New cationic lipid with nitrogen-containing heterocyclic head group and use thereof

Abstract

The present application belongs to the field of drug delivery. Provided is a new cationic lipid with a nitrogen-containing heterocyclic head group, which has a structure as shown in general formula (1), wherein the definition of each symbol is consistent with that described in the text. The cationic lipid of the present application contains a nitrogen-containing heterocyclic head group, a linker arm, a degradable linkage group, and multiple hydrophobic hydrocarbon tail chains, wherein the multiple hydrophobic hydrocarbon tail chains increase the cross-section area of the lipid tail region, thereby facilitating the formation of a cone-shaped structure. The structure can effectively promote the fusion and disruption of LNPs with an endosome membrane, thereby promoting the escape of drugs such as nucleic acids from an endosome into the cytoplasm to exert the functions. The degradable linkage group enables an LNP-pharmaceutical composition prepared therefrom to be degraded in the endosome in a timely manner, thereby promoting the endosomal escape of LNPs. The cationic lipid of the present application can also have an amino acid or a derivative thereof as a linker arm, wherein the raw materials are simple and readily obtainable. An LNP-mRNA composition prepared therefrom has the advantages of low toxicity, high biocompatibility, a high cell transfection efficiency, etc.

Description

A novel cationic lipid containing a nitrogen-containing heterocyclic head group and its applications Technical Field

[0001] This application belongs to the field of drug delivery, specifically relating to a cationic lipid containing a nitrogen heterocyclic head group as a pharmaceutical carrier, as well as lipid compositions, lipid pharmaceutical compositions, their formulations and applications containing the cationic lipid. Background Technology

[0002] Lipid nanoparticles (LNPs) are a four-component spherical vesicle carrier system composed of ionizable cationic lipids, helper lipids, cholesterol, and PEGylated lipids. The core component, the ionizable cationic lipid, protonates and becomes positively charged under acidic conditions, binding to nucleic acids through electrostatic interactions and promoting endosome escape. At physiological pH, it is electrically neutral, a characteristic that significantly reduces the cytotoxicity and non-specific serum protein adsorption caused by the continuous positive charge of traditional cationic liposomes. After entering the cell via endocytosis, LNPs are encapsulated within acidic endosomes. At this point, the core ionizable cationic lipid, protonated and positively charged, escapes by disrupting the endosome membrane, releasing drugs (such as nucleic acids) into the cytoplasm. During this process, the helper lipids and cholesterol jointly maintain the LNP structure and regulate membrane fluidity, while the PEGylated lipids reduce serum protein adsorption and delay clearance through steric hindrance, thus prolonging in vivo circulation time. LNPs have advantages such as ease of preparation, modular design, good biocompatibility and high payload capacity, and can efficiently deliver a variety of drugs (including small molecules, peptides and nucleic acids) intracellularly. Their safety and efficacy have been verified by the success of COVID-19 mRNA vaccines.

[0003] Although the prior art WO2016210190A1 discloses cationic lipids containing nitrogen-containing heterocyclic head groups, its structure contains only two linear hydrophobic tail chains. This structure is not conducive to the formation of a cone-shaped structure that facilitates endosome escape, resulting in poor endosome escape efficiency and ultimately reducing transfection efficiency. Therefore, there is a need in the art to develop novel cationic lipids containing nitrogen-containing heterocyclic head groups to simultaneously achieve efficient transfection and safe, low-toxicity transfection. Summary of the Invention

[0004] This application provides a novel cationic lipid containing a nitrogen-containing heterocyclic head group (the cationic lipid shown in formula (1)) and its preparation method, a lipid composition containing the cationic lipid, a lipid drug composition containing the lipid composition and its formulation, a liposome or lipid nanoparticle containing the lipid composition and its formulation, and an LNP-nucleic acid drug composition containing, in particular, the lipid composition and its formulation, which has the advantages of high delivery efficiency, safety and low toxicity, and high biocompatibility, and can improve the therapeutic and / or preventive effects of drugs.

[0005] The above-mentioned objectives of this application are achieved through the following technical solutions:

[0006] One embodiment of this application provides a cationic lipid:

[0007] A cationic lipid, characterized in that it has the structure shown in general formula (1):

[0008] Or its salts, tautomers, stereoisomers, deuterated derivatives or solvates;

[0009] Where, N r It is a nitrogen-containing heterocyclic group;

[0010] t is an integer from 1 to 6;

[0011] L a L b Each is an independent linker or a binary linker L. d The L d Selected from any one of *-C(=O)O-, *-C(=O)NH-, and -C(=O)-; L c For -Z1-(CH2) tc -Z2-*, where Z1 is a connector or -(CH2) te -L e -, tc and te are each independent integers from 1 to 6, L e Each occurrence is independently selected from -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -CH(OH)-, -O-, -NH-, -O(CH2). s O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH-, -NHC(=O)S-, -C(=S)-, -OC(=S-, -C(=S)O-, -OC(=S)O-, -NHC(=S-, -C(=S)NH-, -NHC(=S)NH-, -OC(=S)NH- and -NHC(=S)O-, where s is 1, 2 or 3; Z2 is selected from any one of -OC(=O)-*, -NHC(=O)-*, -C(=O)NH-*, -OC(=O)NH-*; the aforementioned L d L c The asterisk end in the Z2 structure points to the central carbon atom in formula (1);

[0012] Each X is independently -N<, -CR a < or connect key; when X is -N< or -CRa When X is a connector, both a and b are 2; when X is a connector, both a and b are 1; each R a Independently H or C 1-6 alkyl;

[0013] R m For H or C 1-12 Alkyl; each R g Independently defined as -B1-L1-R1; where each B1 is independently defined as C. 1-12 alkylene or hydroxyl-substituted C 1-12 Alkylene; each L1 is independently selected from the following: linking bond, -CH(OH)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, -NH-, -O(CR h R h ) s O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, -NR h C(=O)-、-C(=O)NR h -、-NR h C(=O)NR h -、-OC(=O)NR h -、-NR h C(=O)O-、-SC(=O)NR h -、-NR h C(=O)S-, -C(=S)-, -OC(=S)-, -C(=S)O-, -OC(=S)O-, -NR h C(=S)-、-C(=S)NR h -、-NR h C(=S)NR h -、-OC(=S)NR h -and-NR h Any one of C(=S)O-, where each R h Independently a hydrogen atom or a carbon atom 1-12 Alkyl group, s is 2, 3 or 4; each R1 is independently C 1-30 Straight-chain hydrocarbon groups or C 1-30 Branched hydrocarbon group.

[0014] This application also provides a lipid composition, wherein the embodiment is as follows:

[0015] A lipid composition comprising a cationic lipid having the structure shown in formula (1).

[0016] This application also provides a lipid pharmaceutical composition, the embodiments of which are as follows:

[0017] A lipid pharmaceutical composition comprising a lipid composition and a drug, wherein the lipid composition comprises a cationic lipid having the structure shown in formula (1), and the drug is selected from any one of nucleic acid drugs, gene vaccines, antitumor drugs, small molecule drugs, polypeptide drugs or protein drugs.

[0018] This application also provides a lipid pharmaceutical composition formulation, the implementation of which is as follows:

[0019] A lipid pharmaceutical composition formulation comprising the aforementioned lipid pharmaceutical composition and a pharmaceutically acceptable diluent or excipient.

[0020] This application also provides a liposome or lipid nanoparticle, which is implemented as follows:

[0021] A liposome or lipid nanoparticle containing a lipid composition, wherein the lipid composition contains a cationic lipid with the structure shown in formula (1).

[0022] Compared with the prior art, this application has the following beneficial effects:

[0023] The novel nitrogen-containing heterocyclic head group cationic lipid of this application has more hydrophobic hydrocarbon tail chains, increasing the cross-section of the lipid tail region and making it easier to form a cone-shaped structure. This structure can effectively promote the fusion and disruption of LNPs with endosomal membranes, thereby facilitating the escape of nucleic acid and other drugs from the endosomal into the cytoplasm to exert their effects, thus enhancing delivery efficiency.

[0024] The novel cationic lipid containing a nitrogen-containing heterocyclic head group of this application contains multiple tertiary amine groups, enabling efficient nucleic acid delivery at lower dosages. This not only reduces raw material costs but also means that excellent drug delivery performance can still be achieved in formulations with reduced cationic lipid dosages, laying the foundation for reducing potential formulation toxicity.

[0025] The novel cationic lipids with nitrogen-containing heterocyclic head groups of this application use nitrogen-containing heterocyclic head groups and natural amino acids or their derivatives (such as lysine, glutamic acid, aspartic acid, etc.) as linking arms. This structural design significantly improves the biocompatibility of cationic lipids by utilizing endogenous substances in organisms. On the other hand, the raw materials such as amino acids and their derivatives are simple and readily available, and can be obtained naturally or synthesized simply, which has the advantages of being simple, safe and low in production cost.

[0026] The novel cationic lipids containing nitrogen-containing heterocyclic head groups of this application contain one or more degradable linkers between the nitrogen-containing heterocycle and the hydrophobic tail. The presence of these degradable linkers allows the LNPs prepared from them to degrade in a timely manner, solving the problem of endosomal accumulation in LNPs prepared from non-degradable cationic lipids in the prior art, which leads to endosomal acidification and drug escape obstruction, thereby improving delivery efficiency. Simultaneously, these degradable linkers are stable at physiological pH and can be enzymatically hydrolyzed after entering cells to form low-toxicity small molecules (such as acids, alcohols, amines, and amino acids) that can be rapidly metabolized by cells, thus greatly reducing the long-term cytotoxicity of the lipids. Attached Figure Description

[0027] Figure 1 shows the cationic lipid E6-1 prepared in Example 6. 1 H NMR spectrum.

[0028] Figure 2 shows the mass spectrometry (MS) of the cationic lipid E6-1 prepared in Example 6.

[0029] Figure 3 shows the high performance liquid chromatography (HPLC) test results of the cationic lipid E6-1 prepared in Example 6.

[0030] Figure 4 shows the cytotoxicity test results of the LNP-mRNA drug composition L-6-1 prepared in Example 31.

[0031] Figure 5 shows the imaging results of mice after injection of the LNP-mRNA drug composition L-6-1 prepared in Example 31.

[0032] Implementation

[0033] Terminology Explanation

[0034] In this application, unless otherwise described, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patent and other publication disclosures cited herein are incorporated herein in their entirety by reference. In the event of any conflict between any description or interpretation of terms herein and any document incorporated herein by reference, the description and interpretation of the terms below shall prevail.

[0035] In this application, when the structure involved has isomers, it can be any one of them unless otherwise specified. For example, for a structure with cis-trans isomers, it can be either the cis or trans structure; for a structure with E / Z isomers, it can be either the E or Z structure; and when it is optically active, it can be either levorotatory or dextrorotatory.

[0036] In this application, the definition of a numerical range includes not only ranges marked with a hyphen (e.g., 0-12), but also ranges marked with a wavy line (e.g., (0~12)) and ranges marked with "to / to" (e.g., 0 to 12, 1 to 12). In this application, unless otherwise specified, integer ranges marked as intervals can represent groups of all integers within that range, and the range includes two endpoints. For example, the integer range 0-12 represents the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. The numerical ranges in this application, including but not limited to ranges of integers, non-integers, percentages, and fractions, all include two endpoints unless otherwise specified.

[0037] The numerical values ​​used in this application, when referring to "about" or "around," generally refer to a range of ±10%, which may be increased to ±15% in some cases, but not exceeding ±20%. A preset value is used as the base. For example, if the molar percentage of steroid lipids in a solution containing solvent is approximately 40%, it can generally be considered that the molar percentage of steroid lipids is between 30% and 50%.

[0038] In this application, unless otherwise specified, "any" includes any one, any two, or any two or more.

[0039] In this application, unless otherwise specified, the terms “comprising,” “including,” and “containing,” as well as similar expressions, shall be interpreted in an open and inclusive sense as “including but not limited to.”

[0040] In this application, two or more objects are "independently preferred". When there are multiple levels of preferred options, it is not required that they are all selected from the same level of preferred options. One can be a wide range of preferred options and the other a narrow range of preferred options, or one can be the widest range and the other any preferred option, or they can be selected from the same level of preferred options.

[0041] In this application, "each occurrence independently constitutes" not only refers to different groups being able to independently constitute any option in the definition, but also indicates that when appearing at different positions within the same group, they can also independently constitute any option in the definition, such as "each occurrence independently constitutes a linking bond, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, -NH-, -O(CR)-, -OC(=O ... 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-, where R c Each time it appears, it is independently a hydrogen atom or a carbon atom. 1-12 alkyl group, in the -NR group c C(=O)NR c In the "-", there are two R's c Each is independently a hydrogen atom or a carbon atom. 1-12 Alkyl, i.e., two Rs c They can be the same or different.

[0042] In this application, the divalent linking group, such as alkylene group, alkylene group, arylene group, amide bond, etc., can be either of the two linking ends when it is linked to other groups, unless otherwise specified. For example, when an amide bond is used as the divalent linking group between Group A and Group B, it can be Group A-C(=O)NH-Group B or Group B-NHC(=O)-Group A.

[0043] In the structural formula of this application, when the end group of the linking group and the substituents contained in the linking group are easily confused, the following is adopted: To mark the positions where other groups are attached in the linker, such as in the structural formula. In, the method adopted The two positions in the divalent linker that connect to other groups are marked. The two structural formulas mentioned above represent -CH(CH2CH2CH3)2- and -CH2CH2CH(CH3)2-CH2CH2-, respectively.

[0044] In this application, the range of carbon atoms in a group is indicated by a subscript at the subscript position of C, representing the number of carbon atoms in the group, for example, C 1-12 Indicates "having 1 to 12 carbon atoms", C 1-30 It indicates "having 1 to 30 carbon atoms". "Substituted C" 1-12 "alkyl" refers to C 1-12 Compounds obtained by substituting hydrogen atoms of alkyl groups. "C" 1-12 "Substituted alkyl" refers to compounds in which the hydrogen atoms of an alkyl group are substituted, resulting in compounds with 1-12 carbon atoms. For example, when a group can be selected from C... 1-12 When alkylene is used, it can be selected from any number of carbon atoms in the range indicated by the subscript, i.e., selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, ... 10 C 11 C 12Any alkylene group. In this application, unless otherwise specified, subscripts in the form of intervals indicate any integer selected from the range, which includes both endpoints.

[0045] In this application, "base" can be referred to as "bond" without changing its meaning. For example, an ether group (-O-) can also be called an ether bond, and an ester group (-OC(=O)- or -C(=O)O-) can also be called an ester bond.

[0046] In this application, the "carbon chain length" between two groups refers to the shortest number of carbons excluding the group itself. For example, the carbon chain length between the two ester bonds of -OC(=O)CH2CH2OC(=O)- and -OC(=O)CH(CH3)CH2OC(=O)- is 2 (also referred to as C2). The carbon chain length between the tertiary amine and the ester bond of >NCH2CH2CH2CH2CH2OC(=O)- is C5 (also referred to as C5).

[0047] The heteroatoms used in this application are not specifically limited, and include, but are not limited to, O, S, N, P, Si, F, Cl, Br, I, B, etc.

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

[0049] In this application, "substituted" means any of the aforementioned groups (e.g., alkyl, alkylene, or alkylene groups) in which at least one hydrogen atom is substituted by a bond connected to a non-hydrogen atom, such as, but not limited to: halogen atoms such as F, Cl, Br, and I; oxo groups (=O); hydroxyl groups (-OH); alkyloxy groups (-OR). d , where R d C 1-12 Alkyl group; Carboxyl group (-COOH); Amine group (-NR) c R c Two Rs c Each independently represents H and C. 1-12 Alkyl); C 1-12 Alkyl and cycloalkyl. In some embodiments, the substituent is C. 1-12 Alkyl group. In other embodiments, the substituent is cycloalkyl. In other embodiments, the substituent is a halogenated group, such as a fluorinated group. In other embodiments, the substituent is an oxogroup. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0050] In this application, "optional" or "optionally" (e.g., optionally substituted) means that the event described thereafter may or may not occur, and the description includes instances where the event or condition occurs and instances where the event or condition does not occur. For example, "optionally substituted hydrocarbon group" means that the hydrocarbon group may or may not be substituted, and the description includes substituted hydrocarbon groups and unsubstituted hydrocarbon groups.

[0051] In this application, "carbon chain linker" refers to a linker whose main chain atoms are all carbon atoms, while the side chain portion allows heteroatoms or heteroatom-containing groups to replace hydrogen atoms of the main chain carbons. When the "main chain atom" is a heteroatom, it is also called a "main chain heteroatom," such as AS-CH2-B, AO-CH2-B, etc. (Atomic spacing denoted as 4) is considered to contain heteroatoms in the main chain. Carbon chain linkers can be divided into alkylene groups and carbon chain linkers with heteroatoms in the side groups; the carbon chain linkers with heteroatoms in the side groups include, but are not limited to, oxo (=O), thio (=S), amino (connected to the main chain carbon via a carbon-nitrogen double bond), ether-bonded oxoalkylene groups, thioether-bonded thioalkylene groups, tertiary amino-bonded nitrogen-bonded groups, etc. The main chain of a "carbon chain linker" is entirely composed of carbon atoms, and the side groups of the carbon chain are allowed to contain heteroatoms. That is, it is formed by connecting methylene or substituted methylene groups. The substituted methylene group can be one monovalent substituent, two monovalent substituents, or one divalent substituent (such as divalent oxygen, which together with divalent methylene forms a three-membered ring). The substituted methylene group can be a single hydrogen atom (e.g., -CH(CH3)-), two hydrogen atoms (e.g., -(CH3)C(OCH3)-), or both hydrogen atoms (e.g., carbonyl, thiocarbonyl, -C(=NH)-, -C(=N)-. + H2)-), can also be a cyclic side group (such as H2)-), and can also be a ring-shaped side group (e.g. The atomic spacing is denoted as 1).

[0052] In this application, a compound or a group can be simultaneously substituted and hybridized, for example, a hydrogen atom can be substituted by a nitrophenyl group, or -CH2-CH2-CH2- can be replaced by -CH2-S-CH(CH3)-.

[0053] In this application, "linking bond" refers to a bond that only serves a connecting function and does not contain any atoms. When a group is defined as a linking bond, it means that the group may not exist.

[0054] In this application, a "group" containing at least one atom refers to a free radical formed by the loss of one or more atoms from a compound. A group formed after the loss of a portion of a compound is also called a residue. The valence state of a group is not particularly limited, but can be categorized as monovalent, divalent, trivalent, tetravalent, ..., 100valent groups, etc. Groups with a valence of 2 or higher are collectively referred to as linking groups. Linking groups can also contain only one atom, such as oxygen or thio groups.

[0055] In this application, "hydrocarbon" refers to hydrocarbons composed of carbon atoms and hydrogen atoms.

[0056] In this invention, hydrocarbons are classified into two types according to the type of hydrocarbon group: aliphatic hydrocarbons and aromatic hydrocarbons. Hydrocarbons without a benzene ring or any structure of a benzene ring with substituted hydrocarbon groups are defined as aliphatic hydrocarbons. Hydrocarbons containing at least one benzene ring or a benzene ring with substituted hydrocarbon groups are defined as aromatic hydrocarbons. Aromatic hydrocarbons may contain aliphatic hydrocarbon groups, such as toluene, diphenylmethane, and 2,3-dihydroindene.

[0057] In this invention, hydrocarbons are classified into two types based on their degree of saturation: saturated hydrocarbons and unsaturated hydrocarbons. All aromatic hydrocarbons are unsaturated hydrocarbons. Saturated aliphatic hydrocarbons are also called alkanes. The degree of unsaturation of unsaturated aliphatic hydrocarbons is not particularly limited. Examples include, but are not limited to, alkenes (containing double bonds), alkynes (containing triple bonds), and dienes (containing conjugated double bonds). When the aliphatic portion of an aromatic hydrocarbon is saturated, it is also called an aromatic alkane, such as toluene.

[0058] In this application, there are no particular restrictions on the structure of the hydrocarbon, which can be a straight-chain structure without side groups, a branched structure with side groups, a cyclic structure, a dendritic structure, a comb-like structure, a hyperbranched structure, etc. Unless otherwise defined, straight-chain structures without side groups, branched structures with side groups, and cyclic structures are preferred, corresponding to straight-chain hydrocarbons, branched hydrocarbons, and cyclic hydrocarbons, respectively. Hydrocarbons without cyclic structures are collectively referred to as open-chain hydrocarbons, including but not limited to straight-chain structures without side groups and branched structures with side groups. 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.

[0059] In this application, "hydrocarbon group" refers to a residue formed after a hydrocarbon loses at least one hydrogen atom. Based on the number of hydrogen atoms lost, hydrocarbon groups can be classified as monovalent (losing one hydrogen atom), divalent (losing two hydrogen atoms, also called a hydrocarbon subgroup), trivalent (losing three hydrogen atoms), and so on. When n hydrogen atoms are lost, the valence state of the resulting hydrocarbon group is n. Unless otherwise specified, "hydrocarbon group" in this application specifically refers to a monovalent hydrocarbon group. Unless otherwise explicitly stated in this specification, hydrocarbon groups are optionally substituted.

[0060] In this application, the source of the hydrocarbon group is not particularly limited. For example, it can originate from aliphatic or aromatic hydrocarbons, saturated or unsaturated hydrocarbons, straight-chain hydrocarbons, branched-chain hydrocarbons, or cyclic hydrocarbons, as well as hydrocarbons or heterocyclic hydrocarbons, etc. From the perspective of saturation, it can originate from alkanes, alkenes, alkynes, dienes, etc.; for cyclic hydrocarbons, it can originate from alicyclic or aromatic hydrocarbons, monocyclic or polycyclic hydrocarbons; for heterocyclic hydrocarbons, it can originate from alicyclic heterocyclic hydrocarbons or aromatic heterocyclic hydrocarbons.

[0061] In this application, "alkyl" refers to a hydrocarbon group formed from an alkane. Unless otherwise specified, it refers to a hydrocarbon group formed by losing a hydrogen atom at any position, and can be straight-chain or branched, substituted or unsubstituted. Specifically, propyl refers to either n-propyl or isopropyl, and propylene refers to either 1,3-propylene, 1,2-propylene, or isopropylene. Unless otherwise expressly stated in this specification, alkyl groups are optionally substituted.

[0062] In this application, "unsaturated hydrocarbon group" refers to the hydrocarbon group formed by the loss of hydrogen atoms from an unsaturated hydrocarbon. Hydrocarbon groups formed by the loss of hydrogen atoms from an unsaturated carbon atom in an unsaturated hydrocarbon can be classified as alkenyl, alkynyl, dienyl, etc., with examples such as propenyl and propynyl. Hydrocarbon groups formed by the loss of hydrogen atoms from a saturated carbon atom in an unsaturated hydrocarbon are, depending on the type of unsaturated bond, called alkenyl, alkynyl, dienyl, etc., specifically such as allyl and propynyl.

[0063] In this application, "alkenyl" or "alkenyl group" means a substituted or unsubstituted straight-chain or branched alkenyl group comprising two or more carbon atoms (e.g., 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 notation "C" is used to indicate this. 2-15 "Alkenyl" means a substituted or unsubstituted straight-chain or branched alkenyl group comprising 2-15 carbon atoms and at least one carbon-carbon double bond; that is, an alkenyl group may include one, two, three, four or more carbon-carbon double bonds. Unless otherwise specifically stated, alkenyl as used herein refers to both unsubstituted and substituted alkenyl groups. Unless otherwise expressly stated in this specification, alkenyl groups are optionally substituted.

[0064] In this application, "alkynyl" or "alkynyl group" means an optionally substituted straight-chain or branched hydrocarbon comprising two or more carbon atoms (e.g., 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 symbol "C" is used. 2-15"Alynyl" means a substituted or unsubstituted straight-chain or branched alkynyl group comprising 2-15 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds. Unless otherwise specified, the alkynyl group referred to herein means both unsubstituted and substituted alkynyl groups. Unless otherwise expressly stated in this specification, the alkynyl group is optionally substituted.

[0065] In this application, "hydroalkyl group" or "hydroalkyl group chain" refers to a straight or branched divalent hydrocarbon chain in which the remainder of a molecule is attached to a free radical group, consisting only of carbon and hydrogen, and may be saturated or unsaturated. For example, a hydroalkyl group (C1) having one to twenty-four carbon atoms. 1-24 Hydroxyl group (C1-C2), having one to twelve carbon atoms. 1-12 (Hydroxyyl groups), specifically, for example, methylene, ethylene, propylene, n-butylene, vinylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. Unless otherwise expressly stated in this specification, hydroxyyl groups are optionally substituted.

[0066] In this application, "alkylene" refers to a divalent alkyl group, including open-chain alkylene and divalent cycloalkyl. Open-chain alkylene refers to a divalent alkyl group that does not contain a cyclic structure, and divalent cycloalkyl refers to a divalent alkyl group that contains a cyclic structure. Unless otherwise expressly stated in this specification, alkylene is optionally substituted.

[0067] In this application, "molecular weight" represents the mass of a compound molecule, and "average molecular weight" represents the mass of a general formula compound component in a macroscopic substance. Unless otherwise specified, "average molecular weight" generally refers to "number-average molecular weight" M. n The number-average molecular weight can refer to the molecular weight of either polydisperse blocks or monodisperse blocks or substances. Unless otherwise specified, the unit of measurement for "molecular weight" and "average molecular weight" is Daltons (Da). The "degree of polymerization" can also characterize the molecular weight of a 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 or number-mean value of the number of repeating units.

[0068] In this application, percentages, "about", generally refer to ±0.5%.

[0069] In this application, the degradable linker is a chemical group containing disulfide, ester, amide, carbamate, urea, or their derivatives. The degradation pathways of the degradable linker include, but are not limited to, oxidative degradation, hydrolysis, and enzymatic degradation. In this application, disulfide, ester, amide, carbamate, and urea groups are all degradable linkers.

[0070] In this application, "hydroxyl protecting group" includes all groups that can be used as protecting groups for the hydroxyl group in general. Preferably, the hydroxyl protecting group is an alkyl acyl (e.g., acetyl, tert-butyryl), aralkyl acyl (e.g., benzyl), benzyl, triphenylmethyl, trimethylsilyl, tert-butyldimethylsilyl, allyl, acetal, or ketal. The removal of the acetyl group is generally carried out under alkaline conditions, most commonly by ammonolysis of NH3 / MeOH and methanololysis catalyzed by methanol anion; benzyl is easily removed by palladium-catalyzed hydrogenolysis in neutral solution at room temperature, or by reduction cleavage with metallic sodium in ethanol or liquid ammonia; triphenylmethyl is generally removed by catalytic hydrogenolysis; trimethylsilyl is usually removed using reagents containing fluoride ions (e.g., tetrabutylamine fluoride / anhydrous THF); tert-butyldimethylsilyl ether is relatively stable and can withstand the ester hydrolysis conditions of alcoholic potassium hydroxide and mild reducing conditions (e.g., Zn / CH3OH), and can be removed by fluoride ions (e.g., Bu4N). + F - It can be removed in tetrahydrofuran solution, or it can be removed with aqueous acetic acid at room temperature.

[0071] In this application, "carboxyl protecting group" refers to a protecting group that can be converted into a carboxyl group through hydrolysis or deprotection reaction. The carboxyl protecting group is preferably alkyl (e.g., methyl, ethyl, tert-butyl) or aralkyl (e.g., benzyl), more preferably tert-butyl (tBu), methyl (Me), or ethyl (Et). In this application, "protected carboxyl group" refers to the group formed after the carboxyl group is protected by a suitable carboxyl protecting group, preferably methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, or benzyloxycarbonyl. The carboxyl protecting group can be removed by hydrolysis under acid or base catalysis, and occasionally by thermal decomposition. For example, tert-butyl can be removed under mild acidic conditions, and benzyl can be removed by hydrogenolysis. The reagent for removing the carboxyl protecting group is selected from TFA, H2O, LiOH, NaOH, KOH, MeOH, EtOH, and combinations thereof, preferably a combination of TFA and H2O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. The protected carboxyl group is deprotected to produce the corresponding free acid, the deprotection is carried out in the presence of a base, and the base and the free acid formed by the deprotection form a pharmaceutically acceptable salt.

[0072] In this application, "amino protecting group" includes all groups that can be used as protecting groups for amino groups in general, such as aryl C. 1-6 Alkyl, C 1-6 Alkoxy C 1-6 Alkyl, C 1-6 Alkoxycarbonyl, aryloxycarbonyl, C 1-6Alkyl sulfonyl, aryl sulfonyl, or silyl groups are preferred. The amino protecting group is preferably Boc tert-butoxycarbonyl, Moz p-methoxybenzyloxycarbonyl, or Fmoc 9-fluorene-methoxycarbonyl. The reagent for removing the amino protecting group is selected from TFA, H2O, LiOH, MeOH, EtOH, and combinations thereof, preferably combinations of TFA and H2O, LiOH and MeOH, or LiOH and EtOH. The reagent for removing the Boc protecting group is TFA or HCl / EA; TFA is preferred. The deprotecting agent used in the Fmoc protecting group removal reaction is a solution of N,N-dimethylformamide (DMF) containing 20% ​​piperidine.

[0073] In this application, "cationic lipid" refers to lipids that contain a positive charge or are ionizable in their entirety. "Cation" refers to a corresponding structure that is permanently or non-permanently positively charged in response to certain conditions (e.g., pH). Therefore, cations include both permanent cations and cationizable cations. A permanent cation is a compound, group, or atom that carries a positive charge at any pH or hydrogen ion activity in its environment. Typically, a positive charge arises from the presence of quaternary ammonium atoms. When a compound carries multiple such positive charges, it can be called a permanent cation. A cationizable cation is a compound, group, or atom that carries a positive charge at lower pH and no charge at higher pH in its environment. Additionally, in non-aqueous environments where pH cannot be determined, cationizable compounds, groups, or atoms carry a positive charge at high hydrogen ion concentrations and no charge at low hydrogen ion concentrations or activity. It depends on the individual properties of the cationizable or polycationizable compound, particularly the pKa of the corresponding cationizable group or atom, which determines whether it carries a charge or no charge at the stated pH or hydrogen ion concentration. In a diluted aqueous environment, the so-called Henderson-Hasselbalch equation, well known to those skilled in the art, can be used to estimate the fraction of positively charged cationizable compounds, groups, or atoms. For example, in some embodiments, if a compound or portion is cationizable, it is preferably positively charged at a pH of about 1 to 9, preferably 4 to 9, 5 to 8, or even 6 to 8, more preferably at a pH equal to or below 9, equal to or below 8, equal to or below 7, and most preferably at physiological pH (e.g., about 7.3 to 7.4), i.e., under physiological conditions, particularly under physiological conditions of cells in vivo. In other embodiments, it is preferred that the cationizable compound or portion is primarily neutral at physiological pH (e.g., about 7.0-7.4) but becomes positively charged at lower pH values. In some embodiments, the preferred range of the pKa of the cationizable compound or portion is about 5 to about 7.

[0074] In this application, lipid nanoparticles, cationic peptides, proteins, polysaccharides, lipids, or polymers are uncharged, neutrally charged, or electrically neutral under physiological conditions, particularly under in vivo cellular conditions. Cationic peptides or proteins preferably contain a large number of cationic amino acids, such as Arg, His, Lys, or Orn (especially more cationic amino acids than anionic amino acid residues like Asp or Glu) or contain components primarily composed of cationic amino acid residues. The term "cationic" can also refer to "polycationic" components / chemical cationic components / compounds, and can also refer to cationic lipids capable of carrying a positive charge. For example, cationic lipids contain one or more positively charged amine groups, and preferably cationic lipids are ionizable, allowing them to exist in a positively charged or neutral form depending on pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles (LNPs) under different pH conditions. This charge state can influence plasma protein uptake, blood clearance and tissue distribution, and the ability to form non-bilayer structures crucial for intracellular nucleic acid delivery.

[0075] In this application, "polyethylene glycol lipid" refers to a molecule that includes both a lipid portion and a polyethylene glycol portion.

[0076] In this application, "neutral lipid" refers to any of a number of lipid substances that exist in the form of a neutral or neutral zwitterion at a selected pH, preferably phospholipids, which may be synthetic or of natural origin.

[0077] In this application, "steroid lipids" refers to steroids or steroid analogues.

[0078] In this application, "amino acid residue" includes amino acids in which a hydrogen atom has been removed from the amino group and / or a hydroxyl group has been removed from the carboxyl group and / or a hydrogen atom has been removed from the thiol group and / or the amino group and / or the carboxyl group and / or the thiol group are protected. Loosely speaking, an amino acid residue may be referred to as an amino acid. The source of the amino acids in this application is not particularly limited unless specifically specified; they can be of natural origin, non-natural origin, or a mixture of both. The structural type of the amino acids in this application is not particularly limited unless specifically specified; they can refer to L-type, D-type, or a mixture of both. In one embodiment of this application, 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 this application, 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.

[0079] The variations in this application refer to any of the following chemical change processes that can transform the target reactive group into a structural form: oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, deprotonation, substitution, deprotection, or alteration of the leaving group.

[0080] In this application, "variable form of reactive group" refers to a reactive group that remains active (still a reactive group) after undergoing at least one chemical change process, such as oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, deprotonation, substitution, deprotection, or change of leaving group, or an inactive form after being protected.

[0081] In this application, "micro-modification" refers to a chemical modification process that can be completed through a simple chemical reaction. This simple chemical reaction process mainly refers to deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and the transformation of leaving groups. "Micro-change form," corresponding to "micro-modification," refers to a structural form that can form the target reactive group after undergoing simple chemical reaction processes such as deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and the transformation of leaving groups. The transformation of the leaving group includes, for example, the transformation from an ester form to an acyl chloride form.

[0082] In this application, "any suitable linker," "any suitable reactive group," etc., refers to a structure that conforms to the basic principles of chemical structure and enables the preparation method of this application to be successfully implemented. A chemical structure described in this way can be considered to have a clear and definite scope.

[0083] In this application, when at least two structural types are listed, the “arbitrary combination” of the listed structural types refers to the combination of any two or more structures among the aforementioned related structural types; and there is no limitation on the number of structural units. The number of any structural unit can be zero, one or more. When the number of structural units of the same type is greater than one, they can be structural units with the same or different chemical structures, and the total number of constituting units is at least two. Examples include any combination of alkylene, divalent cycloalkyl, divalent cycloalkenyl, divalent cycloynyl, divalent cyclodienyl, aromatic, carbon-carbon double bond, carbon-carbon triple bond, conjugated carbon-carbon double bond, divalent alicyclic linker, divalent aromatic heterocyclic linker, and carbon chain linker with heteroatom-containing side groups, such as -Ph-CH2-Ph-(aromatic-alkylene-aromatic), -CH2-Ph-CH2CH2-(alkylene-aromatic-alkylene, wherein the number of alkylene groups is 2 and they have different chemical structures), or the structure where the benzene ring is replaced by a hexane, diazahexane, or 1-(2-pyridyl)hexahydro-1H-1,4-diazaphene. Another example is cycloalkenyl hydrocarbon group = cycloalkenyl + alkylene group = cycloalkenyl as a substituent of the hydrocarbon group, and cyclodienyl hydrocarbon group = cyclodienyl as a substituent of the hydrocarbon group.

[0084] In this application, "N / P ratio" refers to the molar ratio of nitrogen atoms in cationic lipids to phosphate in nucleic acids.

[0085] In this application, "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").

[0086] In this application, "RNA" refers to ribonucleic acid, which 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 cap structures, chain-terminating nucleosides, stem-loops, polyadenylated sequences, and / or polyadenylation signals. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translation of mRNA encoding a specific polypeptide, for example, in vivo translation of mRNA within mammalian cells, can produce the encoded polypeptide. RNA may be selected from the non-restrictive 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.

[0087] In this application, antisense oligonucleotides or small interfering RNA (siRNA) can inhibit the expression of target genes and target proteins in vitro or in vivo.

[0088] In this application, FLuc mRNA can express luciferase protein, which emits bioluminescence in the presence of luciferin substrate. Therefore, FLuc is often used in mammalian cell culture to measure gene expression and cell activity.

[0089] In this application, "inhibition of target gene expression" refers to the ability of 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 exposed to nucleic acid that inhibits target gene expression. The expression of the target gene in the test sample or test animal is compared to 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 exposed to or treated with nucleic acid. The expression of the target gene in the control sample can be specified as a value of 100%. In a particular embodiment, inhibition of target gene expression is achieved when the target gene expression level in the test sample is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the target gene expression level in the control sample or control mammal.

[0090] In this application, the methods for determining the expression level of target genes include, but are not limited to, dot blot, northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme action, and phenotypic determination.

[0091] In this application, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, outside the body, or in vivo.

[0092] In this application, "antigen" typically refers to a substance that can be recognized by the immune system, preferably by the adaptive immune system, and capable of triggering an antigen-specific immune response, for example, by forming antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen can be or may contain a peptide or protein that can be presented to T cells by the MHC. In the sense of this application, an antigen can be the translation product of a provided nucleic acid molecule (preferably mRNA as defined herein). In this context, fragments, variants, and derivatives of peptides and proteins containing at least one epitope are also understood as antigens.

[0093] In this application, "delivery" means providing an entity to a target. For example, delivering a drug and / or a therapeutic and / or preventative agent to a subject, said subject being tissues and / or cells of a human and / or other animal.

[0094] In this application, "pharmaceutically acceptable carrier" refers to a diluent, excipient, vehicle, or medium that is administered co-administered with a therapeutic agent and is suitable, to the extent of reasonable medical judgment, for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of this application include, but are not limited to, sterile liquids such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous solutions of glucose and glycerol may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. The compositions may also contain small amounts of wetting agents, emulsifiers, or pH buffers as needed. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Specifically, excipients include, but are not limited to, anti-adhesion agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), modifiers, emulsifiers, fillers (diluents), film-forming agents or coatings, flavorings, fragrances, flow enhancers, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration. More specifically, excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dicalcium hydrogen phosphate, calcium stearate, croscarmellose sodium, croscarmellose polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, phenylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium glycolate starch, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, and xylitol.

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

[0096] In this application, "treatment" refers to the handling and care of a patient in order to combat a disease, obstacle, or symptom, intended to include delaying the progression of the disease, obstacle, or symptom, alleviating or mitigating symptoms and complications, and / or curing or eliminating the disease, obstacle, or symptom. The patient to be treated is preferably a mammal, especially a human.

[0097] 1. Cationic lipids

[0098] One implementation method of this application:

[0099] A cationic lipid, characterized in that it has the structure shown in formula (1):

[0100] Or its salts, tautomers, stereoisomers, deuterated derivatives or solvates;

[0101] Where, N r It is a nitrogen-containing heterocyclic group;

[0102] t is an integer from 1 to 6;

[0103] L a L b Each is an independent linker or a binary linker L. d The L d Selected from any one of *-C(=O)O-, *-C(=O)NH-, and -C(=O)-; L c For -Z1-(CH2) tc -Z2-*, where Z1 is a connector or -(CH2) te -L e -, tc and te are each independent integers from 1 to 6, L e Each occurrence is independently selected from -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -CH(OH)-, -O-, -NH-, -O(CH2). s O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH-, -NHC(=O)S-, -C(=S)-, -OC(=S-, -C(=S)O-, -OC(=S)O-, -NHC(=S-, -C(=S)NH-, -NHC(=S)NH-, -OC(=S)NH- and -NHC(=S)O-, where s is 1, 2 or 3; Z2 is selected from any one of -OC(=O)-*, -NHC(=O)-*, -C(=O)NH-*, -OC(=O)NH-*; the aforementioned L d Lc The asterisk end in the Z2 structure points to the central carbon atom in formula (1);

[0104] Each X is independently -N<, -CR a < or connect key; when X is -N< or -CR a When X is a connector, both a and b are 2; when X is a connector, both a and b are 1; each R a Independently H or C 1-6 alkyl;

[0105] R m For H or C 1-12 Alkyl; each R g Independently defined as -B1-L1-R1; where each B1 is independently defined as C. 1-12 alkylene or hydroxyl-substituted C 1-12 Alkylene; each L1 is independently selected from the following: linking bond, -CH(OH)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, -NH-, -O(CR h R h ) s O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, -NR h C(=O)-、-C(=O)NR h -、-NR h C(=O)NR h -、-OC(=O)NR h -、-NR h C(=O)O-、-SC(=O)NR h -、-NR h C(=O)S-, -C(=S)-, -OC(=S)-, -C(=S)O-, -OC(=S)O-, -NR h C(=S)-、-C(=S)NR h -、-NR h C(=S)NR h -、-OC(=S)NR h -and-NR h Any one of C(=S)O-, where each R h Independently a hydrogen atom or a carbon atom 1-12 Alkyl group, s is 2, 3 or 4; each R1 is independently C 1-30 Straight-chain hydrocarbon groups or C 1-30 Branched hydrocarbon group.

[0106] 1.1.Nr

[0107] In this application, Nr is a nitrogen-containing heterocyclic group.

[0108] In one specific embodiment of this application, the Nr is selected from... Any one of them; preferably Any one of them; more preferably or

[0109] 1.2.L a L b

[0110] In one specific embodiment of this application, the L a L b The same applies to connecting bonds, *-C(=O)O- or *-C(=O)NH-.

[0111] 1.3.X

[0112] In one specific embodiment of this application, the X is the same, and can be -N<, -CH<, or a linker.

[0113] 1.4.a, b

[0114] In one specific embodiment of this application, when both a and b in the aforementioned embodiments are 2, the a R g Similarly, the b R g Same, preferred of all R g They are all the same.

[0115] 1.5.B1

[0116] In one specific embodiment of this application, in the foregoing embodiment, in B1, the C hydroxyl-substituted... 1-12 Alkyl groups are represented as *-CH2CH(OH)(CH2). P -, p is an integer from 0 to 10, preferably an integer from 1 to 10, and the asterisk end in the structure points to the central carbon atom in formula (1); the C 1-12 The alkylene group is any one of methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, and octylene; more preferably, all B1 groups are identical.

[0117] 1.6.L1

[0118] In one specific embodiment of this application, each occurrence of L1 is independently one of the following: a linking bond, -CH(OH)-, -OC(=O)-, -C(=O)O-, -C(=O)NH-, -OC(=O)O-, and -NHC(=O)O-; more preferably, all occurrences of L1 are the same.

[0119] 1.7L c

[0120] In one specific embodiment of this application, L in the foregoing embodiments c In the context, Z1 is the connecting key, and L... c -(CH2) tc -Z2-*;Preferred L c -(CH2) tc OC(=O)-*、-(CH2) tc NHC (=O)-* or -(CH2) tc C(=O)NH-*; more preferably -(CH2) 1-4 OC(=O)-*、-(CH2) 1-4 NHC (=O)-* or -(CH2) 1-4 C(=O)NH-*, the most preferred are -(CH2)2OC(=O)-*, -(CH2)3NHC(=O)-*, -(CH2)3C(=O)NH-* or -CH2C(=O)NH-*.

[0121] 1.8.R1

[0122] In this application, R1 is independently C each time it appears. 1-30 Straight-chain hydrocarbon groups or C 1-30 Branched hydrocarbon group.

[0123] In one specific embodiment of this application, in the foregoing embodiment, the C 1-30 The straight-chain hydrocarbon group is C 1-30 Straight-chain alkyl, C 2-30 Straight-chain alkenyl, C 2-30 Any one of the straight-chain alkynyl groups; preferably C 1-25 Straight-chain alkyl, C 2-25 Straight-chain alkenyl or C 2-25 Straight-chain alkynyl group; more preferably... Any one of the following, where tp is an integer from 1 to 10 and tq is an integer from 0 to 12.

[0124] In one specific embodiment of this application, in the foregoing embodiment, the C 1-30 Branched hydrocarbon groups are selected from C 1-30 Branched alkyl groups, C 2-30 Branched alkenyl groups, C 2-30 Each of the branched alkynyl groups can be independently represented as Where tm is an integer between 0 and 12; R e R f Each independently is C 1-15Alkyl, C 2-15 alkenyl and C 2-15 Any one of the alkynyl groups; R e R f More preferably, each is independently selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, vinyl, propenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptenyl, octenyl, nonynyl, and decynyl; R e R f The preferred choice is that each of the following is independently selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl.

[0125] In one specific embodiment of this application, R1 is more preferably selected from any of the following structures:

[0126] More preferably, tm in the aforementioned structure is 0, 1, or 2.

[0127] 1.9.R g

[0128] In this application, R g Each occurrence is independently -B1-L1-R1.

[0129] In one specific embodiment of this application, the R g Choose from any of the following structures:

[0130] 1.10. Examples of General Structural Formulas

[0131] In one specific embodiment of this application, the structure of the cationic lipid satisfies the following general formula (1-A), (1-B), or (1-C):

[0132] L in preferred formula (1-A), (1-B) or (1-C) a L b The same, is a connecting bond, *-C(=O)O- or *-C(=O)NH-; L c -(CH2) tc OC(=O)-*、-(CH2) tc NHC (=O)-* or -(CH2) tcC(=O)NH-*, tc is an integer from 1 to 6; t is an integer from 1 to 4, preferably t is 1, 2, or 4; the asterisk points to the central carbon atom in formula (1-A), (1-B), or (1-C).

[0133] In one specific embodiment of this application, the structure of the cationic lipid preferably satisfies general formulas (2-1)-(2-3):

[0134] More preferably, the structure of the cationic lipid satisfies any one of the following general formulas:

[0135] More preferably, the structure of the cationic lipid satisfies any one of the following general formulas:

[0136] 1.11. Specific structural examples

[0137] In one specific embodiment of this application, the structure of preferred formula (1) is selected from any of the following:

[0138] Alternatively, the structure of the cationic lipid may be any of the following:

[0139] 2. Preparation of cationic lipids

[0140] 2.1. In one specific embodiment of this application, the preparation process of the cationic lipid represented by formula (1) is as follows:

[0141] The intermediates / raw materials involved in the preparation process of the cationic lipids in this application include, but are not limited to, Nr0, Nr-C-1, Nr-C-2, IM-C'-1, IM-C'-2, IM-C, IM-N-1 and IM-N-2.

[0142] The preparation process of the cationic lipids in this application involves an initial raw material Nr0 containing a nitrogen-containing heterocycle and containing one reactive group F0; wherein F0 is a protected or unprotected -OH, -COOH, or -NH2.

[0143] In one specific embodiment of this application, Nr0 is selected from any of the following structures:

[0144] The preparation of the cationic lipids in this application may involve small molecule intermediates IM-C'-1 and IM-C'-2 containing three identical or different reactive groups. The structure of IM-C'-1 is shown as follows: The structure of IM-C'-2 is represented as follows Among them, F q The active group and its protected form are selected from any one of -OH, -COOH, -NH2, and -SH, capable of reacting directly or after deprotection with F0 in Nr0. Functional group pairs F1, F2 and F3, F4 are each independently selected from any one of protected or unprotected -OH, -NH2, -COOH and any other suitable reactive group. The definitions of the remaining symbols are consistent with those in general formula (1). IM-C'-1 and IM-C'-2 can be obtained by purchase or by in-house synthesis.

[0145] In this application, IM-C'-1 is selected from any of the following structures:

[0146] In this application, IM-C'-2 is selected from any of the following structures:

[0147] The preparation of the cationic lipids in this application may involve small molecule intermediates IM-N-1 and IM-N-2 containing a hydrophobic tail chain with a reactive group. The structure of IM-N-1 is represented as F5-B1-L1-R1, and the structure of IM-N-2 is represented as F5-R1. F5 is a reactive group capable of reacting with F1 or F2 in IM-C'-1 or F3 or F4 in IM-C'-2, preferably an alkenyl, acrylate, -CHO, -OH, -COOH, -NH2, or -COCl group. -Cl or -Br, and the definitions of the remaining symbols are consistent with those in general formula (1). IM-N-1 or IM-N-2 can be purchased or synthesized in-house, preferably through simple single-step or stepwise reactions such as esterification, amidation, alkylation, addition, or substitution. For example, in Example 3, S3-1 With S3-2 The small molecule intermediate IM-N-1 was obtained via a condensation reaction.

[0148] In this application, IM-N-1 is selected from any of the following structures:

[0149] In this application, IM-N-2 is selected from any of the following structures:

[0150] The preparation of the cationic lipids in this application may involve intermediates Nr-C-1 and Nr-C-2, which contain a nitrogen-containing heterocycle and two reactive groups. The structure of Nr-C-1 is shown as follows: The structure of Nr-C-2 is represented as follows: Among them, functional group pairs F1, F2 and functional group pairs F3, F4 are selected from any one of protected or unprotected -OH, -NH2, -COOH, and the definitions of the remaining symbols are consistent with those in general formula (1). Nr-C-1 can be obtained by a single-step or stepwise reaction of Nr0 with IM-C'-1, and Nr-C-2 can be obtained by a single-step or stepwise reaction of Nr0 with IM-C'-2. For example, in Example 1, S1-1 With S1-2 The Nr-C-1 intermediate can be obtained by condensation reaction followed by removal of the Boc protecting group.

[0151] In this application, Nr-C-1 is selected from any of the following structures:

[0152] In this application, Nr-C-2 is selected from any of the following structures:

[0153] The preparation process of the cationic lipid in this application may involve an intermediate IM-C containing one reactive group and two or four hydrocarbon tail chains, the structure of which can be represented as follows: Among them, F q The protected or unprotected reactive group is a group that can react with FO in Nr0 directly or after deprotection, and is selected from any one of -OH, -NH2, and -SH; the definitions of the other symbols are consistent with those in general formula (1). IM-C can be obtained by a single-step or stepwise reaction of IM-N-2 with IM-C'-2, or by a single-step or stepwise reaction of any suitable IM-N-1 or IM-N-2 with IM-C'-1.

[0154] In this application, IM-C is selected from any of the following structures:

[0155] In one embodiment of this application, the cationic lipid can be prepared by any of the following methods:

[0156] Method (1): React any suitable Nr0 with any suitable IM-C'-1 to obtain Nr-C-1, and then react Nr-C-1 with two or four identical or different IM-N-1 or IM-N-2 through a single step or stepwise reaction to obtain Nr-C-1.

[0157] Method (2): React any suitable Nr0 with any suitable IM-C'-2 to obtain Nr-C-2, and then react Nr-C-2 with two or four identical or different IM-N-1 or IM-N-2 through a single step or stepwise reaction to obtain Nr-C-2;

[0158] Method (3): React any suitable IM-C'-1 or IM-C'-2 mentioned above with two or four identical or different IM-N-1 or IM-N-2 to obtain IM-C, and then react IM-C with Nr0 to obtain IM-C.

[0159] 2.2. Description of relevant raw materials and / or steps in the preparation process

[0160] 2.2.1. The reaction process involves the "protection" and "deprotection" of relevant functional groups.

[0161] In this application, the reaction process also involves the "protection" and "deprotection" processes of relevant functional groups. To prevent the functional group from affecting the reaction, it is usually protected. Furthermore, when there are two or more functional groups, only the target functional group is selectively reacted, thus protecting the other functional groups. 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 groups bonded to the specified functional group under appropriate conditions.

[0162] In this application, the definitions of "carboxyl protecting group" and "amino protecting group" are consistent with those in the "Terminology Explanation" section, and will not be repeated here.

[0163] In this application, the hydroxyl group protected by the hydroxyl protecting group is not particularly limited, and can be, for example, an alcohol hydroxyl group, a phenolic hydroxyl group, etc. The amino group protected by the amino protecting group is not particularly limited, and can be, for example, derived from primary amines, secondary amines, hydrazines, amides, etc. The amino group in this application is not particularly limited, and includes, but is not limited to, primary amino groups, secondary amino groups, tertiary amino groups, and quaternary ammonium ions.

[0164] In this application, the deprotection of the protected hydroxyl group is related to the type of hydroxyl protecting group. There is no particular limitation on the type of hydroxyl protecting group; taking the protection of terminal hydroxyl groups with silyl ethers or tert-butyl groups as examples, the corresponding deprotection methods include:

[0165] A: Deprotection of the silyl ether protecting group

[0166] Compounds used for this type of hydroxyl protection include trimethylsilyl ether, triethylsilyl ether, dimethyl tert-butylsilyl ether, and tert-butyldiphenylsilyl ether. Deprotection of these silyl ethers is achieved using fluoride-containing compounds, preferably tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid, and potassium fluoride, more preferably tetrabutylammonium fluoride and potassium fluoride. The amount of fluoride-containing reagent is 5 to 20 times the molar equivalent of the protected hydroxyl group, preferably 8 to 15 times the initiator. If the amount of fluoride is less than 5 times the molar equivalent of the protected hydroxyl group, incomplete deprotection will occur; if the amount of deprotecting reagent is greater than 20 times the molar equivalent of the protected hydroxyl group, excess reagent or compound will cause purification problems, may be introduced into subsequent steps, and thus cause side reactions. There are no particular restrictions on the reaction solvent, as long as it can dissolve the reactants and products. Aprotic solvents are preferred, more preferably tetrahydrofuran and dichloromethane. The reaction temperature is preferably 0 to 30°C. When the temperature is below 0°C, the reaction rate is slow and the protecting group cannot be completely removed.

[0167] B: Deprotection of the tert-butyl protecting group

[0168] The deprotection of the tert-butyl group is carried out under acidic conditions, preferably with a solution pH of 0 to 4. The acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and nitric acid are preferred, with hydrochloric acid being more preferred. The reaction solvent is not particularly limited, as long as it can dissolve both the reactants and products; water is preferred. The reaction temperature is preferably 0 to 30°C.

[0169] In this application, the raw materials used in each preparation method can be purchased or synthesized by the applicant.

[0170] The intermediates and final products prepared in this application can be purified by methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, thin-film dialysis, or supercritical extraction. The structure and molecular weight of the final products can be characterized using methods including but not limited to nuclear magnetic resonance (NMR), electrophoresis, UV-Vis spectrophotometry, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism spectroscopy, and mass spectrometry.

[0171] 3. Lipid compositions, preparation of lipid compositions, lipid pharmaceutical compositions, formulations of lipid pharmaceutical compositions

[0172] 3.1. Lipid composition

[0173] In this application, a lipid composition contains any of the cationic lipids with the structure shown in general formula (1) described above.

[0174] In one specific embodiment of this application, the preferred lipid composition, in addition to containing cationic lipids with the structure shown in general formula (1), also contains one or more of phospholipids, steroid lipids, and polyethylene glycol lipids, selected from any of the following:

[0175] Case (1): It also contains phospholipids;

[0176] Case (2): Also contains steroid lipids;

[0177] Case (3): It also contains polyethylene glycol lipids;

[0178] Case (4): Also contains phospholipids and steroid lipids;

[0179] Case (5): Also contains phospholipids and polyethylene glycol lipids;

[0180] Case (6): Also contains steroid lipids and polyethylene glycol lipids;

[0181] Case (7): Also contains phospholipids, steroid lipids and polyethylene glycol lipids;

[0182] Case (8): It also contains phospholipids, steroid lipids, polyethylene glycol lipids and another cationic lipid;

[0183] Case (9): It also contains phospholipids, steroid lipids, polyethylene glycol lipids and anionic lipids.

[0184] In one specific embodiment of this application, the phospholipids in the lipid composition are preferably 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-distearateoyl-sn-glycerol-3-phosphate choline, 1,2-diundecanoyl-sn-glycerol-3-phosphate choline, or 1-palmitoyl-2-oleoyl -sn-glycerol-3-phosphate choline, 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-cholesterolylhemisuccino-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinanoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidanoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate choline, Acyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine, 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoicoyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenicoyl-sn-glycerol-3-phosphate ethanolamine, Sodium acyl-sn-glycerol-3-phosphate-rac-(1-glycerol) salt, dioleoylphosphatidylserine, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearate-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, sphingomyelin, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine, and combinations thereof.

[0185] In one specific embodiment of this application, the steroid lipids in the lipid composition are preferably any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatine, ursolic acid, and α-tocopherol, or combinations thereof.

[0186] In one specific embodiment of this application, the polyethylene glycol lipid in the lipid composition is preferably polyethylene glycol-1,2-dimyristoylglycerol, polyethylene glycol-distearylphosphatidylethanolamine, PEG-cholesterol, polyethylene glycol-diacylglycerol, or polyethylene glycol-dialkoxypropyl. Specifically, it includes any one or a combination of polyethylene glycol 500-dispalmitoylphosphatidylcholine, polyethylene glycol 2000-dispalmitoylphosphatidylcholine, polyethylene glycol 500-distearylphosphatidylethanolamine, polyethylene glycol 2000-distearylphosphatidylethanolamine, polyethylene glycol 500-1,2-dioleoylphosphatidylethanolamine, polyethylene glycol 2000-1,2-dioleoylphosphatidylethanolamine, and polyethylene glycol 2000-2,3-dimyristoylglycerol, or the polyethylene glycol lipid is selected from any one or a combination thereof.

[0187] Wherein, n1 is an integer between 25 and 300, and more preferably n1 is any one of 44, 45, 46, 47, and 48.

[0188] In one specific embodiment of this application, another cationic lipid in the lipid composition is preferably 1,2-dioleoyl-3-trimethylammonium-propane (methyl sulfate), 1,2-bis(octadecenoxy-3-methylammonium propane) chloride, 1-[2-(oleoyloxy)ethyl]-2-oleo-3-(2-hydroxyethyl)imidazoline chloride, 1,2-dioleoyl-3-dimethylamino-propane, 2,3-bis(tetradecanoyloxy)propyltrimethylazone chloride, bisdecyldimethylammonium chloride, bisdecyldimethylammonium bromide, N,N-dioleo-N,N-dimethylammonium chloride, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)prop-1-ammonium, or 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dimethylammonium chloride. The following are included in the following formulations: ketone, 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol), methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester, 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine, ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 8-[(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino]octanoate (heptadecane-9-yl) ester, and ((2-(2-hydroxyethoxy)ethyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), and combinations thereof.

[0189] In one specific embodiment of this application, the anionic lipid in the lipid composition is preferably any one of 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt, 1,2-dimyristoyl-sn-glycerol-3-phosphate sodium salt, bis(monooleoylglycerol) phosphate ammonium salt, and cardiolipin, or combinations thereof.

[0190] In one specific embodiment of this application, it is preferred that any of the aforementioned lipid compositions contains 20-80% of the cationic lipids represented by formula (1), 5-15% of phospholipids, 25-55% of steroid lipids and 0.5-10% of polyethylene glycol lipids, wherein the percentages are the molar percentages of each lipid in the total lipids.

[0191] In one specific embodiment of this application, it is preferred that the cationic lipids account for 30-65% of the total lipids in any of the aforementioned lipid compositions; more preferably, it is any one of 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, and 55%.

[0192] In one specific embodiment of this application, preferably, in any of the aforementioned lipid compositions, the phospholipids account for 7.5-13% of the total lipids in molar percentage; more preferably, it is any one of 8%, 9%, 10%, 11%, or 12%.

[0193] In one specific embodiment of this application, preferably, in any of the aforementioned lipid compositions, the steroid lipids account for 35-50% of the total lipids in molar percentage, more preferably any one of 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, and 50%.

[0194] In one specific embodiment of this application, in any of the aforementioned lipid compositions, polyethylene glycol lipids account for 0.5-5% of the total lipids; more preferably 1-3%; and even more preferably any one of 1.5%, 1.6%, 1.7%, 1.8%, and 1.9%.

[0195] 3.2. Preparation of lipid compositions

[0196] In this application, the lipid composition is obtained by mixing the lipid components in an organic solvent, preferably ethanol.

[0197] 4. Lipid-based pharmaceutical compositions and their formulations

[0198] 4.1. Lipid-based pharmaceutical compositions

[0199] In one embodiment of this application, a lipid pharmaceutical composition comprises any of the lipid compositions and pharmaceuticals described above, wherein the lipid composition comprises any of the cationic lipids with a nitrogen-containing heterocyclic head group as shown in general formula (1) described above, and the pharmaceuticals are selected from any of nucleic acid pharmaceuticals, gene vaccines, antitumor pharmaceuticals, small molecule pharmaceuticals, polypeptide pharmaceuticals or protein pharmaceuticals.

[0200] In one specific embodiment of this application, the nucleic acid drug in the lipid drug composition is preferably selected from any one of RNA, DNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir, and ribozyme, wherein the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA, and siRNA; more preferably, the nucleic acid drug is any one of DNA, mRNA, miRNA, and siRNA.

[0201] In one specific embodiment of this application, the lipid pharmaceutical composition is preferably used to prepare a drug, wherein the drug is selected from any one of antitumor agents, antiviral agents, antifungal agents, and vaccines.

[0202] In one specific embodiment of this application, the N / P ratio of the lipid composition to the nucleic acid is preferably (0.1-100):1, more preferably (0.2-30):1, and most preferably (0.5-20):1.

[0203] 4.2. Liposomes or lipid nanoparticles and their preparation

[0204] 4.2.1. Liposomes or lipid nanoparticles

[0205] In one specific embodiment of this application, a liposome or lipid nanoparticle contains any of the lipid compositions described above.

[0206] In one specific embodiment of this application, the aforementioned lipid nanoparticles are preferably LNP-drug compositions, LPP-drug compositions, or PNP-drug compositions; preferably LNP-drug compositions; more preferably LNP-nucleic acid drug compositions; and even more preferably LNP-mRNA drug compositions.

[0207] 4.2.2. Preparation of liposomes or lipid nanoparticles

[0208] In one specific embodiment of this application, liposomes containing the cationic lipids shown in formula (1) can be prepared by conventional methods in the art, including but not limited to thin film dispersion, ultrasonic dispersion, reverse phase evaporation, freeze drying, freeze-thaw and injection methods, preferably thin film dispersion, ultrasonic dispersion and / or reverse phase evaporation.

[0209] In one specific embodiment of this application, lipid nanoparticles can be prepared by conventional methods in the art, including but not limited to vortex mixing, high-shear homogenization ultrasonication, thin film hydration extrusion, and microfluidic methods.

[0210] In one specific embodiment of this application, liposomes are prepared using a thin-film dispersion method, which includes the following steps:

[0211] (1) Weigh cationic lipids, steroid lipids, phospholipids and polyethylene glycol lipids, dissolve them thoroughly in an organic solvent, shake well, remove the organic solvent by rotary evaporation under reduced pressure to form an oil film, and then use a vacuum pump to remove the organic solvent.

[0212] (2) Add phosphate buffer solution containing cryoprotectant, sonicate in water bath to form a semi-transparent emulsion;

[0213] (3) The emulsion is added to a high-pressure homogenizer and then pressed. After pressing, the emulsion is added to a liposome extruder and passed through a membrane to form liposomes.

[0214] (4) Optionally, the liposomes are dried in a freeze dryer to form liposome powder;

[0215] Preferably, the organic solvent is dichloromethane, chloroform, and / or methanol, more preferably chloroform and methanol; preferably, the rotary evaporation speed is 30-300 rpm, more preferably 50-200 rpm, and most preferably 100-170 rpm; preferably, the rotary evaporation temperature is 10-200℃, more preferably 20-100℃, and most preferably 40-80℃.

[0216] In one specific embodiment of this application, lipid nanoparticles containing the cationic lipids shown in formula (1) are preferably prepared by vortex mixing or microfluidic methods, as follows:

[0217] (1) Dissolve each lipid component in an organic solvent to obtain a lipid composition soluble in an organic phase; the organic phase is preferably ethanol;

[0218] (2) Add the nucleic acid drug to the buffer solution to obtain an aqueous solution; the aqueous phase is preferably citrate buffer or sodium acetate buffer.

[0219] (3) The organic phase solution and the aqueous phase solution are mixed by microfluidic equipment or vortex to form a lipid nanoparticle composition, and purified by ultrafiltration to remove organic solvents and free nucleic acid molecules.

[0220] 4.3. Lipid-based pharmaceutical composition formulations

[0221] In one specific embodiment of this application, a lipid pharmaceutical composition formulation contains a lipid pharmaceutical composition (4.1) or liposomes or lipid nanoparticles (4.2), and further contains a pharmaceutically acceptable diluent or excipient, wherein the diluent or excipient is preferably any one of deionized water, ultrapure water, phosphate buffer, and physiological saline, more preferably phosphate buffer or physiological saline, and most preferably physiological saline.

[0222] In this application, the lipid pharmaceutical composition formulation is prepared by equilibrating the lipid pharmaceutical composition, liposomes, or lipid nanoparticles in the diluent or excipient. Preferably, the equilibration time is 0.1–12 h, more preferably 0.2–6 h, and even more preferably 0.5–3 h; preferably, the compounding time is 0.1–12 h, more preferably 0.2–5 h, and even more preferably 0.5–2 h.

[0223] The following describes in further detail the preparation methods of cationic lipids, lipid compositions, and lipid pharmaceutical composition formulations, as well as the bioactivity testing of lipid pharmaceutical compositions, with reference to specific embodiments. These specific embodiments are for further detailed explanation of this application and are not intended to limit the scope of protection of this application. In the embodiments for preparing cationic lipids, the final product is characterized by NMR and its molecular weight is confirmed by mass spectrometry.

[0224] Example 1: Cationic lipids (E1-1)

[0225] The preparation process is as follows:

[0226] Step a: Under nitrogen protection, 1-(3-di-tert-butoxycarbonylaminohexanoic acid (S1-1, 1.38 g, 4.0 mmol), 1-hydroxyethyl-4-methylpiperazine (S1-2, 0.69 g, 4.8 mmol), and 4-dimethylaminopyridine (DMAP, 0.12 g, 1.0 mmol) dissolved in dichloromethane (50 mL) were added to a round-bottom flask, and the reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the solution was concentrated to obtain the crude product. In a dry and clean round-bottom flask, a solution of trifluoroacetic acid / dichloromethane (1:2, v / v) was prepared, and the dichloromethane solution of the crude product was slowly added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried with anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S1-3 (0.87 g).

[0227] Step b: S1-3 (0.41 g, 1.5 mmol) and N,N-diisopropylethylamine (DIPEA, 0.58 g, 4.5 mmol) were sequentially added to a methanol solution of 1,2-epoxytetradecane (S1-4, 1.91 g, 9.0 mmol), and the reaction mixture was placed in a reflux apparatus (90 °C) and stirred for 24 h. After the reaction was completed, the reaction mixture was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E1-1 (1.36 g). 1 H NMR(400MHz, CDCl3)δ:4.25-4.18(m,2H,pip-CH2CH2-),3.78-3.63(m,4H,-CH(OH)-),3.27(s,1H,-OC(=O)CH<),2.65-2.35(m,12H;2H,>CH(CH2)3CH2-;2 H,pip-CH2CH2-;8H,pip-H),2.32(s,3H,pip-CH3),2.14-1.90(m,8H,-CH(OH)CH2N<),1.70-1.21(m,94H,-CH2CH2CH2-,-CH2CH3),0.86(t,12H,-CH2CH3). MS(ESI):m / z=1121.1([M+H] + ).

[0228] Example 2.1: Cationic Lipid (E2-1)

[0229] The preparation process is as follows:

[0230] Step a: Under nitrogen protection, DCC (2.72 g, 13.2 mmol) was added to a round-bottom flask containing 6-heptenic acid (S2-2, 0.77 g, 6.0 mmol), n-heptanol (S2-1, 0.84 g, 7.2 mmol), and DMAP (0.18 g, 1.5 mmol) dissolved in dichloromethane (50 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the product was concentrated. The crude product was purified by column chromatography to obtain S2-3 (1.10 g).

[0231] Step b: Compound S2-3 (0.90 g, 4.0 mmol) was dissolved in 20 mL of dichloromethane. Under ice bath conditions, m-chloroperoxybenzoic acid (m-CPBA, 1.03 g, 6.0 mmol) was added, and the mixture was stirred for 15 min. The ice bath was then removed, and the reaction was allowed to proceed overnight. After the reaction was complete, excess saturated sodium bisulfite solution was added to concentrate the reaction solution. The residue was washed successively with ethyl acetate (20 mL), saturated sodium bicarbonate solution (20 mL), and saturated sodium chloride solution (20 mL). The residue was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain compound S2-4 (0.76 g).

[0232] Step c: S1-3 (0.14 g, 0.5 mmol) and DIPEA (0.19 g, 1.5 mmol) were added sequentially to a methanol solution of S2-4 (0.72 g, 3.0 mmol), and the reaction mixture was placed in a reflux apparatus (90 °C) and stirred for 24 h. After the reaction was completed, the reaction mixture was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E2-1 (0.50 g). 1 H NMR(400MHz, CDCl3)δ:4.26-4.18(m,2H,pip-CH2CH2-),4.05(t,8H,-C(=O)OCH2CH2CH2-),3. 78-3.63(m,4H,-CH(OH)-),3.27(s,1H,-OC(=O)CH<),2.65-2.35(m,12H;2H,>CH(CH2)3CH2-;2 H,pip-CH2CH2-;8H,pip-H),2.33(s,3H,pip-CH3),2.32-2.27(m,8H,-CH2C(=O)O-),2.15-1. 90(m,8H,-CH(OH)CH2N<),1.71-1.21(m,70H,-CH2CH2CH2-,-CH2CH3),0.88(t,12H,-CH2CH3). MS(ESI):m / z=1241.0([M+H] + ).

[0233] Example 2.2: Cationic Lipids (E2-2)

[0234] The preparation process is as follows:

[0235] Step a: Under nitrogen protection, DCC (2.72 g, 13.2 mmol) was added to a round-bottom flask containing octanoic acid (S2-5, 0.86 g, 6.0 mmol) dissolved in dichloromethane (50 mL), 5-hexen-1-ol (S2-6, 0.72 g, 7.2 mmol), and DMAP (0.18 g, 1.5 mmol). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the product was concentrated. The crude product was purified by column chromatography to obtain S2-7 (1.11 g).

[0236] Step b: Compound S2-7 (0.90 g, 4.0 mmol) was dissolved in 20 mL of dichloromethane. m-CPBA (1.03 g, 6.0 mmol) was added under ice bath conditions. The mixture was stirred for 15 min, then the ice bath was removed, and the reaction was allowed to proceed overnight. After the reaction was complete, excess saturated sodium bisulfite solution was added to concentrate the reaction solution. The residue was washed successively with 20 mL of ethyl acetate, 20 mL of saturated sodium bicarbonate solution, and 20 mL of saturated sodium chloride solution. The residue was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain compound S2-8 (0.76 g).

[0237] Step c: S1-3 (0.14 g, 0.5 mmol) and DIPEA (0.19 g, 1.5 mmol) were added sequentially to a methanol solution of S2-8 (0.73 g, 3.0 mmol), and the reaction mixture was placed in a reflux apparatus (90 °C) and stirred for 24 h. After the reaction was completed, the reaction mixture was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E2-2 (0.51 g). 1 H NMR(400MHz, CDCl3)δ:4.25-4.17(m,2H,pip-CH2CH2-),4.06(t,8H,-C(=O)OCH2CH2CH2-),3. 78-3.63(m,4H,-CH(OH)-),3.27(s,1H,-OC(=O)CH<),2.66-2.35(m,12H;2H,>CH(CH2)3CH2-;2 H,pip-CH2CH2-;8H,pip-H),2.33(s,3H,pip-CH3),2.30-2.27(m,8H,-CH2C(=O)O-),2.14-1. 90(m,8H,-CH(OH)CH2N<),1.73-1.21(m,70H,-CH2CH2CH2-,-CH2CH3),0.87(t,12H,-CH2CH3). MS(ESI):m / z=1241.0([M+H] + ).

[0238] Example 3: Cationic lipid (E3-1)

[0239] The preparation process is as follows:

[0240] Step a: Under nitrogen protection, DCC (3.63 g, 17.6 mmol) was added to a round-bottom flask containing 6-bromohexanoic acid (S3-1, 1.55 g, 8.0 mmol), 2-nonen-1-ol (S3-2, 1.36 g, 9.6 mmol), and DMAP (0.24 g, 2.0 mmol) dissolved in dichloromethane (80 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the product was concentrated. The crude product was purified by column chromatography to obtain S3-3 (2.08 g).

[0241] Step b: Under nitrogen protection, compound S1-3 (0.27 g, 1.0 mmol) was dissolved in acetonitrile (30 mL). S3-3 (1.91 g, 6.0 mmol) and DIPEA (0.65 g, 5.0 mmol) were added sequentially with slow stirring. The reaction mixture was stirred at room temperature for 20 h. After the reaction was complete, the reaction solution was concentrated, dissolved in dichloromethane, and extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E3-1 (1.00 g). 1 H NMR (400MHz, CDCl3) δ: 5.80-5.73(m,4H,-C(=O)OCH2CH=CH-), 5.60-5.51(m,4H,-C(=O)OCH2CH=CH-), 4.25-4. 18(m,2H,pip-CH2CH2-),4.51(d,8H,-C(=O)OCH2CH=CH-),3.28(s,1H,-OC(=O)CH<),2.65-2.35(m,20H;8H,-N (CH2)2-;2H,>CH(CH2)3CH2-;2H,pip-CH2CH2-;8H,pip-H),2.33(s,3H,pip-CH3),2.31-2.27(m,8H,-CH2C(=O )O-), 2.07-2.03(m,8H,-CH=CHCH2CH2-), 1.71-1.21(m,62H,-CH2CH2CH2-,-CH2CH3), 0.87(t,12H,-CH2CH3). MS(ESI):m / z=1225.0([M+H] + ).

[0242] Example 4: Cationic lipid (E4-1)

[0243] The preparation process is as follows:

[0244] Under ice bath conditions, S1-3 (0.54 g, 2.0 mmol) was dissolved in dichloromethane solution (30 mL). With vigorous stirring, (9Z,12Z)-octadec-9,12-dienal (S4-1, 1.58 g, 6.0 mmol) was added, followed by sodium triacetoxyborohydride (NaBH(OAc)3, 1.27 g, 6.0 mmol) in three portions over 10 minutes. The reaction mixture was heated to room temperature and stirred for 2 hours. After the reaction was complete, NaOH aqueous solution (1 M, 10 mL) was added to the reaction mixture. After stirring for 15 minutes, the mixture was diluted with water. The reaction mixture was extracted twice with dichloromethane (20 mL * 2). The organic phases were combined, washed with brine (30 mL), dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E4-1 (2.26 g, 93.6%). 1 H NMR(400MHz, CDCl3)δ:5.40-5.32(m,16H,-CH=CHCH2CH=CH-),4.25-4.18(m,2H,pip-CH2CH 2-),3.27(s,1H,-OC(=O)CH<),2.77(t,8H,-CH=CHCH2CH=CH-),2.65-2.35(m,20H;8H,-N(CH 2)2-;2H,>CH(CH2)3CH2-;2H,pip-CH2CH2-;8H,pip-H),2.33(s,3H,pip-CH3),2.07-2.02(m ,16H,-CH=CHCH2CH2-),1.71-1.21(m,70H,-CH2CH2CH2-,-CH2CH3),0.89(t,12H,-CH2CH3). MS(ESI):m / z=1209.2([M+H] + ).

[0245] Example 5: Cationic Lipids (E5-1)

[0246] The preparation process is as follows:

[0247] Step a: Under ice bath conditions, linolenic acid (S5-1, 2.39 g, 9.0 mmol), triethylamine (2.59 g, 25.7 mmol), and acryloyl chloride (S5-2, 1.23 g, 13.5 mmol) were added sequentially to a dichloromethane solution (70 mL). The reaction solution was stirred at 20 °C for 2 h. After the reaction was completed, the precipitated solid in the reaction solution was removed by filtration. The filtrate was washed sequentially with water and 5% hydrochloric acid. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain compound (9Z,12Z)-9,12-dieneoctadecyl acrylate (S5-3, 2.14 g).

[0248] Step b: Dissolve S1-3 (0.27 g, 1.0 mmol) in isopropanol, add sufficient anhydrous potassium carbonate while stirring, and stir at room temperature until the reaction solution becomes alkaline. Add S5-3 (1.93 g, 6.0 mmol) to the reaction solution, and place the reaction solution in a reflux apparatus (90 °C) and continue stirring for 36 h. After the reaction is complete, concentrate the reaction solution, and purify the crude product by column chromatography to obtain cationic lipid E5-1 (1.19 g). 1 H NMR(400MHz, CDCl3)δ:5.40-5.31(m,16H,-CH=CHCH2CH=CH-),4.25-4.19(m,2H,pip-CH2CH2-),4.05(t,8H,- C(=O)OCH2CH2CH2-),3.27(s,1H,-OC(=O)CH<),2.77(t,8H,-CH=CHCH2CH=CH-),2.65-2.35(m,20H;8H,-N(CH 2)2-;2H,>CH(CH2)3CH2-;2H,pip-CH2CH2-;8H,pip-H),2.34(s,3H,pip-CH3),2.32-2.27(m,8H,-CH2C(=O)O -), 2.06-2.02(m,16H,-CH=CHCH2CH2-), 1.71-1.21(m,78H,-CH2CH2CH2-,-CH2CH3), 0.87(t,12H,-CH2CH3). MS(ESI):m / z=1553.4([M+H] + ).

[0249] Example 6.1: Cationic Lipids (E6-1)

[0250] The preparation process is as follows:

[0251] Step a: Under nitrogen protection, DCC (3.63 g, 17.6 mmol) was added to a round-bottom flask containing S3-1 (1.55 g, 8.0 mmol) dissolved in dichloromethane (70 mL), undecyl alcohol (S6-1, 1.65 g, 9.6 mmol), and DMAP (0.24 g, 2.0 mmol). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the product was concentrated. The crude product was purified by column chromatography to obtain S6-2 (2.27 g).

[0252] Step b: Under nitrogen protection, compound S1-3 (0.27 g, 1.0 mmol) was dissolved in acetonitrile (30 mL), and S6-2 (2.09 g, 6.0 mmol) and DIPEA (0.65 g, 5.0 mmol) were added sequentially with slow stirring. The reaction was carried out at room temperature for 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, and extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E6-1 (1.11 g). 1 H NMR(400MHz, CDCl3)δ:4.26-4.17(m,2H,>CHC(=O)OCH2-),4.05(t,8H,-CH2C(=O)OC H2-),3.26(t,1H,>CHC(=O)O-),2.69-2.45(m,10H;8H,pip-H;2H,pip-CH2-),2.44-2 .32(m,10H;4H,>CHN(CH2)2-;6H,-CH2N(CH2)2-),2.32-2.24(m,11H;8H,-CH2C(=O)O -;3H,pip-CH3),1.67-1.19(m,84H,-CH2CH2CH2-,-CH2CH3),0.89(t,12H,-CH2CH3). MS(ESI):m / z=1346.9([M+H] + ).

[0253] Example 6.2: Cationic Lipids (E6-2)

[0254] The preparation process is as follows:

[0255] Step a: Under nitrogen protection, DCC (3.63 g, 17.6 mmol) was added to a round-bottom flask containing S3-1 (1.55 g, 8.0 mmol) dissolved in dichloromethane (80 mL), 1-nonanol (S6-3, 1.38 g, 9.6 mmol), and DMAP (0.24 g, 2.0 mmol). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the product was concentrated. The crude product was purified by column chromatography to obtain S6-4 (2.11 g).

[0256] Step b: Under nitrogen protection, compound S1-3 (0.27 g, 1.0 mmol) was dissolved in acetonitrile (30 mL), and S6-4 (1.93 g, 6.0 mmol) and DIPEA (0.65 g, 5.0 mmol) were added sequentially with slow stirring. The reaction was carried out at room temperature for 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, and extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E6-2 (1.01 g). 1 H NMR(400MHz, CDCl3)δ:4.25-4.18(m,2H,pip-CH2CH2-),4.05(t,8H,-C(=O)OCH2C H2CH2-),3.26(s,1H,-OC(=O)CH<),2.65-2.35(m,20H;8H,-N(CH2)2-;2H,>CH(CH 2)3CH2-;2H,pip-CH2CH2-;8H,pip-H),2.33(s,3H,pip-CH3),2.32-2.27(m,8H,- CH2C(=O)O-),1.72-1.21(m,86H,-CH2CH2CH2-,-CH2CH3),0.87(t,12H,-CH2CH3). MS(ESI): m / z = 1233.1([M+H]) + ).

[0257] Example 7: Cationic Lipids (E7-1)

[0258] The preparation process is as follows:

[0259] S1-3 (0.27 g, 1.0 mmol) was dissolved in isopropanol, and sufficient anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction solution became alkaline. Dodecyl 2-acrylate (S7-1, 1.44 g, 6.0 mmol) was added to the reaction solution, and the reaction solution was placed in a reflux apparatus (90 °C) and stirred for another 36 h. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E7-1 (0.95 g). 1 H NMR(400MHz, CDCl3)δ:4.25-4.18(m,2H,pip-CH2CH2-),4.06(t,8H,-C(=O)OCH2C H2CH2-),3.27(s,1H,-OC(=O)CH<),2.65-2.35(m,20H;8H,-N(CH2)2-;2H,>CH(CH 2)3CH2-;2H,pip-CH2CH2-;8H,pip-H),2.33(s,3H,pip-CH3),2.32-2.27(m,8H,- CH2C(=O)O-),1.71-1.21(m,86H,-CH2CH2CH2-,-CH2CH3),0.88(t,12H,-CH2CH3). MS(ESI): m / z = 1233.1([M+H]) + ).

[0260] Example 8: Cationic Lipids (E8-1)

[0261] The preparation process is as follows:

[0262] Step a: 5-Bromopentanol (S8-1, 0.84 g, 5.0 mmol) was dissolved in 50 mL of dichloromethane, DMAP (1.29 g, 10 mmol) was added, followed by the addition of phenyl p-nitrochloroformate (1.11 g, 5.5 mmol) in portions. The mixture was stirred at room temperature for 3 h. S6-1 (0.96 g, 5.6 mmol) was then added to the reaction mixture, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was diluted with dichloromethane and washed with saturated brine. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain 5-bromopentyl undecyl carbonate (S8-2, 1.32 g).

[0263] Step b: Under nitrogen protection, compound S1-3 (0.14 g, 0.5 mmol) was dissolved in acetonitrile (20 mL). S8-2 (1.10 g, 3.0 mmol) and DIPEA (0.32 g, 2.5 mmol) were added sequentially with slow stirring. The reaction mixture was stirred at room temperature for 20 h. After the reaction was complete, the reaction solution was concentrated, dissolved in dichloromethane, and extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E8-1 (0.58 g). 1 H NMR(400MHz, CDCl3)δ:4.24-4.18(m,2H,pip-CH2CH2-),4.15-4.10(m,16H,-CH2OC(=O)O-),3.27(s,1H,-OC(=O)CH<),2.65-2.35(m,20H;8H,-N(CH 2)2-;2H,>CH(CH2)3CH2-;2H,pip-CH2CH2-;8H,pip-H),2.33(s,3H,pip-CH3),1.71-1.21(m,102H,-CH2CH2CH2-,-CH2CH3),0.87(t,12H,-CH2CH3). MS(ESI):m / z=1409.2([M+H] + ).

[0264] Example 9: Cationic Lipids (E9-1)

[0265] The preparation process is as follows:

[0266] Step a: S6-1 (2.06 g, 12.0 mmol) was dissolved in dichloromethane (60 mL), and triethylamine (5 mL) and N,N'-carbonyldiimidazole (CDI, 1.94 g, 12.0 mmol) were added sequentially. The reaction was carried out at 50 °C for 1.5 h. Then 5-amino-1-pentanol (S9-1, 1.85 g, 18.0 mmol) was added, and the reaction was continued at 50 °C for 16 h. After the reaction was completed, the reaction solution was cooled to room temperature and washed sequentially with 5% citric acid (30 mL * 2) and saturated brine (30 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain the crude product. Dichloromethane (30 mL) was added to the crude product, stirred for 10 min, filtered, and the residue was washed with a small amount of dichloromethane and concentrated to obtain compound S9-2 (2.37 g).

[0267] Step b: S9-2 (1.81 g, 6.0 mmol) was dissolved in dichloromethane (80 mL), and triphenylphosphine (PPh3, 2.36 g, 9.0 mmol) was added. Carbon tetrabromide (CBr4, 2.98 g, 9.0 mmol) was added in portions under ice bath conditions, and the reaction was carried out for 20 min under ice bath conditions. After the reaction was complete, 10 mL of methanol was added to quench the reaction, and the product was directly concentrated to obtain the crude product. The crude product was purified by column chromatography to obtain product S9-3 (1.28 g).

[0268] Step c: Under nitrogen protection, compound S1-3 (0.14 g, 0.5 mmol) was dissolved in acetonitrile (20 mL). S9-3 (1.09 g, 3.0 mmol) and DIPEA (0.32 g, 2.5 mmol) were added sequentially with slow stirring. The reaction mixture was stirred at room temperature for 20 h. After the reaction was complete, the reaction solution was concentrated, dissolved in dichloromethane, and extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E9-1 (0.57 g). 1 H NMR(400MHz, CDCl3)δ:4.25-4.16(m,2H,pip-CH2CH2-),4.13-4.10(m,8H,-NHC(= O)OCH2-),3.27(s,1H,-OC(=O)CH<),3.15(t,2H,-CH2NHC(=O)O-),2.66-2.35(m, 20H;8H,-N(CH2)2-;2H,>CH(CH2)3CH2-;2H,pip-CH2CH2-;8H,pip-H),2.33(s,3H ,pip-CH3),1.71-1.21(m,102H,-CH2CH2CH2-,-CH2CH3),0.87(t,12H,-CH2CH3). MS(ESI): m / z = 1405.3 ([M+H]) + ).

[0269] Example 10.1: Cationic Lipids (E10-1)

[0270] The preparation process is as follows:

[0271] Step a: Under nitrogen protection, DCC (3.63 g, 17.6 mmol) was added to a round-bottom flask containing S10-1 (1.78 g, 8.0 mmol) dissolved in dichloromethane (60 mL), 4-octyne-1-ol (S10-2, 1.21 g, 9.6 mmol), and DMAP (0.24 g, 2.0 mmol). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the product was concentrated. The crude product was purified by column chromatography to obtain S10-3 (2.16 g).

[0272] Step b: Under nitrogen protection, compound S1-3 (0.27 g, 1.0 mmol) was dissolved in acetonitrile (40 mL). S10-3 (1.99 g, 6.0 mmol) and DIPEA (0.65 g, 5.0 mmol) were added sequentially with slow stirring. The reaction mixture was stirred at room temperature for 20 h. After the reaction was complete, the reaction solution was concentrated, dissolved in dichloromethane, and extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E10-1 (1.04 g). 1 H NMR(400MHz, CDCl3)δ:4.25-4.18(m,2H,pip-CH2CH2-),4.16(t,8H,-C(=O)OCH2CH2CH2-),3.27(t,1H,-OC(=O)CH<),2.65-2.35( m,20H;8H,-N(CH2)2-;2H,>CH(CH2)3CH2-;2H,pip-CH2CH2-;8H,pip-H),2.32-2.23(m,19H;16H,-CH2C≡CCH2-;3H,pip-CH3),2.14 -2.09(m,8H,-CH2C(=O)O-),1.83-1.77(m,8H,-C(=O)OCH2CH2CH2-),1.65-1.56(m,12H;8H,-N(CH2CH2)2-;4H,>CHCH2CH2CH2-), 1.53-1.46(m,10H;8H,-CH2CH2C(=O)O-;2H,>CHCH2CH2CH2-), 1.37-1.21(m,32H,-CH2CH2CH2-,-CH2CH3), 0.96(t,12H,-CH2CH3). MS(ESI):m / z=1273.9([M+H] + ).

[0273] Example 10.2: Cationic Lipids (E10-2)

[0274] The preparation process is as follows:

[0275] Step a: Under nitrogen protection, DCC (3.63 g, 17.6 mmol) was added to a round-bottom flask containing S10-1 (1.78 g, 8.0 mmol) dissolved in dichloromethane (80 mL), 4-decyn-1-ol (S10-4, 1.48 g, 9.6 mmol), and DMAP (0.24 g, 2.0 mmol). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the product was concentrated. The crude product was purified by column chromatography to obtain S10-5 (2.36 g).

[0276] Step b: Under nitrogen protection, compound S1-3 (0.27 g, 1.0 mmol) was dissolved in acetonitrile (40 mL). S10-5 (2.15 g, 6.0 mmol) and DIPEA (0.65 g, 5.0 mmol) were added sequentially with slow stirring. The reaction mixture was stirred at room temperature for 20 h. After the reaction was complete, the reaction solution was concentrated, dissolved in dichloromethane, and extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E10-2 (1.13 g). 1 H NMR(400MHz, CDCl3)δ:4.26-4.18(m,2H,pip-CH2CH2-),4.16(t,8H,-C(=O)OCH2CH2CH2-),3.26(t, 1H,-OC(=O)CH<),2.67-2.36(m,20H; 8H,-N(CH2)2-; 2H,>CH(CH2)3CH2-; 2H,pip-CH2CH2-; 8H,pip- H),2.31-2.23(m,19H;16H,-CH2C≡CCH2-;3H,pip-CH3),2.18-2.09(m,8H,-CH2C(=O)O-),1.85-1.7 7(m,8H,-C(=O)OCH2CH2CH2-),1.65-1.21(m,54H,-CH2CH2CH2-,-CH2CH3),0.91(t,12H,-CH2CH3). MS(ESI):m / z=1385.0([M+H] + ).

[0277] Example 11: Cationic Lipids (E11-1)

[0278] The preparation process is as follows:

[0279] Step a: S1-3 (2.38 g, 6.6 mmol) was dissolved in dichloromethane (50 mL), and then DCC (1.36 g, 6.6 mmol) was slowly added. After stirring for 30 min, DMAP (0.07 g, 0.6 mmol) and S25-5 (1.03 g, 6.0 mmol) were added sequentially, and the reaction was continued with stirring for 2 h. After the reaction was completed, the mixture was filtered and concentrated to obtain the crude product. In a dry and clean round-bottom flask, a trifluoroacetic acid / dichloromethane (1:2, v / v) solution was prepared. The dichloromethane solution of the crude product was slowly added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the mixture was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S11-2 (1.42 g).

[0280] Step b: Under ice bath conditions, S4-1 (3.17 g, 12.0 mmol), DL-proline (0.41 g, 3.6 mmol), and N-chlorosuccinimide were added to acetonitrile (50 mL), and the mixture was stirred at 0 °C for 2 h. After the reaction was complete, the reaction solution was diluted with anhydrous ethanol (30 mL), and sodium borohydride (0.53 g, 16.8 mmol) was added. The reaction was then stirred at 0 °C for another 4 h. After the reaction was complete, the reaction solution was quenched with water (10 mL), extracted with methyl tert-butyl ether, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain compound (9Z,12Z)-2-chloro-octadec-9,12-dien-1-ol (S11-3, 3.39 g), which was used directly in the next step.

[0281] Step c: At room temperature, S11-3 (3.01 g, 10.0 mmol) and NaOH aqueous solution (5 M, 20 mL) were added to 1,4-dioxane (20 mL), and the reaction mixture was stirred at 35 °C for 2 h. After the reaction was completed, the reaction mixture was separated by a separatory funnel, the organic phase was washed with saturated brine, dried with anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain S11-4 (1.95 g).

[0282] Step d: S11-2 (0.30 g, 1.0 mmol) and DIPEA (0.39 g, 3.0 mmol) were added sequentially to a methanol solution of S11-4 (1.58 g, 6.0 mmol), and the reaction mixture was placed in a reflux apparatus (90 °C) and stirred for 24 h. After the reaction was completed, the reaction mixture was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E11-1 (1.10 g). 1H NMR(400MHz, CDCl3)δ:5.40-5.31(m,16H,-CH=CHCH2CH=CH-),3.78-3.63(m,5H;4H,-CH(OH)-;1H, -NHC(=O)CH<),3.47-3.29(m,2H,-CH2NHC(=O)-),3.26-3.06(m,2H,>CH(CH2)3CH2-),2.77(t,8H,- CH=CHCH2CH=CH-),2.67-2.36(m,12H;4H,pip-CH2CH2-;8H,pip-H),2.14-2.02(m,24H;8H,-CH(OH )CH2N<;16H,-CH=CHCH2CH2-),1.73-1.21(m,75H,-CH2CH2CH2-,-CH2CH3),0.87(t,12H,-CH2CH3). MS(ESI):m / z=1356.3([M+H] + ).

[0283] Example 12.1: Cationic Lipid (E12-1)

[0284] The preparation process is as follows:

[0285] S11-2 (0.30 g, 1.0 mmol) was dissolved in isopropanol, and sufficient anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction solution became alkaline. S7-1 (1.44 g, 6.0 mmol) was then added to the reaction solution, and the reaction solution was placed in a reflux apparatus (90 °C) and stirred for another 36 h. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E12-1 (0.99 g). 1 H NMR(400MHz, CDCl3)δ:4.05(t,8H,-C(=O)OCH2CH2CH2-),3.77-3.63(m,1H,-N HC(=O)CH<),3.47-3.29(m,2H,-CH2NHC(=O)-),2.65-2.35(m,22H;8H,-N(CH2 )2-;2H,>CH(CH2)3CH2-;4H,pip-CH2CH2-;8H,pip-H),2.32-2.27(m,8H,-CH2C(=O)O-),1.73-1.21(m,91H,-CH2CH2CH2-,-CH2CH3),0.90(t,12H,-CH2CH3). MS(ESI): m / z = 1260.8 ([M+H]) + ).

[0286] Example 12.2: Cationic Lipids (E12-2)

[0287] The preparation process is as follows:

[0288] S11-2 (0.30 g, 1.0 mmol) was dissolved in isopropanol, and sufficient anhydrous potassium carbonate was added with stirring. The mixture was stirred at room temperature until the reaction solution became alkaline. N-Dodecylacrylamide (S12-1, 1.43 g, 6.0 mmol) was added to the reaction solution, and the reaction solution was placed in a reflux apparatus (90 °C) and stirred for another 36 h. After the reaction was completed, the reaction solution was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E12-2 (0.99 g). 1 H NMR (400MHz, CDCl3) δ: 3.78-3.63(m,1H,-NHC(=O)CH<), 3.47-3.29(m,2H,-CH2NHC(=O)-), 3.28(dd,8H,-CH2C(=O)NHCH2-), 2.82-2.35(m,30H; 8H,- N(CH2)2-;2H,>CH(CH2)3CH2-;4H,pip-CH2CH2-;8H,pip-H;8H,-CH2C(=O)NH-),1.70-1.21(m,91H,-CH2CH2CH2-,-CH2CH3),0.87(t,12H,-CH2CH3). MS(ESI):m / z=1256.2([M+H] + ).

[0289] Example 13: Cationic Lipids (E13-1)

[0290] The preparation process is as follows:

[0291] Step a: Glycerol (S13-1, 0.74 g, 8.0 mmol) was adsorbed onto an equal weight of silica gel by vigorous stirring. Then, nonanoic acid (S13-2, 2.53 g, 16.0 mmol), immobilized lipase R. miehei (0.20 g), and molecular sieve (0.55 g) were added sequentially to the formulation. The mixture was then suspended in diethyl ether (50 mL) and stirred at room temperature for 48 h. After the reaction was completed, the lipase and silica gel were separated by filtration, concentrated, and the crude product was recrystallized in methanol to obtain compound S13-3 (2.42 g).

[0292] Step b: Under nitrogen protection, DCC (1.85 g, 9.0 mmol) was added to a round-bottom flask containing S13-3 (1.87 g, 5.0 mmol), S13-4 (0.49 g, 2.0 mmol), and DMAP (0.12 g, 1.0 mmol) dissolved in dichloromethane (50 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the solution was concentrated to obtain the crude product. In a dry and clean round-bottom flask, a solution of trifluoroacetic acid / dichloromethane (1:2, v / v) was prepared, and the dichloromethane solution of the crude product was slowly added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S13-5 (1.31 g).

[0293] Step c: S13-5 (0.94 g, 1.1 mmol) was dissolved in dichloromethane (20 mL), and then DCC (0.23 g, 1.1 mmol) was slowly added. After stirring for 30 min, DMAP (0.01 g, 0.1 mmol) and S13-6 (0.16 g, 1.0 mmol) were added sequentially, and the reaction was continued for 2 h. After the reaction was completed, the mixture was filtered, concentrated, and the crude product was dissolved in dichloromethane, washed once with water, and extracted twice with dichloromethane (10 mL * 2). The organic phases were combined and dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E13-1 (0.79 g). 1 HNMR(400MHz, CDCl3)δ:5.38-5.31(m,2H,-C(=O)OCH<),4.65-4.58(m,1H,-C(=O)NHCH<),4.38-4.29(m,4H,-CH(CH2O C(=O))2-),4.19-4.13(m,4H,-CH(CH2OC(=O))2-),2.91-2.82(m,2H,-CH2C(=O)NH-),2.72-2.52(m,4H,-N(CH2)2-),2 .41-2.38(m,2H,>CHCH2CH2C(=O)O-),2.35(s,3H,>NCH3),2.31-2.27(m,8H,-CH2C(=O)O-),2.02-1.97(m,2H,>CHCH2C H2C(=O)O-), 1.83-1.73(m,1H,-NHC(=O)CH2CH<), 1.71-1.21(m,52H,-CH2CH2CH2-,-CH2CH3), 0.86(t,12H,-CH2CH3). MS(ESI):m / z=994.7([M+H] + ).

[0294] Example 14: Cationic Lipids (E14-1)

[0295] The preparation process is as follows:

[0296] Step a: Freshly activated magnesium granules (0.66 g, 27.6 mmol) and 5 mL of anhydrous diethyl ether were added sequentially to a clean round-bottom flask. 9-Bromo-1-nonene bromide (S14-1, 4.55 g, 22.2 mmol) was dissolved in 30 mL of anhydrous diethyl ether. This bromide-ether solution was then added dropwise to the magnesium granules under ice bath conditions. After the addition was complete, the reaction mixture was allowed to react at 35 °C for 1 h, and then cooled in an ice bath. Ethyl formate (0.74 g, 10.0 mmol) was mixed with 10 mL of anhydrous diethyl ether and slowly added dropwise to the reaction mixture with stirring. After the reaction began to reflux, the remaining ethyl formate ether solution was quickly added, and the reaction mixture was stirred again at room temperature for 1 h. Then, 5 mL of acetone was added dropwise, followed by quenching with ice water (10 mL). The reaction mixture was treated with 10% (v / v) hydrated H2SO4 (50 mL) until the solution became homogeneous. The aqueous phase was extracted with diethyl ether (30 mL * 2), the organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain S14-2 (2.52 g).

[0297] Step b: S14-2 (1.96 g, 7.0 mmol) was dissolved in a mixed solution of dichloromethane (15 mL) and acetonitrile (15 mL), and then ruthenium chloride (RuCl3, 0.26 g, 1.2 mmol) was added. The mixture was cooled to 10 °C, and an aqueous solution of sodium periodate (0.15 g, 0.7 mmol) was added dropwise. The mixture was stirred at 10 °C for 20 h. After the reaction was complete, the reaction solution was diluted with water, and the organic and aqueous phases were separated. Saturated brine was added to the organic layer with stirring, and then 3% sodium sulfide solution was added dropwise for decolorization. The organic and aqueous phases were separated, and the organic phase was dried with anhydrous magnesium sulfate, filtered, and concentrated to obtain compound S14-3 (1.90 g).

[0298] Step c: Under a nitrogen atmosphere, tert-butyldimethylchlorosilane (TBDMSCl, 0.83 g, 5.5 mmol) was added to a round-bottom flask containing S14-3 (1.58 g, 5.0 mmol) dissolved in DMF (30 mL) and imidazole (0.85 g, 12.5 mmol). The reaction mixture was stirred overnight at 50 °C. After the reaction was completed, the mixture was cooled to room temperature, diluted with water, extracted three times with ethyl acetate, the organic phases were combined and washed once with saturated brine, the organic layer was dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain a diacid with hydroxyl groups protected by TBS (S14-4, 1.96 g).

[0299] Step d: Under nitrogen protection, DCC (3.71 g, 18.0 mmol) was added to a round-bottom flask containing 2-nonen-1-ol (S14-5, 1.42 g, 10.0 mmol), S14-4 (1.72 g, 4.0 mmol), and DMAP (0.24 g, 2.0 mmol) dissolved in dichloromethane (70 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the filtrate was concentrated. The residue was dissolved in tetrahydrofuran (20 mL), and then 20 mL of TBAF tetrahydrofuran solution (1 M) was added. The reaction was carried out overnight to remove TBS protection. After the reaction was completed, the mixture was concentrated and extracted. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to give compound S14-6 (1.73 g).

[0300] Step e: Under nitrogen protection, DCC (1.02 g, 5.0 mmol) was added to a round-bottom flask containing S14-6 (1.55 g, 2.8 mmol), S13-4 (0.27 g, 1.1 mmol), and DMAP (0.07 g, 0.6 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, and the solution was concentrated to obtain the crude product. In a dry and clean round-bottom flask, a solution of trifluoroacetic acid / dichloromethane (1:2, v / v) was prepared, and the dichloromethane solution of the crude product was slowly added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S14-7 (1.04 g).

[0301] Step f: S14-7 (0.82 g, 0.7 mmol) was dissolved in dichloromethane (15 mL), and then DCC (0.14 g, 0.7 mmol) was slowly added. After stirring for 30 min, DMAP (0.01 g, 0.1 mmol) and S13-6 (0.09 g, 0.6 mmol) were added sequentially, and the reaction was continued to be stirred for 2 h. After the reaction was completed, the mixture was filtered, concentrated, and the crude product was dissolved in dichloromethane, washed once with water, and then extracted twice with dichloromethane (10 mL * 2). The organic phases were combined and dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E14-1 (0.66 g). 1H NMR (400MHz, CDCl3) δ: 5.82-5.71(m,4H,-C(=O)OCH2CH=CH-), 5.56-5.54(m,4H,-C(=O)OCH2CH=CH-), 4.87(m,2H,-C(=O)OCH<), 4. 65-4.58(m,1H,-C(=O)NHCH<),4.55(t,8H,-C(=O)OCH2CH=CH-),2.91-2.82(m,2H,-CH2C(=O)NH-),2.72-2.52(m,4H,-N(CH2)2-),2 .40-2.39(m,2H,>CHCH2CH2C(=O)O-),2.35(s,3H,>NCH3),2.32-2.25(m,8H,-CH2C(=O)O-),2.06-1.97(m,10H;8H,-CH=CHCH2CH2- ;2H,>CHCH2CH2C(=O)O-),1.83-1.73(m,1H,-NHC(=O)CH2CH<),1.71-1.21(m,84H,-CH2CH2CH2-,-CH2CH3),0.91(t,12H,-CH2CH3). MS(ESI):m / z=1379.1([M+H] + ).

[0302] Example 14.2: Cationic Lipids (E14-2)

[0303] The preparation process is as follows:

[0304] Step a: Under nitrogen protection, DCC (3.71 g, 18.0 mmol) was added to a round-bottom flask containing S6-3 (1.44 g, 10.0 mmol), S14-4 (1.72 g, 4.0 mmol), and DMAP (0.24 g, 2.0 mmol) dissolved in dichloromethane (60 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the filtrate was concentrated. The residue was dissolved in tetrahydrofuran (20 mL), and then 20 mL of TBAF tetrahydrofuran solution (1 M) was added. The reaction was carried out overnight to remove TBS protection. After the reaction was completed, the mixture was concentrated and extracted. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain compound S14-8 (1.76 g).

[0305] Step b: Under nitrogen protection, DCC (1.02 g, 5.0 mmol) was added to a round-bottom flask containing S14-8 (1.56 g, 2.8 mmol), S13-4 (0.27 g, 1.1 mmol), and DMAP (0.07 g, 0.6 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, and the solution was concentrated to obtain the crude product. In a dry and clean round-bottom flask, a solution of trifluoroacetic acid / dichloromethane (1:2, v / v) was prepared, and the dichloromethane solution of the crude product was slowly added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S14-9 (1.06 g).

[0306] Step c: S14-9 (0.82 g, 0.7 mmol) was dissolved in dichloromethane (15 mL), and then DCC (0.14 g, 0.7 mmol) was slowly added. After stirring for 30 min, DMAP (0.01 g, 0.1 mmol) and S13-6 (0.09 g, 0.6 mmol) were added sequentially, and the reaction was continued for 2 h. After the reaction was completed, the mixture was filtered, concentrated, and the crude product was dissolved in dichloromethane, washed once with water, and then extracted twice with dichloromethane (10 mL * 2). The organic phases were combined and dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E14-2 (0.66 g). 1 H NMR(400MHz, CDCl3)δ:4.87(m,2H,-C(=O)OCH<),4.65-4.56(m,1H,-C(=O)NHCH<),4.05(t,8H,-C(=O)OCH2CH2 CH2-),2.91-2.82(m,2H,-CH2C(=O)NH-),2.72-2.52(m,4H,-N(CH2)2-),2.40-2.38(m,2H,>CHCH2CH2C(=O)O- ),2.35(s,3H,>NCH3),2.32-2.27(m,8H,-CH2C(=O)O-),2.13-1.97(m,4H;2H,-N(CH2)2-;2H,>CHCH2CH2C(=O) O-),1.83-1.73(m,1H,-NHC(=O)CH2CH<),1.70-1.21(m,108H,-CH2CH2CH2-,-CH2CH3),0.89(t,12H,-CH2CH3). MS(ESI):m / z=1387.2([M+H] + ).

[0307] Example 15: Cationic Lipids (E15-1)

[0308] The preparation process is as follows:

[0309] Step a: Under nitrogen protection, DCC (3.71 g, 18.0 mmol) was added to a round-bottom flask containing 3-decyn-1-ol (S15-1, 1.54 g, 10.0 mmol), S14-4 (1.72 g, 4.0 mmol), and DMAP (0.24 g, 2.0 mmol) dissolved in dichloromethane (60 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the filtrate was concentrated. The residue was dissolved in tetrahydrofuran (20 mL), and then 20 mL of TBAF tetrahydrofuran solution (1 M) was added. The reaction was carried out overnight to remove TBS protection. After the reaction was completed, the mixture was concentrated and extracted. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain compound S15-2 (1.84 g).

[0310] Step b: Under nitrogen protection, DCC (1.02 g, 5.0 mmol) was added to a round-bottom flask containing S15-2 (1.62 g, 2.8 mmol), S13-4 (0.27 g, 1.1 mmol), and DMAP (0.07 g, 0.6 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, and the solution was concentrated to obtain the crude product. In a dry and clean round-bottom flask, a solution of trifluoroacetic acid / dichloromethane (1:2, v / v) was prepared, and the dichloromethane solution of the crude product was slowly added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S15-3 (1.10 g).

[0311] Step c: S15-3 (0.85 g, 0.7 mmol) was dissolved in dichloromethane (15 mL), and then DCC (0.14 g, 0.7 mmol) was slowly added. After stirring for 30 min, DMAP (0.01 g, 0.1 mmol) and S13-6 (0.09 g, 0.6 mmol) were added sequentially, and the reaction was continued for 2 h. After the reaction was completed, the mixture was filtered, concentrated, and the crude product was dissolved in dichloromethane, washed once with water, and then extracted twice with dichloromethane (10 mL * 2). The organic phases were combined and dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E15-1 (0.69 g). 1H NMR(400MHz, CDCl3)δ:4.87(m,2H,-C(=O)OCH<),4.65-4.58(m,1H,-C(=O)NHCH<),4.05(t,8H,-C(=O)OCH2CH 2CH2-),2.91-2.82(m,2H,-CH2C(=O)NH-),2.72-2.52(m,4H,-N(CH2)2-),2.40-2.38(m,2H,>CHCH2CH2C(=O) O-),2.32-2.23(m,27H;16H,-CH2C≡CCH2-;3H,>NCH3;8H,-CH2C(=O)O-),2.01-1.97(m,2H,>CHCH2CH2C(=O)O -),1.85-1.74(m,1H,-NHC(=O)CH2CH<),1.71-1.21(m,84H,-CH2CH2CH2-,-CH2CH3),0.87(t,12H,-CH2CH3). MS(ESI):m / z=1427.1([M+H] + ).

[0312] Example 16: Cationic Lipids (E16-1)

[0313] The preparation process is as follows:

[0314] Step a: Under ice bath conditions, TBDMSCl (2.72 g, 18.0 mmol) and imidazole (2.04 g, 30.0 mmol) were added sequentially to a round-bottom flask containing diethyl 3-hydroxyglutarate (S16-1, 2.64 g, 15.0 mmol) dissolved in dichloromethane (80 mL). The reaction mixture was brought to room temperature and stirred for 17 h. After the reaction was completed, the mixture was filtered, the filtrate was concentrated, and the crude product was purified by column chromatography to obtain compound S16-2 (3.00 g).

[0315] Step b: Under ice bath conditions, a THF solution (1M, 7.2mL, 7.2mmol) of diisobutylaluminum hydride (DIBAL-H) ​​was added to a round-bottom flask containing S16-2 (2.61g, 9.0mmol) dissolved in dichloromethane (30mL). The reaction mixture was brought to room temperature and stirred for 17h. After the reaction was completed, the reaction was quenched with MeOH, filtered, and the filtrate was concentrated. The crude product was purified by column chromatography to obtain compound S16-3 (1.22g).

[0316] Step c: Under nitrogen protection, DCC (3.71 g, 18.0 mmol) was added to a round-bottom flask containing S16-4 (2.80 g, 10.0 mmol), S16-3 (0.94 g, 4.0 mmol), and DMAP (0.24 g, 2.0 mmol) dissolved in dichloromethane (80 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the filtrate was concentrated. The residue was dissolved in tetrahydrofuran (20 mL), and then 20 mL of TBAF tetrahydrofuran solution (1 M) was added. The reaction was carried out overnight to remove TBS protection. After the reaction was completed, the mixture was concentrated and extracted. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain compound S16-5 (2.03 g).

[0317] Step d: Under nitrogen protection, DCC (1.02 g, 5.0 mmol) was added to a round-bottom flask containing S16-5 (1.77 g, 2.8 mmol), S13-4 (0.27 g, 1.1 mmol), and DMAP (0.07 g, 0.6 mmol) dissolved in dichloromethane (40 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the solution was concentrated to obtain the crude product. In a dry and clean round-bottom flask, a solution of trifluoroacetic acid / dichloromethane (1:2, v / v) was prepared, and the dichloromethane solution of the crude product was slowly added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S16-6 (1.20 g).

[0318] Step e: S16-6 (0.92 g, 0.7 mmol) was dissolved in dichloromethane (15 mL), and then DCC (0.14 g, 0.7 mmol) was slowly added. After stirring for 30 min, DMAP (0.01 g, 0.1 mmol) and S13-6 (0.09 g, 0.6 mmol) were added sequentially, and the reaction was continued for 2 h. After the reaction was completed, the mixture was filtered, concentrated, and the crude product was dissolved in dichloromethane, washed once with water, and then extracted twice with dichloromethane (10 mL * 2). The organic phases were combined and dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E16-1 (0.72 g). 1H NMR(400MHz, CDCl3)δ:5.46-5.29(m,16H,-CH=CH-),5.17-5.08(m,2H,-C(=O)OCH<),4.65-4.58(m,1H,-C(=O)NHCH<),4.19-4.01(m,8H,-C (=O)OCH2-),2.91-2.82(m,2H,-CH2C(=O)NH-),2.77(t,8H,-CH=CHCH2CH=CH-),2.72-2.52(m,4H,-N(CH2)2-),2.40-2.38(m,2H,>CHCH2CH2 C(=O)O-),2.35(s,3H,>NCH3),2.32-2.27(m,8H,>CH(CH2)2OC(=O)CH2-),2.10-1.97(m,18H;16H,-CH=CHCH2-;2H,>CHCH2CH2C(=O)O-),1. 97-1.89(m,8H,-C(=O)OCH2CH2-), 1.83-1.73(m,1H,-NHC(=O)CH2CH<), 1.71-1.19(m,68H,-CH2CH2CH2-,-CH2CH3), 0.87(t,12H,-CH2CH3). MS(ESI):m / z=1539.2([M+H] + ).

[0319] Example 17: Cationic Lipids (E17-1)

[0320] The preparation process is as follows:

[0321] S16-6 (1.54 g, 1.1 mmol) was dissolved in dichloromethane (20 mL), and DCC (0.23 g, 1.1 mmol) was slowly added. After stirring for 30 min, DMAP (0.01 g, 0.1 mmol) and 4-(4-methyl-1-piperazinyl)butyric acid (S17-1, 0.19 g, 1.0 mmol) were added sequentially, and the reaction was continued for 2 h. After the reaction was completed, the mixture was filtered, concentrated, and the crude product was dissolved in dichloromethane, washed once with water, and extracted twice with dichloromethane (10 mL * 2). The organic phases were combined and dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E17-1 (1.23 g). 1H NMR(400MHz, CDCl3)δ:5.46-5.28(m,16H,-CH=CH-),5.17-5.09(m,2H,-C(=O)OCH<),4.65-4.58(m,1H,-C(=O)NHCH<),4.19-4.02(m,8H,-C (=O)OCH2-),2.91-2.81(m,2H,-CH2C(=O)NH-),2.77(t,8H,-CH=CHCH2CH=CH-),2.67-2.36(m,12H;2H,>CHCH2CH2C(=O)O-;2H,pip-CH2CH2 -;8H,pip-H),2.35(s,3H,>NCH3),2.31-2.27(m,8H,>CH(CH2)2OC(=O)CH2-),2.10-1.97(m,18H;16H,-CH=CHCH2-;2H,>CHCH2CH2C(=O)O-) ,1.97-1.89(m,8H,-C(=O)OCH2CH2-),1.83-1.80(m,2H,pip-CH2CH2-),1.71-1.19(m,64H,-CH2CH2CH2-,-CH2CH3),0.87(t,12H,-CH2CH3). MS(ESI):m / z=1568.2([M+H] + ).

[0322] Example 18: Cationic Lipids (E18-1)

[0323] The cationic lipid E18-1 (1.17g) was prepared by replacing raw material S16-6 in Example 17 with raw material S15-3 (1.42g, 1.1mmol) and following the same reaction steps. 1H NMR(400MHz, CDCl3)δ:4.87(m,2H,-C(=O)OCH<),4.65-4.58(m,1H,-C(=O)NHCH<),4.05(t,8H,-C(=O)OCH2C H2CH2-),2.91-2.82(m,2H,-CH2C(=O)NH-),2.67-2.36(m,12H;2H,>CHCH2CH2C(=O)O-;2H,pip-CH2CH2-;8H, pip-H),2.33-2.23(m,27H;16H,-CH2C≡CCH2-;3H,>NCH3;8H,-CH2C(=O)O-),2.00-1.96(m,2H,>CHCH2CH2C(= O)O-),1.83-1.80(m,2H,pip-CH2CH2-),1.71-1.21(m,80H,-CH2CH2CH2-,-CH2CH3),0.87(t,12H,-CH2CH3). MS(ESI):m / z=1456.1([M+H] + ).

[0324] Example 19.1: Cationic Lipids (E19-1)

[0325] The raw material S16-6 in Example 17 was replaced with raw material S14-7 (1.37 g, 1.1 mmol) and prepared according to the same reaction steps to obtain cationic lipid E19-1 (1.12 g). 1H NMR (400MHz, CDCl3) δ: 5.82-5.71(m,4H,-C(=O)OCH2CH=CH-), 5.56-5.54(m,4H,-C(=O)OCH2CH=CH-), 4.87(m,2H,-C(=O)OCH<), 4. 65-4.58(m,1H,-C(=O)NHCH<),4.55(t,8H,-C(=O)OCH2CH=CH-),2.91-2.82(m,2H,-CH2C(=O)NH-),2.67-2.36(m,12H;2H,>CHCH2C H2C(=O)O-;2H,pip-CH2CH2-;8H,pip-H),2.35(s,3H,>NCH3),2.32-2.25(m,8H,-CH2C(=O)O-),2.06-1.97(m,10H;8H,-CH=CHCH2C H2-;2H,>CHCH2CH2C(=O)O-),1.83-1.80(m,2H,pip-CH2CH2-),1.71-1.21(m,80H,-CH2CH2CH2-,-CH2CH3),0.91(t,12H,-CH2CH3). MS(ESI):m / z=1408.1([M+H] + ).

[0326] Example 19.2: Cationic Lipids (E19-2)

[0327] The cationic lipid E19-2 (1.13g) was prepared by replacing raw material S16-6 in Example 17 with raw material S14-9 (1.37g, 1.1mmol) and following the same reaction steps. 1H NMR(400MHz, CDCl3)δ:4.87(m,2H,-C(=O)OCH<),4.65-4.56(m,1H,-C(=O)NHCH<),4.05(t,8H,-C(=O)OCH 2CH2CH2-),2.91-2.82(m,2H,-CH2C(=O)NH-),2.67-2.36(m,12H;2H,>CHCH2CH2C(=O)O-;2H,pip-CH2CH2 -;8H,pip-H),2.34(s,3H,>NCH3),2.32-2.27(m,8H,-CH2C(=O)O-),2.13-1.97(m,2H,>CHCH2CH2C(=O)O- ),1.83-1.80(m,2H,pip-CH2CH2-),1.70-1.21(m,104H,-CH2CH2CH2-,-CH2CH3),0.89(t,12H,-CH2CH3). MS(ESI):m / z=1416.2([M+H] + ).

[0328] Example 20: Cationic Lipids (E20-1)

[0329] The preparation process is as follows:

[0330] Step a: Under nitrogen protection, DCC (1.81 g, 8.8 mmol) was added to a round-bottom flask containing S1-1 (1.38 g, 4.0 mmol) dissolved in dichloromethane (50 mL), 1-methyl-4-(hydroxyethyl)piperidine (S20-1, 0.69 g, 4.8 mmol), and DMAP (0.12 g, 1.0 mmol). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the solution was concentrated to obtain the crude product. In a dry and clean round-bottom flask, a solution of trifluoroacetic acid / dichloromethane (1:2, v / v) was prepared, and the dichloromethane solution of the crude product was slowly added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S20-2 (0.86 g).

[0331] Step b: S20-2 (0.41 g, 1.5 mmol) and DIPEA (0.58 g, 4.5 mmol) were added sequentially to a methanol solution of S1-4 (1.91 g, 9.0 mmol), and the reaction mixture was placed in a reflux apparatus (90 °C) and stirred for 24 h. After the reaction was completed, the reaction mixture was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E20-1 (1.32 g). 1 H NMR (400MHz, CDCl3) δ: 4.09-4.00(m,2H,-C(=O)OCH2-), 3.78-3.63(m,4H,-CH(OH)-), 3.27(s,1H,-OC(=O)CH<), 2.72-2.35(m,6H; 4H,-N(CH2)2-; 2H,-CH(CH2)3CH2-),2.32(s,3H,>NCH3),2.14-1.90(m,8H,-CH(OH)CH2N<),1.70-1.21(m,101H,-CH2CH2CH2-,-CH2CH3),0.90(t,12H,-CH2CH3). MS(ESI):m / z=1121.1([M+H] + ).

[0332] Example 21: Cationic Lipids (E21-1)

[0333] The preparation process is as follows:

[0334] Step a: Under nitrogen protection, DCC (1.81 g, 8.8 mmol) was added to a round-bottom flask containing S1-1 (1.38 g, 4.0 mmol), S21-1 (0.55 g, 4.8 mmol), and DMAP (0.12 g, 1.0 mmol) dissolved in dichloromethane (50 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the solution was concentrated to obtain the crude product. In a dry and clean round-bottom flask, a solution of trifluoroacetic acid / dichloromethane (1:2, v / v) was prepared, and the dichloromethane solution of the crude product was slowly added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S21-2 (0.78 g).

[0335] Step b: S21-2 (0.36 g, 1.5 mmol) and DIPEA (0.58 g, 4.5 mmol) were added sequentially to a methanol solution of S1-4 (1.91 g, 9.0 mmol), and the reaction mixture was placed in a reflux apparatus (90 °C) and stirred for 24 h. After the reaction was completed, the reaction mixture was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E21-1 (1.31 g). 1 H NMR (400MHz, CDCl3) δ: 4.25-4.18(m,2H,-C(=O)OCH2-), 3.78-3.63(m,4H,-CH(OH)-), 3.27(s,1H,-OC(=O)CH<), 2.86(t,2H,-C(=O)OCH2CH2-), 2.65-2. 35(m,6H;2H,>CH(CH2)3CH2-;4H,-N(CH2CH2)2-),2.14-1.90(m,8H,-CH(OH)CH2N<),1.73-1.21(m,98H,-CH2CH2CH2-,-CH2CH3),0.88(t,12H,-CH2CH3). MS(ESI):m / z=1092.1([M+H] + ).

[0336] Example 22: Cationic Lipid (E22-1)

[0337] The preparation process is as follows:

[0338] Step a: Under nitrogen protection, DCC (1.85 g, 9.0 mmol) was added to a round-bottom flask containing S13-3 (0.49 g, 2.0 mmol), S18-3 (1.17 g, 5.0 mmol), and DMAP (0.12 g, 1.0 mmol) dissolved in dichloromethane (40 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration, and the solution was concentrated to obtain the crude product. In a dry and clean round-bottom flask, a solution of trifluoroacetic acid / dichloromethane (1:2, v / v) was prepared, and the dichloromethane solution of the crude product was slowly added dropwise under ice bath conditions. The reaction was carried out at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S22-1 (1.28 g).

[0339] Step b: S22-1 (0.93 g, 1.1 mmol) was dissolved in dichloromethane (20 mL), and then DCC (0.23 g, 1.1 mmol) was slowly added. After stirring for 30 min, DMAP (0.01 g, 0.1 mmol) and S13-6 (0.16 g, 1.0 mmol) were added sequentially, and the reaction was continued for 2 h. After the reaction was completed, the mixture was filtered, concentrated, and the crude product was dissolved in dichloromethane, washed once with water, and then extracted twice with dichloromethane (10 mL * 2). The organic phases were combined and dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E22-1 (0.78 g). 1 H NMR (400MHz, CDCl3) δ: 5.37-5.32(m,2H,-C(=O)OCH<), 4.86-4.84(m,1H,-C(=O)NHCH<), 4.38-4.29(m,4 H,-CH(CH2OC(=O))2-),4.19-4.13(m,4H,-CH(CH2OC(=O))2-),3.03-2.82(m,4H;2H,>CHCH2C(=O)O-;2H ,-CH2C(=O)NH-),2.72-2.52(m,4H,-N(CH2)2-),2.35(s,3H,>NCH3),2.31-2.26(m,8H,-CH2C(=O)O-),1 .83-1.73(m,1H,-NHC(=O)CH2CH<), 1.71-1.21(m,52H,-CH2CH2CH2-,-CH2CH3), 0.87(t,12H,-CH2CH3). MS(ESI):m / z=980.7([M+H] + ).

[0340] Example 23: Cationic Lipids (E23-1)

[0341] The cationic lipid E23-1 (0.80 g) was prepared by replacing raw material S16-6 in Example 17 with raw material S13-5 (0.94 g, 1.1 mmol) and following the same reaction steps. 1H NMR(400MHz, CDCl3)δ:5.37-5.31(m,2H,-C(=O)OCH<),4.65-4.58(m,1H,-C(=O)NHCH<),4.38-4.29(m,4H,-CH(CH2OC (=O))2-),4.19-4.13(m,4H,-CH(CH2OC(=O))2-),2.91-2.82(m,2H,-CH2C(=O)NH-),2.67-2.37(m,12H;2H,>CHCH2CH 2C(=O)O-;2H,pip-CH2CH2-;8H,pip-H),2.35(s,3H,>NCH3),2.31-2.27(m,8H,-CH2C(=O)O-),2.02-1.96(m,2H,>CHC H2CH2C(=O)O-),1.83-1.79(m,2H,pip-CH2CH2-),1.70-1.21(m,48H,-CH2CH2CH2-,-CH2CH3),0.86(t,12H,-CH2CH3). MS(ESI):m / z=1023.7([M+H] + ).

[0342] Example 24: Cationic Lipid (E24-1)

[0343] The preparation process is as follows:

[0344] Step a: Dissolve S24-1 (0.86 g, 5.5 mmol) in dichloromethane (40 mL), then slowly add DCC (1.13 g, 5.5 mmol), stir for 30 min, then add DMAP (0.06 g, 0.5 mmol) and S1-1 (1.73 g, 5.0 mmol) sequentially, and continue stirring for 2 h. After the reaction is complete, filter, concentrate, dissolve the crude product in dichloromethane, wash once with water, and extract twice with dichloromethane (20 mL * 2). Combine the organic phases and dry with anhydrous magnesium sulfate, filter, concentrate, and obtain the crude product. Prepare a trifluoroacetic acid / dichloromethane (1:2, v / v) solution in a dry, clean round-bottom flask, and slowly add the above crude product dichloromethane solution dropwise under ice bath conditions, reacting at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried with anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S24-2 (1.07 g).

[0345] Step b: S24-2 (0.43 g, 1.5 mmol) and DIPEA (0.58 g, 4.5 mmol) were added sequentially to a methanol solution of S1-4 (1.91 g, 9.0 mmol), and the reaction mixture was placed in a reflux apparatus (90 °C) and stirred for 24 h. After the reaction was completed, the reaction mixture was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E24-1 (1.35 g). 1 H NMR(400MHz, CDCl3)δ:3.80-3.61(m,5H;4H,-CH(OH)-;1H,-NHC(=O)CH<),3.51-3.32(m,4H;2H,>NCH2CH3;2H,-CH2NH(C=O)-),3.28-3.02(m,2H,>CH(CH 2)3CH2-),2.82-2.61(m,4H,CH3CH2N(CH2)2-),2.21-1.85(m,8H,-CH(OH)CH2N<),1.81-1.12(m,104H,-CH2CH2CH2-,-CH2CH3),0.86(t,12H,-CH2CH3). MS(ESI):m / z=1133.6([M+H] + ).

[0346] Example 25: Cationic Lipids (E25-1)

[0347] The preparation process is as follows:

[0348] S24-2 (0.43 g, 1.5 mmol) and DIPEA (0.58 g, 4.5 mmol) were added sequentially to a methanol solution of S1-4 (2.18 g, 9.0 mmol), and the reaction mixture was placed in a reflux apparatus (90 °C) and stirred for 24 h. After the reaction was completed, the reaction mixture was concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E25-1 (1.48 g). 1HNMR(400MHz, CDCl3)δ:4.04(t,8H,-C(=O)OCH2-),3.78-3.63(m,5H;4H,-CH(OH)-;1H,-NH C(=O)CH<),3.47-3.29(m,4H;2H,>NCH2CH3;2H,-CH2NH(C=O)-),3.26-3.06(m,2H,>CH(CH2 )3CH2-),2.72-2.52(m,4H,CH3CH2N(CH2)2-),2.35-2.27(m,8H,-CH2C(=O)O-),2.14-1.90 (m,8H,-CH(OH)CH2N<),1.70-1.23(m,80H,-CH2CH2CH2-,-CH2CH3),0.89(t,12H,-CH2CH3). MS(ESI):m / z=1254.1([M+H] + ).

[0349] Example 26: Cationic Lipids (E26-1)

[0350] The preparation process is as follows:

[0351] Step a: Dissolve S26-1 (0.63 g, 5.5 mmol) in dichloromethane (40 mL), then slowly add DCC (1.13 g, 5.5 mmol), stir for 30 min, then add DMAP (0.06 g, 0.5 mmol) and S1-1 (1.73 g, 5.0 mmol) sequentially, and continue stirring for 2 h. After the reaction is complete, filter, concentrate, dissolve the crude product in dichloromethane, wash once with water, and extract twice with dichloromethane (20 mL * 2). Combine the organic phases and dry with anhydrous magnesium sulfate, filter, concentrate, and obtain the crude product. Prepare a trifluoroacetic acid / dichloromethane (1:2, v / v) solution in a dry, clean round-bottom flask, and slowly add the above crude product dichloromethane solution dropwise under ice bath conditions, reacting at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried with anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S26-2 (0.91 g).

[0352] Step b: Under nitrogen protection, compound S26-2 (0.24 g, 1.0 mmol) was dissolved in acetonitrile (30 mL). S3-3 (1.91 g, 6.0 mmol) and DIPEA (0.65 g, 5.0 mmol) were added sequentially with slow stirring. The reaction mixture was stirred at room temperature for 20 h. After the reaction was complete, the reaction solution was concentrated, dissolved in dichloromethane, and extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E26-1 (0.97 g). 1 H NMR (400MHz, CDCl3) δ: 5.81-5.72(m,4H,-C(=O)OCH2CH=CH-), 5.60-5.51(m,4H,-C(=O)OCH2CH=CH-), 4.52(d ,8H,-C(=O)OCH2CH=CH-),3.42(q,2H,-C(=O)NHCH2CH2-),3.14-3.07(m,1H,-NHC(=O)CH<),2.66-2.35(m,16 H;8H,-N(CH2)2-;2H,>CH(CH2)3CH2-;2H,-C(=O)NHCH2CH2-;4H,-N(CH2CH2)2-),2.32-2.27(m,8H,-CH2C(=O )O-), 2.07-2.02(m,8H,-CH=CHCH2CH2-), 1.76-1.21(m,66H,-CH2CH2CH2-,-CH2CH3), 0.87(t,12H,-CH2CH3). MS(ESI):m / z=1195.0([M+H] + ).

[0353] Example 27: Cationic Lipids (E27-1)

[0354] The preparation process is as follows:

[0355] Under ice bath conditions, S26-4 (0.48 g, 2.0 mmol) was dissolved in dichloromethane solution (40 mL). S4-1 (1.58 g, 6.0 mmol) was added with vigorous stirring, followed by NaBH(OAc)3 (1.27 g, 6.0 mmol) in three portions over 10 minutes. The reaction mixture was heated to room temperature and stirred for 2 hours. After the reaction was complete, NaOH aqueous solution (1 M, 10 mL) was added, and the mixture was stirred for 15 minutes before dilution with water. The reaction solution was extracted twice with dichloromethane (20 mL x 2), the organic phases were combined, washed with brine (20 mL), dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E27-1 (2.18 g, 92.5%). 1 H NMR(400MHz, CDCl3)δ:5.41-5.31(m,16H,-CH=CHCH2CH=CH-),3.40(q,2H,-C(=O)NHCH2CH2 -),3.15-3.07(m,1H,-NHC(=O)CH<),2.77(t,8H,-CH=CHCH2CH=CH-),2.65-2.35(m,16H;8H ,-N(CH2)2-;2H,>CH(CH2)3CH2-;2H,-C(=O)NHCH2CH2-;4H,-N(CH2CH2)2-),2.07-2.02(m, 16H,-CH=CHCH2CH2-), 1.73-1.21(m,74H,-CH2CH2CH2-,-CH2CH3), 0.87(t,12H,-CH2CH3). MS(ESI):m / z=1179.2([M+H] + ).

[0356] Example 28: Cationic Lipids (E28-1)

[0357] The preparation process is as follows:

[0358] Step a: Dissolve S28-1 (0.70 g, 5.5 mmol) in dichloromethane (40 mL), then slowly add DCC (1.13 g, 5.5 mmol), stir for 30 min, then add DMAP (0.06 g, 0.5 mmol) and S1-1 (1.73 g, 5.0 mmol) sequentially, and continue stirring for 2 h. After the reaction is complete, filter, concentrate, dissolve the crude product in dichloromethane, wash once with water, and extract twice with dichloromethane (20 mL * 2). Combine the organic phases and dry with anhydrous magnesium sulfate, filter, concentrate, and obtain the crude product. Prepare a trifluoroacetic acid / dichloromethane (1:2, v / v) solution in a dry, clean round-bottom flask, and slowly add the above crude product dichloromethane solution dropwise under ice bath conditions, reacting at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried with anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S28-2 (0.96 g).

[0359] Step b: Dissolve S28-2 (0.26 g, 1.0 mmol) in isopropanol, add sufficient anhydrous potassium carbonate with stirring, and stir at room temperature until the reaction solution becomes alkaline. Add S5-3 (1.93 g, 6.0 mmol) to the reaction solution, and place the reaction solution in a reflux apparatus (90 °C) and continue stirring for 36 h. After the reaction is complete, concentrate the reaction solution, and purify the crude product by column chromatography to obtain cationic lipid E28-1 (1.19 g). 1 H NMR(400MHz, CDCl3)δ:5.40-5.31(m,16H,-CH=CHCH2CH=CH-),4.05(t,8H,-C(=O)OCH2CH2CH2-),3.50-3.42 (m,2H,-C(=O)NHCH2-),3.14-3.07(m,1H,-NHC(=O)CH<),2.78(t,8H,-CH=CHCH2CH=CH-),2.65-2.35(m,16H; 8H,-N(CH2)2-;2H,>CH(CH2)3CH2-;2H,-C(=O)NHCH2CH2-;4H,-N(CH2CH2)2-),2.32-2.28(m,8H,-CH2C(=O)O -), 2.07-2.02(m,16H,-CH=CHCH2CH2-), 1.71-1.19(m,84H,-CH2CH2CH2-,-CH2CH3), 0.89(t,12H,-CH2CH3). MS(ESI):m / z=1537.4([M+H] + ).

[0360] Example 29: Cationic Lipids (E29-1)

[0361] The preparation process is as follows:

[0362] Under nitrogen protection, compound S28-2 (0.26 g, 1.0 mmol) was dissolved in acetonitrile (30 mL). S6-2 (2.09 g, 6.0 mmol) and DIPEA (0.65 g, 5.0 mmol) were added sequentially with slow stirring, and the reaction was carried out at room temperature for 20 h. After the reaction was complete, the reaction solution was concentrated, dissolved in dichloromethane, and extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E28-1 (1.09 g). 1 H NMR(400MHz, CDCl3)δ:4.06(t,8H,-C(=O)OCH2CH2CH2-),3.50-3.42(m,2H,-C(=O )NHCH2-),3.14-3.08(m,1H,-NHC(=O)CH<),2.65-2.35(m,16H;8H,-N(CH2)2-;2H ,>CH(CH2)3CH2-;2H,-C(=O)NHCH2CH2-;4H,-N(CH2CH2)2-),2.31-2.27(m,8H,-C H2C(=O)O-),1.71-1.21(m,108H,-CH2CH2CH2-,-CH2CH3),0.88(t,12H,-CH2CH3). MS(ESI): m / z = 1329.2 ([M+H]) + ).

[0363] Example 30: Cationic Lipids (E30-1)

[0364] The preparation process is as follows:

[0365] Step a: Dissolve S30-1 (0.70 g, 5.5 mmol) in dichloromethane (30 mL), then slowly add DCC (1.13 g, 5.5 mmol), stir for 30 min, then add DMAP (0.06 g, 0.5 mmol) and S1-1 (1.73 g, 5.0 mmol) sequentially, and continue stirring for 2 h. After the reaction is complete, filter, concentrate, dissolve the crude product in dichloromethane, wash once with water, and extract twice with dichloromethane (20 mL * 2). Combine the organic phases and dry with anhydrous magnesium sulfate, filter, concentrate, and obtain the crude product. Prepare a trifluoroacetic acid / dichloromethane (1:2, v / v) solution in a dry, clean round-bottom flask, and slowly add the above crude product dichloromethane solution dropwise under ice bath conditions, reacting at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried with anhydrous magnesium sulfate, filtered, concentrated, and further purified by column chromatography to obtain compound S30-2 (0.95 g).

[0366] Step b: Under nitrogen protection, compound S30-2 (0.14 g, 0.5 mmol) was dissolved in acetonitrile (20 mL). S8-2 (1.10 g, 3.0 mmol) and DIPEA (0.32 g, 2.5 mmol) were added sequentially with slow stirring. The reaction mixture was stirred at room temperature for 20 h. After the reaction was complete, the reaction solution was concentrated, dissolved in dichloromethane, and extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and the crude product was purified by column chromatography to obtain cationic lipid E30-1 (0.57 g). 1 H NMR(400MHz, CDCl3)δ:4.15-4.11(m,16H,-CH2OC(=O)O-),3.73(t,4H,>O(CH2CH2)2 -),3.38(m,2H,-C(=O)NHCH2-),3.14-3.07(m,1H,-NHC(=O)CH<),2.65-2.36(m,20H ;8H,-N(CH2)2-;2H,>CH(CH2)3CH2-;2H,-C(=O)NHCH2CH2-;4H,-N(CH2CH2)2-;4H,- N(CH2CH2)2-),1.71-1.22(m,104H,-CH2CH2CH2-,-CH2CH3),0.87(t,12H,-CH2CH3). MS(ESI): m / z = 1409.2([M+H]) + ).

[0367] Example 31: Preparation and Physicochemical Properties Testing of LNP-mRNA Drug Compositions

[0368] Example 31.1: Preparation of LNP-mRNA Drug Composition

[0369] In this embodiment, an LNP-mRNA pharmaceutical composition (LNP-mRNA) containing Fluc-mRNA was prepared. The phospholipids contained in it are all DSPC, the steroid lipids contained in it are all cholesterol, and the polyethylene glycol lipids contained in it are all PEG2k-DMG. The difference is that they are cationic lipids.

[0370] The method for preparing LNP-mRNA is as follows:

[0371] Step a: Weigh a certain amount of cationic lipids, DSPC, cholesterol, and polyethylene glycol lipid stock solution. Dissolve the cationic lipids, DSPC, cholesterol, and polyethylene glycol lipids in ethanol according to a certain molar ratio (Formula A: 50:10:38:1.5, Formula B: 35:46.5:16:2.5) to obtain an ethanol phase solution. The control groups LCT-1, LCT-2, and LCT-3 used cationic lipids C-1, C-2, and C-3, respectively; the experimental groups L-1 to L-31 used all the nitrogen-containing heterocyclic head groups from Examples 1-30 of this application; L-32 used cationic lipid C-3; L-31 and L-32 used Formula B; and the remaining groups used Formula A.

[0372] C-1 was prepared according to the method disclosed in patent document WO2016210190A1, C-2 was prepared according to the method disclosed in patent document CN115894281A, and C-3 was obtained by reacting S1-3 from Example 1 of this application with tetradecanoic acid. The structures of C-1, C-2 and C-3 are as follows:

[0373] C-1: C-2: C-3:

[0374] Step b: Add Fluc-mRNA to 10-50 mM citrate buffer (pH=4) to obtain an aqueous solution.

[0375] Step c: Prepare LNP-mRNA by mixing the ethanol phase solution and the aqueous phase solution (1:3 v / v), and wash repeatedly with DPBS ultrafiltration to remove ethanol and free molecules. Finally, pass the solution through a 0.2 μm sterile filter for later use.

[0376] Example 31.2: Physicochemical property testing of LNP-mRNA drug composition

[0377] Encapsulation efficiency determination: The encapsulation efficiency of the LNP-mRNA compositions was determined using the Quant-it Ribogreen RNA quantification kit. The results showed that the lipid compositions (L-1 to L-31) of this application exhibited high encapsulation efficiencies for nucleic acid drugs (mRNA), all within the range of 85%-96%, with most within the range of 90%-96%. The results indicate that the cationic lipids containing nitrogen-containing heterocyclic head groups in each experimental group can effectively encapsulate mRNA, showing encapsulation efficiencies comparable to or superior to C-1, C-2, and C-3. Differences also existed in the encapsulation efficiencies among the cationic lipids containing nitrogen-containing heterocyclic head groups with different structures.

[0378] Particle size determination: In this embodiment, the particle size of LNP-mRNA was determined by dynamic light scattering (DLS). The measured LNP-mRNA showed high size uniformity, with a PDI of less than 0.3 for all samples. The LNP-mRNA prepared by the lipid composition of this application has a particle size in the range of 85-110 nm, which meets the requirements for particle size as a gene vector.

[0379] Table 1: Summary of formulations of various lipid compositions and the particle size and encapsulation efficiency of the LNP-mRNA prepared from them.

[0380] Example 32: Biological activity test of LNP-mRNA drug composition

[0381] (1) Serum stability evaluation

[0382] The LNP-mRNA drug composition was added to a culture medium containing 10% fetal bovine serum (FBS) and stirred at 37°C. Samples were taken periodically to measure the particle size change of the LNP-mRNA, and the serum stability of the LNP-mRNA drug composition was analyzed by testing the particle size change. The experimental results showed that within 7 days, the particle size change in both the control and experimental groups was less than 10%, and the particle size change in all biological experimental groups was less than 5%, indicating that the LNP-mRNA drug composition prepared by cationic lipids in this application has excellent serum stability.

[0383] (2) Cytotoxicity (biocompatibility) studies

[0384] Prepare DMEM high-glucose complete medium containing 10% FBS. Prepare working solutions (0.1, 0.15, 0.2, 0.25, and 0.3 μg / 100 μL) for samples (L-1 to L-32 and L-CT1 to L-CT3) using complete medium, respectively, and store for later use. Seed 293T cells in logarithmic growth phase at 7 × 10³ cells / well, 100 μL / well into 96-well plates. Both control and experimental groups have 6 replicates. After incubation for 24 h in a 5% CO2, 37°C incubator, retain the original medium. Add 100 μL / well of complete medium to the control group and 100 μL / well of working solution to the sample groups. Continue incubation for another 24 h, then add 100 μL / well of medium containing 10% CCK-8 and incubate for 2 h in a 5% CO2, 37°C incubator. Measure the absorbance at 450 nm using a microplate reader. Calculate the relative viability of cells using the following formula:

[0385] Relative activity % = (sample absorbance value - background absorbance value) / (control group absorbance value - background absorbance value) × 100%; where the background absorbance value is the absorbance with only CCK-8 reagent and culture medium added.

[0386] Experimental results showed that the LNP-mRNA drug composition prepared using the cationic lipids of this application did not produce significant cytotoxicity at any of the five concentration gradients, and the cell survival rate was greater than 95%.

[0387] (3) Evaluation of in vitro transfection effect

[0388] To investigate the mRNA transfection rate at the cellular level of the LNP-mRNA drug compositions prepared in Example 31.1 of this application, Luciferase bioluminescence was used for testing. The LNP-mRNA drug compositions were dissolved in culture medium to prepare the required dosage. Using 293T cells as a cell model, 100 μL of cell suspension per well was seeded into 96-well plates with black edges and transparent bottoms at a seeding density of 6000 cells / well. After seeding, the cells were incubated in a cell culture incubator for 24 h. Then, 0.2 μg of mRNA was administered per well. The blank control group received the corresponding dose of free Fluc-mRNA. After 24 hours of transfection, the old culture medium was removed and replaced with a new culture medium containing D-fluorescein sodium (1.5 mg / mL) substrate. After incubation for 5 minutes, bioluminescence was detected using a microplate reader. Stronger fluorescence indicated that more Fluc-mRNA was transported into the cytoplasm and translated into the corresponding fluorescent protein. The experimental results are shown in Table 2, where the relative fluorescence intensity is the ratio of the fluorescence intensity of each group to that of the blank control group. The results show that the LNP-mRNA drug compositions prepared in this application all have excellent in vitro transfection effects, that is, the LNPs in the experimental group are all effective nucleic acid delivery vectors. The transfection efficiency of LNPs prepared by cationic lipids in this application is better than that of L-CT1, L-CT2 and L-CT3 prepared by cationic lipids in the prior art. This may be because the cationic lipids containing nitrogen-containing heterocyclic head groups in this application contain more hydrophobic hydrocarbon tail chains. This structure is more likely to form a conical geometry that is conducive to the escape of nucleic acid endosomes, thereby improving the transfection rate. The experimental groups L-2-1, L-2-2, L-3, L-6-1, L-6-2, L-7, L-8, L-9, L-10-1, L-10-2, L-25, L-29, L-30, and L-31 showed relatively high fluorescence values. At the same time, there were differences in fluorescence intensity among the experimental groups, which may be due to the following differences among the cationic lipids: the type and / or number of degradable linkers, and / or the type of hydrophobic hydrocarbon tail chain, and / or the saturation of the hydrophobic hydrocarbon tail chain, and / or the number of hydroxyl groups, and / or the number of ionizable tertiary amines. Specifically, only E13-1, E14-1, E14-2, E15-1, E16-1, E17-1, and E18-1, which contain tertiary amine groups at the head, showed slightly lower transfection activity than other experimental groups. The type of degradable linker in the hydrocarbon tail chain and its distance from the central tertiary amine atom affect the activity of cationic lipids. The carbon chain length of the degradable linker and tertiary amine in E6-1 is C5, while that in E12-1 and E12-2 is C2. The results show that the transfection rate of E6-1 is better than that of E12-1 and E12-2. In this application, the saturation degree of the hydrocarbon tail chain has no significant effect on the activity of cationic lipids. E6-2, E10-1, and E10-2 all showed excellent transfection effects.Compared to E24-1, E25-1, and E29-1, the transfection efficiency was in the order of E25-1 > E29-1 > E24-1. E25-1 contains hydroxyl groups and degradable ester groups. The hydroxyl groups can interact with the phosphate groups on nucleic acids through hydrogen bonding, thereby improving delivery efficiency. This further illustrates that introducing ester bonds into the hydrophobic hydrocarbon tail chain will show better delivery effect. The cationic lipids used in L-6-1 and L-31 are both E6-1 of this application. The formulation used in L-31 uses less cationic lipid. The results show that the cationic lipids containing nitrogen heterocyclic head groups of this application can achieve excellent encapsulation and transfection even with less cationic lipid. In contrast, L-32 has poorer encapsulation and transfection effects when the amount of cationic lipid C-3 is less.

[0389] Table 2: Cell transfection test results

[0390] (5) Evaluation of in vivo transfection effect

[0391] Lipid nanoparticles L-6-1 were delivered to 6-8 week old female BALB / c mice via tail vein injection at a dose of 10 μg / mouse. In vivo fluorescence imaging was performed at 6, 12, and 24 hours after administration. After imaging at the last time point, the mice were euthanized, and imaging of the major organs (heart, liver, spleen, lung, and kidney) was performed (from left to right in the figure). 10-15 min before imaging, 0.2 mL of D-fluorescein sodium (15 mg / mL) was injected intraperitoneally. The experimental results (Figure 5) show that the lipid nucleic acid drug composition prepared by the cationic lipids of this application can achieve efficient in vivo delivery of nucleic acid drugs, and the LNP-mRNA drug composition delivered into the body is mainly distributed in the liver.

[0392] The above description is merely an embodiment of this application and does not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

[0393] For those skilled in the art, this application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from the spirit and scope of this application and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to this application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to this application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A cationic lipid, characterized in that, The structure is shown in equation (1): Or its salts, tautomers, stereoisomers, deuterated derivatives or solvates; Where, N r It is a nitrogen-containing heterocyclic group; t is an integer from 1 to 6; L a L b Each is an independent linker or a binary linker L. d The L d Selected from any one of *-C(=O)O-, *-C(=O)NH-, and -C(=O)-; L c For -Z1-(CH2) tc -Z2-*, where Z1 is a connector or -(CH2) te -L e -, tc and te are each independent integers from 1 to 6, L e Each occurrence is independently selected from -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -CH(OH)-, -O-, -NH-, -O(CH2). s O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH-, -NHC(=O)S-, -C(=S)-, -OC(=S)O-, -OC(=S)O-, -NHC(=S)NH-, -NHC(=S)NH-, -OC(=S)NH-, and -NHC(=S)O-, where s is 1, 2, or 3; Z2 is selected from any one of -OC(=O)-*, -NHC(=O)-*, -C(=O)NH-*, and -OC(=O)NH-*; the aforementioned L d L c The asterisk end in the Z2 structure points to the central carbon atom in formula (1); Each X is independently -N<, -CR a < or connect key; when X is -N< or -CR a When X is a connector, both a and b are 2; when X is a connector, both a and b are 1; each R a Independently H or C 1-6 alkyl; R m For H or C 1-12 Alkyl; each R g Independently defined as -B1-L1-R1; where each B1 is independently defined as C. 1-12 alkylene or hydroxyl-substituted C 1-12 Alkylene; each L1 is independently selected from the following: linking bond, -CH(OH)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, -NH-, -O(CR h R h ) s O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, -NR h C(=O)-、-C(=O)NR h -、-NR h C(=O)NR h -、-OC(=O)NR h -、-NR h C(=O)O-、-SC(=O)NR h -、-NR h C(=O)S-, -C(=S)-, -OC(=S)-, -C(=S)O-, -OC(=S)O-, -NR h C(=S)-、-C(=S)NR h -、-NR h C(=S)NR h -、-OC(=S)NR h -and-NR h Any one of C(=S)O-, where each R h Independently a hydrogen atom or a carbon atom 1-12 Alkyl group, s is 2, 3 or 4; each R1 is independently C 1-30 Straight-chain hydrocarbon groups or C 1-30 Branched hydrocarbon group.

2. The cationic lipid according to claim 1, characterized in that, The N r Selected from Any one of them; preferably Any one of them; more preferably or 3. The cationic lipid according to claim 1, characterized in that, The L a L b The same applies to connecting bonds, *-C(=O)O- or *-C(=O)NH-.

4. The cationic lipid according to claim 1, characterized in that, The X is the same, and can be -N<, -CH<, or a linker.

5. The cationic lipid according to claim 1, characterized in that, When both a and b are 2, the a R g Similarly, the b R g Same, preferred of all R g They are all the same.

6. The cationic lipid according to claim 1, characterized in that, In B1, the C-hydroxyl-substituted... 1-12 Alkyl groups are represented as *-CH2CH(OH)(CH2). P -, p is an integer from 0 to 10, preferably an integer from 1 to 10, and the asterisk end in the structure points to the central carbon atom in formula (1); The C 1-12 The alkylene group is any one of methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, and octylene; More preferably, all occurrences of B1 are identical.

7. The cationic lipid according to claim 1, characterized in that, Each time L1 appears, it is independently any one of the following: a linking bond, -CH(OH)-, -OC(=O)-, -C(=O)O-, -C(=O)NH-, -OC(=O)O-, and -NHC(=O)O-. More preferably, all occurrences of L1 are the same.

8. The cationic lipid according to claim 1, characterized in that, L c In the context, Z1 is the connecting key, and L... c -(CH2) tc -Z2-*;Preferred L c -(CH2) tc OC(=O)-*、-(CH2) tc NHC (=O)-* or -(CH2) tc C(=O)NH-*; more preferably -(CH2) 1-4 OC(=O)-*、-(CH2) 1-4 NHC (=O)-* or -(CH2) 1-4 C(=O)NH-*, the most preferred are -(CH2)2OC(=O)-*, -(CH2)3NHC(=O)-*, -(CH2)3C(=O)NH-* or -CH2C(=O)NH-*.

9. The cationic lipid according to claim 1, characterized in that, R1 is independently C each time it appears. 1-30 Straight-chain hydrocarbon groups or C 1-30 Branched hydrocarbon groups; The C 1-30 The straight-chain hydrocarbon group is C 1-30 Straight-chain alkyl, C 2-30 Straight-chain alkenyl, C 2-30 Any one of the straight-chain alkynyl groups; preferably C 1-25 Straight-chain alkyl, C 2-25 Straight-chain alkenyl or C 2-25 Straight-chain alkynyl group; more preferably... Any one of the following, where tp is an integer from 1 to 10 and tq is an integer from 0 to 12; The C 1-30 Branched hydrocarbon groups are selected from C 1-30 Branched alkyl groups, C 2-30 Branched alkenyl groups, C 2-30 Each of the branched alkynyl groups can be independently represented as Where tm is an integer between 0 and 12; R e R f Each independently is C 1-15 Alkyl, C 2-15 alkenyl and C 2-15 Any one of the alkynyl groups; R e R f More preferably, each is independently selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, vinyl, propenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptenyl, octenyl, nonynyl, and decynyl; R e R f The most preferred choice is that each of the following is independently selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl; More preferably, R1 is selected from any of the following structures: More preferably, tm in the aforementioned structure is 0, 1, or 2.

10. The cationic lipid according to claim 1, characterized in that, The R g Choose from any of the following structures:

11. The cationic lipid according to claim 1, characterized in that, The structure of the cationic lipid satisfies the following general formula (1-A), (1-B), or (1-C): L in preferred formula (1-A), (1-B) or (1-C) a L b The same, is a connecting bond, *-C(=O)O- or *-C(=O)NH-; L c -(CH2) tc OC(=O)-*、-(CH2) tc NHC (=O)-* or -(CH2) tc C(=O)NH-*, tc is an integer from 1 to 6; t is an integer from 1 to 4, preferably t is 1, 2, or 4; the asterisk points to the central carbon atom in formula (1-A), (1-B), or (1-C).

12. The cationic lipid according to claim 1, characterized in that, Its structure is selected from any of the following structures: Alternatively, the structure of the cationic lipid may be any of the following:

13. A lipid composition, characterized in that, The cationic lipid contained in any one of claims 1-12.

14. The lipid composition according to claim 13, characterized in that, It also contains one or more of phospholipids, steroid lipids, polyethylene glycol lipids, another cationic lipid, and anionic lipids; selected from any of the following: Case (1): It also contains phospholipids; Case (2): Also contains steroid lipids; Case (3): It also contains polyethylene glycol lipids; Case (4): Also contains phospholipids and steroid lipids; Case (5): Also contains phospholipids and polyethylene glycol lipids; Case (6): Also contains steroid lipids and polyethylene glycol lipids; Case (7): Also contains phospholipids, steroid lipids and polyethylene glycol lipids; Case (8): It also contains phospholipids, steroid lipids, polyethylene glycol lipids and another cationic lipid; Case (9): Also contains phospholipids, steroid lipids, polyethylene glycol lipids and anionic lipids; Preferably, the phospholipid is selected from 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-distearateoyl-sn-glycerol-3-phosphate choline, 1,2-diundecanoyl-sn-glycerol-3-phosphate choline, and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline. Base, 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoyl)-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol -3-phosphoethanolamine, 1,2-diphydanoyl-sn-glycerol-3-phosphoethanolamine, 1,2-distearatel-sn-glycerol-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphoethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphoethanolamine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphoethanolamine), 1,2-dioleoyl-s Sodium n-glycerol-3-phosphate-rac-(1-glycerol) salt, dioleoylphosphatidylserine, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearate-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, 1-stearoyl-2-oleoylphosphatidylcholine, sphingomyelin, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine, any one of the following and combinations thereof; Or preferably, the steroid lipids are selected from any one of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatine, ursolic acid, α-tocopherol, and combinations thereof; Preferably, the polyethylene glycol lipid is selected from polyethylene glycol-1,2-dimyristoylglycerol, polyethylene glycol-distearylphosphatidylethanolamine, PEG-cholesterol, polyethylene glycol-diacylglycerol, polyethylene glycol-dialkoxypropyl, specifically including polyethylene glycol 500-dispalmitoylphosphatidylcholine, polyethylene glycol 2000-dispalmitoylphosphatidylcholine, polyethylene glycol 500-distearylphosphatidylethanolamine, polyethylene glycol 2000-distearylphosphatidylethanolamine, polyethylene glycol 500-1,2-dioleoylphosphatidylethanolamine, polyethylene glycol 2000-1,2-dioleoylphosphatidylethanolamine and polyethylene glycol 2000-2,3-dimyristoylglycerol and combinations thereof, or the polyethylene glycol lipid is selected from any one of the following structures and combinations thereof: Wherein, n1 is an integer between 25 and 300, and more preferably n1 is any one of 44, 45, 46, 47, and 48; Or preferably, the other cationic lipid is selected from 1,2-dioleoyl-3-trimethylammonium-propane (methyl sulfate), 1,2-bisoctadecenoxy-3-methylammonium propane chloride, 1-[2-(oleoyloxy)ethyl]-2-oleo-3-(2-hydroxyethyl)imidazoline chloride, 1,2-dioleoyl-3-dimethylamino-propane, 2,3-bis(tetradecanoyloxy)propyltrimethylazinonium chloride, bisdecyldimethylammonium chloride, bisdecyldimethylammonium bromide, N,N-dioleo-N,N-dimethylammonium chloride, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)prop-1-ammonium, 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione, 1,1'-( Any one of (2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethyl)azadiyl)bis(dodecane-2-ol), methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester, 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine, ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 8-[(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino]octanoate (heptadecane-9-yl) ester and ((2-(2-hydroxyethoxy)ethyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) and combinations thereof; Or preferably, the anionic lipid is selected from any one of 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt, 1,2-dimyristoyl-sn-glycerol-3-phosphate sodium salt, bis(monooleoylglycerol) phosphate ammonium salt and cardiolipin, and combinations thereof.

15. The lipid composition according to claim 14, characterized in that, It contains 20-80% cationic lipids, 5-15% phospholipids, 25-55% steroid lipids and 0.5-10% polyethylene glycol lipids, where the percentages are the molar percentages of each lipid in the total lipids. Preferably, the cationic lipids account for 30-65% of the total lipids; more preferably, they are any one of 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, and 55%. Or preferably, the phospholipids account for 7.5-13% of the total lipids in molar percentage; more preferably, they are any one of 8%, 9%, 10%, 11%, and 12%. Or preferably, the steroid lipids account for 35-50% of the total lipids in molar percentage, more preferably any one of 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, and 50%; Alternatively, preferably, the polyethylene glycol lipid accounts for 0.5-5% of the total lipid molar percentage; more preferably 1-3%; and even more preferably any one of 1.5%, 1.6%, 1.7%, 1.8%, and 1.9%.

16. A lipid pharmaceutical composition, characterized in that, The lipid composition and drug comprising any one of claims 13-15, wherein the drug is selected from any one of nucleic acid drugs, gene vaccines, antitumor drugs, small molecule drugs, peptide drugs or protein drugs; wherein the nucleic acid drug is selected from any one of RNA, DNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir and ribozyme; preferably the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA and siRNA; more preferably the nucleic acid drug is any one of DNA, mRNA, miRNA and siRNA.

17. The lipid pharmaceutical composition according to claim 16, characterized in that, The lipid pharmaceutical composition is used to prepare a drug, which is selected from any one of antitumor agents, antiviral agents, antifungal agents, and vaccines.

18. A liposome or lipid nanoparticle, characterized in that, A lipid composition comprising any one of claims 13-15.

19. A lipid pharmaceutical composition formulation, characterized in that, The pharmaceutical composition, liposomes or lipid nanoparticles containing any one of claims 16-18 further contain a pharmaceutically acceptable diluent or excipient, wherein the diluent or excipient is preferably any one of deionized water, ultrapure water, phosphate buffer, and physiological saline, more preferably phosphate buffer or physiological saline, and most preferably physiological saline.

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