP-drug composition preparation according to claim 30, characterized in that, The diluent or excipient is physiological saline.

34. A liposome or lipid nanoparticle, characterized in that, Containing the lipid composition according to any one of claims 23-25.

35. The liposome or lipid nanoparticle according to claim 34, characterized in that, The lipid nanoparticles are LNP-drug compositions, LPP-drug compositions, or PNP-drug compositions.

36. The liposome or lipid nanoparticle according to claim 34, characterized in that, The lipid nanoparticles are LNP-drug compositions.

37. The liposome or lipid nanoparticle according to claim 34, characterized in that, The lipid nanoparticles are LNP-nucleic acid drug compositions.

38. The liposome or lipid nanoparticle according to claim 34, characterized in that, The lipid nanoparticles are LNP-mRNA drug compositions.

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