Degradable cationic lipid containing disulfide bond and use thereof

Abstract

Disclosed in the present invention is a cationic lipid containing a disulfide bond as shown in formula (1), wherein -S-S- is a disulfide bond; L1 and L2 are each independently a degradable divalent linking group; X1 and X2 are each independently a nitrogen-containing heterocyclic ring; G1 and G2 are each independently a linking bond or a trivalent branched group; when G1 is a linking bond, k1 is 1; when G1 is a trivalent branched group, k1 is 2; when G2 is a linking bond, k1 is 1; when G2 is a trivalent branched group, k2 is 2; L3 and L4 are each independently selected from a linking bond or a divalent linking group; and R1 and R2 are each independently a substituted or unsubstituted C5-30 hydrocarbyl group or C5-30 hydrocarbon derivative residue. A lipid composition prepared from the cationic lipid of the present invention can enable an LNP-nucleic acid pharmaceutical composition preparation to more fully exert a drug effect on the premise of having good capability for compounding a nucleic acid and a drug, in-vivo stability and low toxicity, thereby improving the immune or treatment effect.

Description

A degradable cationic lipid containing disulfide bonds and its application Technical Field

[0001] The present invention belongs to the field of drug delivery, and specifically relates to a pharmaceutical carrier cationic lipid, and in particular to a degradable cationic lipid containing a disulfide bond, as well as a lipid composition comprising the cationic lipid, a lipid pharmaceutical composition, and preparations and applications thereof. Background Art

[0002] Lipid nanoparticles (LNPs) are one of the most popular delivery technologies today. The lipid formulation of LNPs can be composed of a variety of lipids including cationic lipids, amphoteric lipids, neutral lipids, steroid lipids, and PEGylated lipids. As a key component in LNP-nucleic acid drug composition preparations, cationic lipids can be complexed with negatively charged nucleic acid drugs through electrostatic interactions. Cationic lipids include cationizable lipids and permanent cationic lipids, among which cationizable lipids can be electrically neutral under physiological conditions, avoiding excessive interference with biological membranes in systemic circulation, and have milder toxicity than permanent cations. After LNPs are taken up by cells, cationizable lipids are converted into positively charged molecules under the acidic conditions of the endosome cavity, increasing their membrane permeability and promoting the endosomal escape of lipid drug compositions. At the same time, the reorganization of the lipid component structure is also conducive to the release of drug molecules from LNPs, thereby improving drug efficacy.

[0003] The specific structure of cationic lipids has an important impact on the nucleic acid drug delivery efficiency of LNP. Whether LNP can fully release drugs in cells is one of the key factors affecting delivery efficiency. The concentration of glutathione (GSH) in cells is significantly higher than that outside cells, which is also one of the main factors causing the reducing environment in cells. In this regard, some existing technologies use disulfide bonds to connect two lipid parts together, and use the cleavage of disulfide bonds in the reducing environment inside cells to achieve destabilization of the LNP structure, thereby promoting the release of encapsulated substances. Cationic lipids with these characteristics disclosed in CN107406396A and CN103930398A improve the expression efficiency of nucleic acid drugs in cells. However, for cationic lipids containing disulfide bonds, the reduction sensitivity of their disulfide bonds may not be fully utilized in actual use. This is because, compared with the cytoplasm containing high concentrations of GSH, the GSH concentration in the endosomal environment is lower and the reducing conditions are poor, resulting in the disulfide bonds not necessarily being able to be effectively broken in the endosome.

[0004] Therefore, when conventional cationic lipids containing disulfide bonds are used for the delivery of nucleic acid drugs, their efficiency is not completely satisfactory. To solve this problem, some substantial improvements need to be made to cationic lipids containing disulfide bonds. Summary of the Invention

[0005] The object of the present invention is to provide a novel disulfide bond-containing cationic lipid, as well as a lipid composition, a lipid pharmaceutical composition and a preparation thereof containing the cationic lipid, for use in the field of drug delivery.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] A cationic lipid containing a disulfide bond, characterized in that the structure is as shown in the general formula (1):

[0008] or a salt, tautomer, stereoisomer, isotope-substituted product or solvate thereof;

[0009] Among them, -SS- is a disulfide bond;

[0010] L1 and L2 are each independently -(CH2) t -L d -(CH2) t -or-(CH2) t -L d -(CH2) t -L d -(CH2) t -, wherein each t is independently an integer from 1 to 4;

[0011] L d is a degradable divalent linking group. Any two L d have the same or different structures;

[0012] X1 and X2 are independently Among them, R d2 、R d3 are each independently a hydrogen atom or an optionally substituted C 1-6 Alkyl, and R d2 、R d3 The number of is independently 1, 2, 3 or 4; s is 1, 2 or 3; n is 1 or 2;

[0013] G1 and G2 are each independently a connecting bond or a trivalent branching group;

[0014] When G1 is a connecting bond, k1 is 1; when G1 is a trivalent branching group, k1 is 2;

[0015] When G2 is a connecting bond, k1 is 1; when G2 is a trivalent branching group, k2 is 2;

[0016] L3, L4 each occur independently selected from a linker, -(CH2) q -, Z, P0, and any combination thereof, wherein q is an integer of 1-10, Z is a divalent linking group containing a heteroatom, and P0 is p is independently 0 or 1 each time it occurs;

[0017] R1 and R2 are each independently an optionally substituted C 5-30 Hydrocarbon or C 5-30 Hydrocarbon derivative residues.

[0018] The present invention also provides another embodiment:

[0019] A lipid composition comprises a disulfide bond-containing cationic lipid having a structure as shown in formula (1).

[0020] The present invention also provides another embodiment:

[0021] A lipid pharmaceutical composition comprises a lipid composition and a drug, wherein the lipid composition comprises a disulfide bond-containing cationic lipid having a structure as shown in formula (1).

[0022] The present invention also provides another embodiment:

[0023] A lipid pharmaceutical composition preparation contains the aforementioned lipid pharmaceutical composition and a pharmaceutically acceptable diluent or excipient.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] Cationic lipid structure of the present invention can be considered as being made up of the following parts by covalent bonding: two cationic lipid fragments each having a polar head (tertiary amine group) and a hydrophobic tail (hydrocarbon chain), and a degradable segment connecting the aforementioned two segments. In particular, the degradable segment contains a disulfide bond and other degradable divalent linking groups. After the disulfide bond breaks in the cell, an original cationic lipid molecule can form two products that still possess the basic structure of cationic lipids. When the LNP structure destabilizes, the destructive ability of the cationic lipid polar head to the endosomal membrane structure is retained, so that the loaded drug can be effectively released into the cytoplasm. Other degradable divalent linking groups in the degradable segment, including but not limited to ester groups, urea groups, thiourea groups, carbamate groups, carbonate groups, oligopeptide linking groups, etc., can significantly improve the degradable ability of cationic lipids, prevent the accumulation of LNP in the endosome and hinder the endosomal escape of the drug, and significantly improve the drug effect of the LNP-nucleic acid pharmaceutical composition preparation. In some embodiments of the present invention, the number of degradable groups is also increased in the portion between the tertiary amine group and the aliphatic hydrocarbon chain, which further improves the intracellular degradability of LNP to a certain extent.

[0026] The lipid composition prepared using the cationic lipid of the present invention, while having good nucleic acid drug complexing ability, systemic circulation stability and low toxicity, can enable the LNP-nucleic acid drug composition preparation to more fully exert its efficacy and improve the immune or therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a diagram of the cationic lipid E1-1 prepared in Example 1.1 1 H NMR spectrum.

[0028] Figure 2 is a diagram of the cationic lipid E1-2 prepared in Example 1.2 1 H NMR spectrum.

[0029] Figure 3 shows the cationic lipid E4-3 prepared in Example 4.3 1 H NMR spectrum.

[0030] Figure 4 shows the cationic lipid E6-1 prepared in Example 6.1 1 H NMR spectrum.

[0031] Figure 5 shows the cationic lipid E7-1 prepared in Example 7.1 1 H NMR spectrum.

[0032] FIG6 shows the high performance liquid chromatography (HPLC) test results of the cationic lipid E7-1 prepared in Example 7.1.

[0033] FIG7 is a mass spectrum (MS) of the cationic lipid E7-1 prepared in Example 7.1.

[0034] FIG8 shows the cytotoxicity test results of the LNP-mRNA pharmaceutical composition L7-1 prepared in Example 11.

[0035] FIG9 shows the imaging results of mice after injection of the LNP-mRNA pharmaceutical composition L7-1 prepared in Example 11.

[0036] Implementation Method

[0037] 1. Terminology

[0038] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly used in the art to which the claimed subject matter belongs. If there are multiple definitions for a term, the definition herein shall prevail.

[0039] Unless otherwise stated, the terms "include," "including," and "containing" and similar expressions should be interpreted in an open and inclusive sense as "including but not limited to" or "including, without limitation."

[0040] “Including but not limited to” a certain range means that items within the range are optional, but the optional items are not limited to the range.

[0041] "Independently" can be used not only to describe objects with different names, but also to describe objects with the same name that appear in different locations. For example, "in-NR c C(=O)O- and -NR c C(=O)NR c -Middle, R c Each occurrence is independently a hydrogen atom or C 1-12 Alkyl" This description indicates that in "-NR c C(=O)NR c -" in the middle, two R c The groups may be the same or different (e.g., one R c is a methyl group, and another R c is a hydrogen atom or an ethyl group), and "-NR c C(=O)NR c -" c Can be used with "-NR c R in "C(=O)O-" c Same or different.

[0042] When at least two items are listed, a "combination" of the listed items refers to a combination of any two or more of the listed items, or a combination of any two or more of the same item. The number of any item in the combination may be one or more, and the specific forms of multiple items of the same name may be the same or different. A combination of linkers is considered to be a new linker formed by the linkers being linked together. The combination of a linker and any linker still represents the linker itself. For example, the description “L is any one of a connecting bond, an alkylene group, -O-, -S-, -C(=O)-, -NH- and a combination thereof” indicates that L can be any one of the listed items, or a combination of any two or more thereof, and the combination can be -CH2-O-CH2- (i.e., a combination of -O- and an alkylene group in the listed items, wherein the specific form of the alkylene group is 2 methylene groups), -CH2-NH-CH2CH2- (i.e., a combination of -NH- and an alkylene group in the listed items, wherein the specific form of the alkylene group is 1 methylene group and 1 ethylene group), -SS- (a combination of 2 -S-), -C(=O)NH- (a combination of 1 -C(=O)- and 1 -NH-), and the like.

[0043] A "connecting bond" does not contain any atoms and only serves as a connection. It can be represented by a dash (such as the "-" at both ends of -C(=O)NH-), or by a Marking (such as In particular, when the definition of a group includes a "bond", it means that the group may not exist and the position where the group is located is occupied by a bond.

[0044] The group may be monovalent, divalent, or polyvalent, where "polyvalent" means a valence of at least 3.

[0045] Groups with a valence greater than or equal to 2 are collectively referred to as "linking groups" and contain at least one atom by default. Linking groups containing only one atom include, but are not limited to, ether groups (-O-), thioether groups (-S-), and the like. The "group" in a divalent linking group can be replaced by "bond" without changing its meaning. For example, a divalent ether group can also be referred to as an ether bond (-O-), a divalent ester group can also be referred to as an ester bond (-OC(=O)- or -C(=O)O-), and a divalent carbamate group can also be referred to as a carbamate bond (-OC(=O)NH- or -NHC(=O)O-). Unless otherwise specified, there is no particular restriction on the connection end of the linking group used to connect other groups. For example, when -C(=O)NH- is used as a divalent linking group between group A and group B, either connection end (left or right end) of -C(=O)NH- can be used to connect A or B, i.e., both AC(=O)NH-B and A-NHC(=O)-B are optional.

[0046] For the connecting bond introduced from the cyclic structure, when it is not marked on a specific ring atom but points to the inside of the ring, it means that the connecting bond can be introduced from any suitable ring atom, and when the marked ring is part of a fused ring structure, the connecting bond can be introduced from any suitable ring atom in the fused ring structure. For the connecting bond and the group introduced by the connecting bond expressed in this way, the number can be one or more, and if there is no special explanation, it is assumed to be one. represents a structure containing any suitable chemical linkage (including but not limited to etc.)

[0047] The "any suitable" in "any suitable linking group", "any suitable reactive group", "any suitable chemical connection method", etc. refers to the basic principles of chemical structure. Chemical structures described in this way are considered to have a clear and definite scope.

[0048] Numerical intervals can be represented by dashes, wavy lines, or "to / to", such as the three intervals of 1-6, 1~6, and 1 to / to 6, all of which represent a group consisting of all numerical values ​​between 1 and 6. Unless otherwise specified, the numerical values ​​include but are not limited to integers, non-integers, percentages, fractions, etc., and the interval includes two endpoints. Unless otherwise specified, the average number includes integers and non-integers by default. For example, "the average number of EO units is selected from 22 to 100" means that the integer 22 and the non-integer 22.2 are both optional values ​​for the average number of EO units. For another example, "an integer in 1-3" means a group consisting of 1, 2, and 3. Unless otherwise specified, the numerical interval representing the number of groups is composed of integers by default, such as -(CH2) 1-4 - represents a group consisting of -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, and express The group composed of.

[0049] For percentages, when the value is given to the Nth decimal place (excluding the percentage sign), "approximately" or "around" generally refers to the basic value ± (0.5*0.1 N )%. For example, "about 1%" is accurate to the 0th decimal place (excluding the percentage sign), and the default value is 1±(0.5*0.1 0 )%, i.e., the range of 0.5%-1.5%; "about 2.2%" is accurate to the first decimal place (excluding the percentage sign), and the default value is 2.2±(0.5*0.1 1 )%, that is, the range of 2.15%-2.25%.

[0050] The numerical ranges marked at the C subscript position may represent the number of carbon atoms in the group, and unless otherwise specified, the carbon number does not include the contribution of substituents. For example, C 1-12 means "having 1 to 12 carbon atoms". For example, C 1-10 Alkylene represents any alkylene group having 1 to 10 carbon atoms, i.e. C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 Any of alkylene, including but not limited to linear C 1-10 Alkylene (e.g. -(CH2)6-) and branched C 1-10 Alkylene (e.g. -(CH2)3-CH(CH3)-(CH2)3-). Another example is "substituted C 1-3 "Alkyl" refers to a C1, C2 or C3 alkyl group containing at least one substituent, such as -CH2Cl, -CH2CH2OH, -CH2CH(OCH3)CH3, which are respectively methyl substituted by -Cl, ethyl substituted by -OH, and propyl substituted by -OCH3; the number of carbon atoms and heteroatoms in the substituent is not particularly limited.

[0051] Unless otherwise specified, stereoisomers of any structure are deemed to be disclosed together, including but not limited to cis / trans isomers, E- / Z-isomers, levorotatory / dextrorotatory isomers, etc. For example, for a compound having cis-trans isomers, even if only the structure of its cis isomer is provided, its trans isomer should also be considered to be provided simultaneously, and vice versa.

[0052] Unless otherwise specified, the unit of measurement for molecular weight is Dalton (Da). The molecular weight of a polymer is by default the average molecular weight, and is generally referred to as the number-average molecular weight (M n ).

[0053] The "stability" and "degradability" of a group are relative concepts.

[0054] A "degradable" linker can break a chemical bond to form two parts that are not directly connected. If the structure of the linker is changed after chemical changes, but it is still a complete linker, then the linker is still classified as "stable". The conditions used to examine the degradability of the linker are not particularly limited, and can be in vivo physiological conditions, in vitro simulated physiological conditions or other conditions, preferably in vivo physiological conditions or in vitro simulated physiological conditions. The physiological conditions are not particularly limited, and include but are not limited to serum, heart, liver, spleen, lung, kidney, bone, muscle, fat, brain, lymph nodes, small intestine, gonads and other parts. They can refer to physiological conditions in cells or extracellular matrix, and can also refer to physiological conditions in normal physiological tissues or pathological physiological tissues (such as tumors, inflammation, etc.). The in vitro simulated physiological conditions are not particularly limited, and include but are not limited to physiological saline, buffer, culture medium, etc. There is no special restriction on the speed of "degradation", for example, rapid degradation under the action of enzymes, slow hydrolysis under physiological conditions, etc. In vivo physiological conditions include physiological conditions during treatment, such as ultraviolet irradiation, heat therapy, etc. Conditions used to investigate the degradability of the linker include, but are not limited to, light, heat, low temperature, enzymes, redox, acidity, alkalinity, physiological conditions, and in vitro simulated physiological conditions, preferably light, heat, enzymes, redox, acidity, alkalinity, and the like. The light conditions include, but are not limited to, visible light, ultraviolet light, infrared light, near-infrared light, mid-infrared light, and the like. The heat conditions refer to temperatures above normal physiological temperatures, typically above 37°C, and typically below 45°C, preferably below 42°C. The low temperature conditions refer to temperatures below human physiological temperature, preferably below 25°C, more preferably ≤10°C, such as refrigeration temperature, freezing temperature, liquid nitrogen treatment temperature, 2-10°C, 4-8°C, 4°C, 0°C, and -20±5°C. The enzymes are not particularly limited, and all enzymes that can be produced under physiological conditions are included, such as peptidases, proteases, and lyases. The redox conditions are not particularly limited, such as redox transitions and hydrogenation-reduction transitions between sulfhydryl groups and disulfide bonds. The acidic and alkaline conditions mentioned above mainly refer to the pH conditions of normal tissues, diseased tissues, organs or tissues in the treatment period, etc. For example, the stomach is acidic, and tumor sites are often acidic. Degradation can occur through metabolic effects in the body (such as physiological effects, such as enzymatic reactions, redox reactions), can occur in specific parts of the body due to microenvironmental stimulation (such as acidity and alkalinity), or can occur under clinical treatment stimulation (such as light, heat, low temperature), etc. It should be noted that some extreme conditions in organic chemistry relative to organisms, such as strong acids, strong bases, high temperatures (such as above 100°C), etc., are not included in the category of "degradation" under these conditions; for example, although ether bonds can be broken under strong acid conditions such as hydrobromic acid, they should be considered stable linking groups.

[0055] A "stable" linker can remain as a complete linker (stably covalently linked to its adjacent groups), allowing chemical changes that do not destroy the integrity of the linker to occur. The chemical changes are not particularly limited, and include but are not limited to isomerization, oxidation, reduction, ionization, protonation, deprotonation, substitution reactions, and the like. The conditions used to examine the stability of the linker are not particularly limited, and include but are not limited to light, heat, low temperature, enzymes, redox, neutral, acidic, alkaline, physiological conditions, in vitro simulated physiological conditions, and the like, preferably light, heat, enzymes, redox, acidic, alkaline, and the like. The stable existence of a linker refers to the fact that the linker can maintain its structural integrity and be stably connected to other parts in its use environment (such as the metabolic cycle in the body) without special stimulation (such as the pH conditions of a special part, light, heat, low temperature during treatment, etc.).

[0056] Linkers are not necessarily "stable" or "degradable." Their stability or degradability is influenced by the compound's structure and the specific environment in which they are used. For example, amide bonds are much more stable than ester bonds under acidic or alkaline conditions, and therefore are considered "stable" linkers. However, peptide bonds, formed by the dehydration condensation of the α-carboxyl group of an amino acid and the α-amino group of another amino acid, can be cleaved when exposed to specific enzymes and are therefore also considered "degradable" linkers. Carbamate and thiocarbamate groups can be both stable and degradable linkers; more generally, carbamate and thiocarbamate groups tend to degrade slowly, while non-peptide amide bonds can remain stable during circulation. For example, while common ester bonds can degrade under acidic and alkaline conditions, ester bonds in specialized structures can also degrade under ultraviolet light. For example, even if certain chemical bonds can be degraded under the action of specific enzymes, if their circulation pathway during clinical use does not pass through or basically does not pass through the specific enzyme environment (such as in the case of targeted drug administration), these chemical bonds can still be considered to be stable.

[0057] All compounds of the general formula should be understood to include their salts. The term "salt" is selected from any one, any two, or any combination of two or more of the acid addition salts formed by the corresponding compound with inorganic and / or organic acids and the base addition salts formed with inorganic and / or organic bases. When the compound of the general formula contains a basic moiety (such as, but not limited to, pyridine or imidazole) and an acidic moiety (such as, but not limited to, carboxylic acid), zwitterions ("inner salts") can be formed and are included in the term "salt" used. "Salt" can be a pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salt or other salt. Salts of compounds of the general formula can be formed by reacting the compound of the general formula with a certain amount (such as an equivalent amount) of an acid or base in a medium such as a salt precipitate or in an aqueous medium and then lyophilizing. Exemplary acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and the like. Exemplary base addition salts include ammonium salts, alkali metal salts (such as sodium salts, lithium salts and potassium salts), alkaline earth metal salts (such as calcium salts and magnesium salts), salts with organic bases (such as organic amines) and salts with amino acids (such as arginine or lysine). Basic nitrogen-containing groups can be quaternized with reagents such as lower alkyl halides (such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides), dialkyl sulfates (such as dimethyl, diethyl, dibutyl and diamyl sulfates), long chain halides (such as decyl, lauryl, tetradecyl and stearyl chlorides, bromides and iodides), arylalkyl halides (such as benzyl and phenethyl bromides) and others. Both the acid addition salts and the base addition salts are preferably pharmaceutically acceptable salts.

[0058] A "solvate" of a compound of the general formula refers to an aggregate comprising one or more molecules of the compound of the general formula and one or more solvent molecules. The solvent may be water, and the corresponding solvate may be referred to as a hydrate, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, and the like. The solvent may also be an organic solvent.

[0059] "Tautomers" refer to isomers of a compound that undergo tautomerism. Tautomerism refers to the process by which hydrogen atoms or protons shift within a molecule, causing a change in the compound's structure, often involving the conversion of single bonds to double bonds. Tautomerism includes, but is not limited to, keto-enol tautomerism, amide-imidic acid tautomerism, lactam-lactim tautomerism, enamine-imine tautomerism, enamine-enamine tautomerism (e.g., enzyme reactions catalyzed by pyridoxal phosphate), proton transfer tautomerism, and valence tautomerism.

[0060] "Stereoisomers" are isomers that have the same atomic bonding order but different three-dimensional structures. Stereoisomers can be divided into enantiomers and diastereomers, with diastereomers further comprising cis-trans isomers and conformers. Enantiomers are molecules that are mirror images of each other and cannot be superimposed.

[0061] "Isotopically substituted" refers to a derivative form of a compound in which one or more atoms in the molecule are replaced by their respective isotopes.

[0062] The heteroatom is not particularly limited and includes, but is not limited to, O, S, N, P, Si, F, Cl, Br, I, B, and the like.

[0063] When a group or compound is "containing a heteroatom", the position of the heteroatom is not particularly limited, including but not limited to the case where the heteroatom is located in the main chain, side chain and / or substituent.

[0064] Relative to a compound, a group formed after losing some atoms or groups is also called a residue.

[0065] When a molecular structure (including compounds, groups, fragments, etc.) is "substituted," it means that the molecular structure contains one or more substituents. A "substituted" structure is also referred to as a "substituted form," as opposed to an "unsubstituted" structure. Unless otherwise specified, a "substituent" can be a monoatomic or polyatomic group, excluding hydrogen atoms, and replaces an existing hydrogen atom in the molecular structure. For substituted hydrocarbon and heteroalkyl groups (including but not limited to alkyl, heteroalkyl, etc.), the substituents include but are not limited to =O, -OR", =NR", =N-OR", -NR"R", -SR", -F, -Cl, -Br, -I, -SiR"R"R", -C(=O)R", -OC(=O)R", -C(=O)OR", -OC(=O)OR", -C(=O)NR"R", -OC(=O)NR"R", -NR"C(=O)R", -NR"C(=O)NR"R", -NR"C(=O)OR", -CN, -NO2, etc.; wherein each R" is independently hydrogen or C containing 0-3 heteroatoms 1-10Alkyl; any two R" can optionally form a ring structure with the nitrogen atom to which they are commonly attached; for example, -NR"R" includes but is not limited to 1-pyrrolidinyl and 4-morpholinyl.

[0066] "Optionally substituted" means that the group or compound being described may be substituted or unsubstituted.

[0067] "Amino" and "amine" include monovalent, divalent, trivalent, tetravalent neutral or cationic forms. "Amino" includes primary amino (-NH2), secondary amino (-NH-), tertiary amino (-N<) and quaternary amino (>N + Amine compounds refer to NH3 substituted with hydrocarbons, including primary amines (single hydrocarbon substitution, such as CH3NH2), secondary amines (double hydrocarbon substitution, such as NH(CH3)2), tertiary amines (trihydrocarbon substitution, such as N(CH3)3) and quaternary amines (tetrahydrocarbon substitution, such as N + (CH3)3). The group formed by the loss of one or more hydrogen atoms from an amine compound is collectively referred to as an "amine group." The hydrogen atoms can be amino, hydrocarbon, or other groups. Cationic amino or amine groups are also called quaternary ammonium groups.

[0068] The term "amide" refers to an amide of the general formula R p -C(=O)-N(R q )2, wherein R p is an organic group, each R q are independently H or an organic group, and R p or R q When it is an organic group, its carbon atom is connected to (C=O) or N in the general formula. q Amides can be divided into primary amides, secondary amides and tertiary amides according to their q All are H, one R of the secondary amide q is H and the other R q For organic groups, the two R q They are all organic groups. Secondary amides are also called N-substituted amides, and tertiary amides are also called N,N-disubstituted amides.

[0069] The term "amide group" refers to a monovalent, divalent, or trivalent group formed by the loss of some atoms or groups from an amide. For example, -C(=O)NH- is a divalent amide group, and -C(=O)N< is a trivalent amide group. Unless otherwise specified, "amide bond" refers to -C(=O)NH-.

[0070] "Acylamide" refers to a group formed by linking an acyl group to an amine group, including primary amide groups (e.g., -C(=O)NH-) and secondary amide groups (e.g., -C(=O)N<). The acyl group can be a carbonyl group, a sulfonyl group, or a phosphoryl group, with carbonyl being the default.

[0071] "Hydrocarbons" refer to compounds composed of the elements carbon and hydrogen. Hydrocarbons that do not contain benzene rings or other aromatic rings are collectively referred to as "aliphatic hydrocarbons," while hydrocarbons that do contain benzene rings or other aromatic rings are collectively referred to as "aromatic hydrocarbons." Aliphatic hydrocarbons can be divided into open-chain hydrocarbons and alicyclic hydrocarbons, with open-chain hydrocarbons further divided into straight-chain hydrocarbons and branched-chain hydrocarbons. Based on the degree of unsaturation, aliphatic hydrocarbons can be divided into saturated aliphatic hydrocarbons and unsaturated aliphatic hydrocarbons, which can be further divided into alkanes, alkenes, and alkynes. Alkenes contain at least one carbon-carbon double bond, and alkynes contain at least one carbon-carbon triple bond.

[0072] "Hydrocarbyl" refers to a hydrocarbon radical formed by the loss of at least one hydrogen atom and may be monovalent, divalent, trivalent, tetravalent, or higher. Unless otherwise specified, a hydrocarbyl radical is assumed to be a monovalent hydrocarbyl radical. Unless otherwise specified, a hydrocarbyl radical is optionally substituted, and the substituents are not particularly limited. When an aliphatic hydrocarbon radical is "substituted," the substituents may or may not contain carbon-carbon double bonds, carbon-carbon triple bonds, and / or aromatic rings.

[0073] "Hydrocarbon derivatives" include aliphatic and aromatic hydrocarbon derivatives, whose structures contain one or more non-hydrocarbon moieties located as side groups, substituents, and / or backbones. Common non-hydrocarbon moieties are heteroatom groups. For example, ethers can be considered aliphatic hydrocarbon derivatives containing -O- groups in their backbones, and halogenated benzenes can be considered aromatic hydrocarbon derivatives containing halogen substituents. "Hydrocarbon derivative residue" refers to a group formed by the loss of at least one hydrogen atom from a hydrocarbon derivative.

[0074] "Alkyl" refers to a hydrocarbon group formed by losing a hydrogen atom at any position of an alkane, and can be straight-chain or branched. Specifically, for example, propyl refers to either n-propyl or isopropyl. Unless otherwise specified, an alkyl group is optionally substituted.

[0075] "Alkenyl" refers to a hydrocarbon group containing at least one carbon-carbon double bond formed by losing a hydrogen atom at any position of an alkene, and may be straight-chain or branched. For example, "C 2-15 "Alkenyl" refers to a straight chain or branched alkenyl group comprising 2 to 15 carbon atoms and at least one carbon-carbon double bond. An alkenyl group may contain one, two, three, four or more carbon-carbon double bonds. Unless otherwise specified, an alkenyl group is optionally substituted.

[0076] "Alkynyl" refers to a hydrocarbon group containing at least one carbon-carbon triple bond formed by losing a hydrogen atom at any position of an alkyne, and may be straight-chain or branched. For example, "C 2-15 "Alkynyl" refers to a straight chain or branched alkynyl group comprising 2 to 15 carbon atoms and at least one carbon-carbon triple bond. Alkynyl groups can contain one, two, three, four or more carbon-carbon triple bonds. Unless otherwise specified, alkynyl groups are optionally substituted.

[0077] "Alkylenylene" is a divalent hydrocarbon group, "alkylene" is a divalent alkyl group, "alkenylene" is a divalent alkenyl group, and "alkynylene" is a divalent alkynyl group. Unless otherwise specified, alkylene, alkylene, alkenylene, and alkynylene groups include open-chain structures and cyclic structures and are optionally substituted.

[0078] "Amino acid residue" includes residues formed by the following changes in amino acids: removal of a hydrogen atom from the amino group, removal of a hydroxyl group from the carboxyl group, removal of a hydrogen atom from the sulfhydryl group, protected amino groups, protected carboxyl groups, protected sulfhydryl groups, and combinations thereof. Amino acids may be of natural origin, non-natural origin, or a mixture of the two. The structure of amino acids may be L-type, D-type, or a mixture of the two. A divalent amino acid residue is preferably a residue formed by removing a hydrogen atom from the amino group and a hydroxyl group from the carboxyl group of the corresponding amino acid. For example, a divalent glycine residue preferably has structure.

[0079] "Oligopeptide" refers to a peptide molecule containing two or more amino acid residues, and preferably the number of amino acid residues constituting the oligopeptide is 2 to 20.

[0080] "Functional group," also known as a "functional group," includes, but is not limited to, reactive groups, protected reactive groups, precursors of reactive groups, and the like. A polyfunctional compound contains at least two functional groups, such as a polyol containing at least two hydroxyl groups, and a polythiol containing at least two sulfhydryl groups. It should be noted that a polyfunctional compound may contain more than one functional group. For example, tris(hydroxymethyl)aminomethane is a triol containing an amino group, and citric acid is a tricarboxylic acid containing a hydroxyl group.

[0081] Unless otherwise specified, the reactive groups involved in the preparation method also include protected forms thereof, and the protected forms can be deprotected in any appropriate step of the actual preparation process to obtain the corresponding active forms.

[0082] Ring atoms are atoms that together form the ring backbone.

[0083] "Minor modification" refers to chemical modifications that can be accomplished through simple chemical reaction processes. These simple chemical reaction processes primarily include protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation, and leaving group modification. Modification of the leaving group, i.e., the conversion of a leaving group from an ester to an acyl chloride, includes, but is not limited to, conversion. "Minor modification" refers to a structural form that can form the desired reactive group through minor modification.

[0084] "Protection" of reactive groups refers to the strategy of reversibly converting the protected reactive group into an inert (non-reactive) group using specific reagents. The portion of a protected group that distinguishes it from the unprotected form is called a "protecting group." For example, -OTBS is a protected form of a hydroxyl group (-OH), where TBS is the protecting group for the hydroxyl group.

[0085] "Deprotection" refers to the process of converting a protected form into an unprotected form.

[0086] "Hydroxy protecting groups" include groups commonly used in the art to protect hydroxyl groups, including but not limited to alkanoyl groups (e.g., acetyl, tert-butyryl), aralkanoyl groups (e.g., benzoyl), benzyl, trityl, trimethylsilyl, tert-butyldimethylsilyl, allyl, acetal, ketal, and the like. Removal of the acetyl group is generally carried out under alkaline conditions, with the most commonly used systems being NH3 / MeOH and NaOMe / MeOH. The benzyl protecting group can be removed by catalytic hydrogenation or reduction with sodium metal / lithium in ethanol or liquid ammonia. The trityl group is generally removed by catalytic hydrogenation. The trimethylsilyl group is typically removed using a fluoride-containing system (e.g., tetrabutylammonium fluoride / anhydrous THF). The tert-butyldimethylsilyl group can be removed using a fluoride-containing system or with aqueous acetic acid at room temperature. Protection of diols includes, but is not limited to, the formation of dioxolanes, dioxanes, cyclic carbonates, cyclic boronates, and the like.

[0087] "Thiol protecting groups" include groups commonly used in the art to protect thiol groups. Similar to hydroxyl groups, thiol groups can be protected in the form of thioethers and thioesters. Thiol protecting groups include, but are not limited to, tert-butyl, benzyl, substituted benzyl, diphenylmethyl, substituted diphenylmethyl, triphenylmethyl, acetyl, benzoyl, tert-butyloxycarbonyl, benzyloxycarbonyl, thioacetal, thioketal, and the like. Thioether deprotection can be achieved by acid-catalyzed reduction with Na / NH3 or with heavy metal ions such as Ag. + 、Hg + The reaction is then treated with hydrogen sulfide. Some hemimercaptoacetals containing S-diphenylmethyl, S-triphenylmethyl, S-2-tetrahydropyranyl, or S-isobutoxymethyl groups can be oxidized to disulfides using (SCN)2, iodine, or thionyl chloride, followed by reduction to thiols. The formation and deprotection of thioesters are similar to those for carboxylates.

[0088] "Carboxyl protecting groups" include groups commonly used in the art to protect carboxyl groups, such as alkyl groups (e.g., tert-butyl (tBu), methyl (Me), or ethyl (Et)) and aralkyl groups (e.g., benzyl (Bn)). "Protected carboxyl groups" refer to groups formed after a carboxyl group is protected by a suitable carboxyl protecting group, including but not limited to methoxycarbonyl, ethoxycarbonyl, tert-butyloxycarbonyl, and benzyloxycarbonyl. Carboxyl protecting groups can be removed by hydrolysis under the catalysis of an acid or base, and occasionally by thermal decomposition. For example, the tert-butyl group can be removed under mild acidic conditions, and the benzyl group can be removed by hydrogenolysis. Reagents for removing carboxyl protecting groups include but are not limited to TFA, H2O, LiOH, NaOH, KOH, MeOH, EtOH, and combinations thereof. The protected carboxyl group can produce the corresponding free acid during the deprotection process. The deprotection is carried out in the presence of a base, and the base and the free acid can form a pharmaceutically acceptable salt.

[0089] "Amino protecting groups" include groups commonly used in the art to protect amino groups, such as aryl C 1-6 Alkyl, C 1-6 Alkoxy C 1-6 Alkyl, C 1-6 Alkoxycarbonyl, aryloxycarbonyl, C 1-6 Alkylsulfonyl, arylsulfonyl, silyl, etc. The amino protecting group is preferably Boc (tert-butyloxycarbonyl), Moz (p-methoxybenzyloxycarbonyl), Cbz (benzyloxycarbonyl), or Fmoc (9-fluorenylmethyleneoxycarbonyl). Reagents for removing amino protecting groups include, but are not limited to, TFA, H2O, LiOH, MeOH, EtOH, and combinations thereof. The reagent for removing Boc protection is preferably TFA. The reagent for removing Fmoc protection is preferably a 20% piperidine solution in N,N-dimethylformamide (DMF).

[0090] "Alkynyl protecting groups" include groups commonly used in the art to protect alkynyl groups, including but not limited to trimethylsilyl (TMS), triethylsilyl, tert-butyldimethylsilyl (TBS), biphenyldimethylsilyl, and the like. TMS-protected alkynyl groups are easily deprotected under alkaline conditions (e.g., K2CO3 / MeOH or KOH / MeOH). TBS-protected alkynyl groups can be deprotected in a solution of tetra-n-butylammonium fluoride in tetrahydrofuran (TBAF / THF).

[0091] There is no particular limitation on the hydroxyl groups that can be protected, for example, the hydroxyl groups in compounds such as alcohols and phenols; there is no particular limitation on the amino groups that can be protected, for example, the amino groups in compounds such as primary amines, secondary amines, hydrazines, and amides.

[0092] "Lipids" are a broad group of organic compounds, including fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, K), monoglycerides, diglycerides, phospholipids and other hydrophobic or amphiphilic small molecules. Lipids include simple esters, complex esters and derived lipids. The simple esters are esters composed of fatty acids and alcohols, which can be divided into three subcategories: fats, oils and waxes. The complex esters are also called "lipids", including phospholipids, sphingolipids, glycolipids, steroids, sterols, and lipoproteins. The derived lipids, including simple lipid derivatives and complex lipid derivatives, have the general properties of lipids. Lipids can be synthetic or derived (isolated or modified) from compounds of natural sources.

[0093] "Lipid nanoparticles" or "LNPs" (lipid nanoparticles) refer to nanoscale (e.g., 1 nm to 1000 nm) particles comprising one or more lipids. LNPs may further comprise at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules).

[0094] A "cationic lipid" can be a lipid that is positively charged at any pH or hydrogen ion activity, or a lipid that is capable of becoming positively charged in response to the pH or hydrogen ion activity of its intended use environment (i.e., a "cationizable lipid"), the latter also including zwitterionic lipids that meet the aforementioned characteristics. In some cases, the positive charge in the cationic lipid is derived from the presence of a quaternary nitrogen atom. For LNPs containing cationizable lipids, preferably about 1% to 100% of the cationizable lipid is converted to a cationic form at a pH of about 1 to 9, more preferably at a pH of 4 to 9, 5 to 8, or 6 to 8, and most preferably at an endosomal pH (e.g., about 5.5 to 6.5).

[0095] "PEGylated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion, and can be further divided into linear PEGylated lipids and non-linear PEGylated lipids based on the structure of the polyethylene glycol portion.

[0096] "Neutral lipids" refer to lipids, including phospholipids, that exist in an overall electrically neutral nonionic or zwitterionic form at a selected pH.

[0097] "Steroid lipids" refer to fused ring systems consisting of three cyclohexanes and one cyclopentane. The main characteristic lipids.

[0098] "Targeting group" refers to a group that provides a stronger affinity for a selected target (e.g., a cell, tissue, organ, body region or compartment, such as a cell, tissue or organ compartment). Some exemplary targeting groups include, but are not limited to, residues of antibodies, antigens, peptides, vitamins, carbohydrates (including but not limited to monosaccharides such as N-acetylgalactosamine (GalNAc)), folic acid, aptamers, receptor ligands, transferrin, biotin, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands.

[0099] "N / P ratio" refers to the molar ratio of cationizable groups (typically tertiary amine groups) in the cationic lipid to phosphate groups in the nucleic acid.

[0100] "Nucleic acid" refers to DNA, RNA or modified forms thereof, containing the purine and pyrimidine bases (adenine "A", cytosine "C", guanine "G", thymine "T", uracil "U") that make up DNA or RNA.

[0101] "RNA" refers to naturally occurring or non-naturally occurring ribonucleic acid. RNA may include one or more modified and / or non-naturally occurring nucleobases, nucleosides, nucleotides, or linkers. RNA may include a cap structure, chain-terminating nucleosides, stem-loops, polyadenylation sequences, and / or polyadenylation signals. RNA includes, but is not limited to, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), single-stranded guide RNA (sgRNA), cas9 mRNA, and the like. Antisense oligonucleotides or small interfering RNA (siRNA) can inhibit the expression of target genes and target proteins in vitro or in vivo. RNA may have a nucleotide sequence encoding a specific polypeptide, such as mRNA, which can be translated to produce the encoded polypeptide. FLuc mRNA can express luciferase protein, which emits bioluminescence in the presence of a luciferin substrate.

[0102] "Inhibiting the expression of a target gene" 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 a target gene) is contacted with a nucleic acid that inhibits the expression of a target gene. The expression of the target gene in a test sample or a test animal is compared with the expression of the target gene in a control sample (e.g., a cell sample in a culture medium expressing a target gene) that is not exposed to or not administered with nucleic acid. The expression of the target gene in the control sample can be designated as a value of 100%. In a specific embodiment, when the target gene expression level in the test sample is lower than 100% relative to the target gene expression level in the control sample or a control mammal, for example, about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0%, the expression of the target gene is inhibited.

[0103] Methods for determining target gene expression levels include, but are not limited to, dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme assays, and phenotypic assays.

[0104] "Transfection" refers to the process of introducing a species (e.g., RNA) into a cell. Transfection can occur in vitro, ex vivo, or in vivo.

[0105] "Antigen" typically refers to a substance that can be recognized by the immune system, preferably by the adaptive immune system, and can trigger 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 can comprise a peptide or protein that can be presented to a T cell by MHC. An antigen can be a translation product of a provided nucleic acid molecule (preferably an mRNA as defined herein). In this context, fragments, variants, and derivatives of peptides and proteins comprising at least one epitope are also understood to be antigens.

[0106] "Delivery" refers to providing an entity to a target. For example, a drug and / or therapeutic agent and / or prophylactic agent is delivered to a subject, which is an organ and / or tissue and / or cell of a human and / or other animal.

[0107] "Pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with a therapeutic agent and, within the scope of sound medical judgment, suitable for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reaction, or other problems or complications commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers that can be used in pharmaceutical compositions 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, and the like. When the pharmaceutical composition is administered intravenously, water is an exemplary carrier. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injections. 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, and the like. The pharmaceutical composition may also contain a small amount of a wetting agent, emulsifier, or pH buffer, as needed. Oral formulations may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Specifically, for example, excipients include, but are not limited to, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), demulcents, emulsifiers, fillers (diluents), film formers, flavorings, fragrances, glidants (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 phosphate, calcium stearate, cross-linked sodium carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, citric acid, cross-linked polyvinyl pyrrolidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, phenylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, and xylitol.

[0108] The pharmaceutical composition can act systemically and / or locally. For this purpose, they can be administered by the approach that is suitable, for example, by injection (such as intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermal administration, or by oral, buccal, nasal, transmucosal, local, in the form of ophthalmic preparations, or by inhalation. The pharmaceutical composition can be administered in the dosage form that is suitable. The dosage form includes but is not limited to tablets, capsules, lozenges, hard candies, powders, sprays, emulsifiable pastes, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

[0109] "Antagomirs," also known as "anti-miRs," are a class of chemically engineered oligonucleotides designed to silence endogenous miRNAs.

[0110] A "vaccine" is a prophylactic or therapeutic material that provides at least one antigen or antigenic function that can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0111] "Treatment" refers to the management and care of a patient for the purpose of combating a disease, disorder or condition, and is intended to include delaying the progression of the disease, disorder or condition, alleviating or relieving symptoms and complications, and / or curing or eliminating the disease, disorder or condition. The patient to be treated is preferably a mammal, especially a human.

[0112] 2. Cationic lipids containing disulfide bonds

[0113] One embodiment of the present invention:

[0114] A cationic lipid containing a disulfide bond, characterized in that the structure is as shown in the general formula (1):

[0115] or a salt, tautomer, stereoisomer, isotope-substituted product or solvate thereof;

[0116] Among them, -SS- is a disulfide bond;

[0117] L1 and L2 are each independently -(CH2) t -L d -(CH2) t -or-(CH2) t -L d -(CH2) t -L d -(CH2) t -, wherein each t is independently an integer from 1 to 4;

[0118] L d is a degradable divalent linking group. Any two Ld have the same or different structures;

[0119] X1 and X2 are independently Among them, R d2 、R d3 are each independently a hydrogen atom or an optionally substituted C 1-6 Alkyl, and R d2 、R d3 The number of is independently 1, 2, 3 or 4; s is 1, 2 or 3; n is 1 or 2;

[0120] G1 and G2 are each independently a connecting bond or a trivalent branching group;

[0121] When G1 is a connecting bond, k1 is 1; when G1 is a trivalent branching group, k1 is 2;

[0122] When G2 is a connecting bond, k1 is 1; when G2 is a trivalent branching group, k2 is 2;

[0123] L3, L4 each occur independently selected from a linker, -(CH2) q -, Z, P0, and any combination thereof, wherein q is an integer of 1-10, Z is a divalent linking group containing a heteroatom, and P0 is p is independently 0 or 1 each time it occurs;

[0124] R1 and R2 are each independently an optionally substituted C 5-30 Hydrocarbon or C 5-30 Hydrocarbon derivative residues.

[0125] 2.1.L d , L1, L2

[0126] In a specific embodiment of the present invention, L da divalent linking group selected from any one of the following structures: disulfide, vinyl ether, ester, thioester, dithioester, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate, carbamate, thiocarbamate, dithiocarbamate, urea, thiourea, acetal, cyclic acetal, thioacetal, azaacetal, azaheterocyclic acetal, azathiaacetal, dithioacetal, hemiacetal, thiohemiacetal, azahemiacetal, ketal, thioketal, azaketal, azaheterocyclic ketal, azathiaketal, imine, hydrazone, acylhydrazone, oxime, thiooxime ether, semicarbazone, thiosemicarbazone, hydrazine, hydrazide, thiocarbohydrazide, azocarbonylhydrazide, thioazocarbonylhydrazide , carbazate, hydrazinothiocarbazide, thiocarbazide, azo bond, isourea, isothiourea, allophanate, thioallocarbamate, guanidine, amidine, aminoguanidine, amidine, imidoester, imidothioester, sulfonate, sulfinate, sulfonylhydrazide, sulfonylurea, maleimide, orthoester, phosphate, phosphite, hypophosphite, phosphonate, phosphosilane ester, silane ester, carbonamide, thioamide, sulfonamide, polyamide, phosphoramide, phosphoramidite, pyrophosphamide, cyclophosphamide, ifosfamide, thiophosphamide, aconitamide, oligopeptide fragment, nucleotide and its derivative backbone, deoxynucleotide and its derivative backbone; or, L d It is a degradable divalent linking group composed of any of the aforementioned divalent linking groups and an adjacent heteroatom group; the adjacent heteroatom group is selected from -O-, -S-, -C(=O), -NR c - and any combination thereof, wherein R c A hydrogen atom or C 1-6 Alkyl; preferably, L d A divalent linking group selected from any one of esters, carbonates, carbamates, thiocarbamates, dithiocarbamates, ureas, and thioureas, or L d It is a divalent linking group composed of an oligopeptide fragment and an adjacent heteroatom group.

[0127] In a specific embodiment of the present invention, L d Selected from -OC(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)NH-, -NHC(=O)O-, -NHC(=S)O-, -NHC(=O)NH-, -NHC(=S)NH-, -L x -(AA) m -L x - any one; among which, -(AA) m - is a divalent oligopeptide linker, m is 2 or 3; L xEach occurrence is independently -C(=O), -NH- or -O-, and forms an amide bond or an ester bond with the adjacent AA; m AAs are each independently an amino acid residue or an amino acid derivative residue, wherein the amino acid is selected from any one of glycine, alanine, β-alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, ornithine and citrulline;

[0128] In a more specific embodiment, -(AA) m - is an oligopeptide divalent linker, the oligopeptide being selected from any one of glycine-phenylalanine, histidine-β-alanine, glycine-glycine, lysine-glycine, lysine-glutamic acid, glycine-glycine-glycine, glycine-glycine-phenylalanine, glycine-phenylalanine-glycine, glycine-lysine-glycine, glycine-histidine-lysine, glycine-cysteine-glutamic acid, glycine-leucine-glycine, valine-citrulline-glycine, valine-alanine-glycine, arginine-glycine-aspartic acid, and glycine-histidine-proline;

[0129] In a more specific embodiment, -(AA) m - is a divalent linking group of a phenylalanine-glycine dipeptide;

[0130] In a more specific embodiment, -(AA) m - is a divalent linker of a glycine-glycine-glycine tripeptide;

[0131] In a more specific embodiment, L1 and L2 are each independently -(CH2) t -OC(=O)-(CH2) t -、-(CH2) t -C(=O)O-(CH2) t -、-(CH2) t -OC(=O)O-(CH2) t -、-(CH2) t -NHC(=O)O-(CH2) t -、-(CH2) t -NHC(=S)O-(CH2) t -、-(CH2) t -NHC(=O)NH-(CH2) t -、-(CH2) t -NHC(=S)NH-(CH2) t -、-(CH2) t-OC(=O)-(CH2) t -C(=O)O-(CH2) t -、-(CH2) t -OC(=O)O-(CH2) t -OC(=O)-(CH2) t -、-(CH2) t -L x -(AA) m -L x -(CH2) t -, and any of its connecting ends is connected to a disulfide bond; preferably, L1 and L2 are each independently -CH2-OC(=O)-CH2-, -(CH2)2-OC(=O)-CH2-, -(CH2)3-OC(=O)-CH2-, -(CH2)3-C(=O)O-(CH2)2-, -(CH2)2-OC(=O)O-(CH2)2-, -(CH2)2-NHC(=O)O-(CH2)2-, -(CH2)2-NHC( =S)NH-(CH2)2-, -(CH2)2-OC(=O)-(CH2)2-C(=O)O-(CH2)2-, -(CH2)3-OC(=O)O-(CH2)2-OC(=O)-CH2-, -(CH2)3-C(=O)-Gly-Phe-O-(CH2)2-, -(CH2)3-C(=O)-(Gly)3-O-(CH2)2-; wherein Gly is a glycine residue and Phe is a phenylalanine residue.

[0132] In a specific embodiment of the present invention, the structure of the glycine residue is The structure of the phenylalanine residue is

[0133] In one embodiment of the present invention, L1 and L2 are the same.

[0134] 2.2.X1, X2

[0135] In a specific embodiment of the present invention, R d2 、R d3 are each independently a hydrogen atom;

[0136] Preferably, X1 and X2 are each independently Any one of them.

[0137] In one embodiment of the present invention, X1 and X2 are the same.

[0138] 2.3.R1, R2

[0139] In a specific embodiment of the present invention, R1 and R2 are the same or different and are each independently C 5-30 Aliphatic hydrocarbon group, C 5-30 Aliphatic hydrocarbon derivative residues, C 7-30 Aromatic hydrocarbon or C 7-30 Aromatic hydrocarbon derivative residue; preferably, R1, R2 are each independently selected from R L 、R B 、R r and R A More preferably, R1 and R2 are each independently R L 、R B or R A ;

[0140] R L C is a linear structure 5-30 Aliphatic hydrocarbon group, containing 0-4 carbon-carbon double bonds or carbon-carbon triple bonds; preferably, R L for Any one or any substituted form of; wherein, p R Each occurrence is independently an integer from 1 to 15; the substituted form contains 1 to 4 substituents; each occurrence of the substituent is independently a linear or branched C 1-4 Alkyl, hydroxyl or halogen atom, preferably methyl or hydroxyl; more preferably, R L Any of the following structures:

[0141] R B C for branched structure 5-30 Aliphatic hydrocarbon or C 5-30 An aliphatic hydrocarbon derivative residue selected from any of the following structures:

[0142] Preferably, R B Any of the following structures:

[0143] R r C containing a ring 5-30 Aliphatic hydrocarbon group, preferably

[0144] R A C 7-30 Aromatic hydrocarbon or C 7-30 Aromatic hydrocarbon derivative residue, preferably

[0145] 2.4.L3, L4

[0146] In a specific embodiment of the present invention, L3 and L4 are each independently a linker, -Z-(CH2)q -、-Z-(CH2) q -Z-(CH2) q -、-Z-P0-(CH2) q -、-Z-P0-Z-(CH2) q -or-P0-(CH2) q -, and the left end is connected to R1 or R2; wherein the structures of any two Z are the same or different;

[0147] In a more specific embodiment, each occurrence of Z is independently selected from -O-, -S-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -NR c -、-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 -、-NR c C(=O)S-、-C(R c )=N-NR c -、-NR c -N=C(R c )-, -CH(OH)-, R c Each occurrence is independently H or methyl; preferably, each occurrence of Z is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -OC(=O)O-, -OC(=O)NH-, -NHC(=O)O-, and -CH(OH)-;

[0148] In a more specific embodiment, P0 is

[0149] In one specific embodiment of the present invention, L3 and L4 are the same.

[0150] In a specific embodiment of the present invention, L3 and L4 are the same and belong to any of the following situations:

[0151] Case (1): L3 and L4 are both connecting bonds;

[0152] Case (2): L3 and L4 are both -C(=O)O-(CH2) q -or-OC(=O)-(CH2) q -;

[0153] Case (3): L3 and L4 are both -C(=O)NH-(CH2) q -or-NHC(=O)-(CH2) q -;

[0154] Case (4): L3 and L4 are both -OC(=O)NH-(CH2) q -or-NHC(=O)O-(CH2) q -;

[0155] Case (5): L3 and L4 are both -OC(=O)-(CH2) q -C(=O)O-(CH2) q -、-OC(=O)-(CH2) q -OC(=O)-(CH2) q -、-C(=O)O-(CH2) q -C(=O)O-(CH2) q -、-C(=O)O-(CH2) q -OC(=O)-(CH2) q -、-OC(=O)-(CH2) q -C(=O)NH-(CH2) q -、-OC(=O)-(CH2) q -NHC(=O)-(CH2) q -、-C(=O)O-(CH2) q -C(=O)NH-(CH2) q -、-C(=O)O-(CH2) q -NHC(=O)-(CH2) q -、-OC(=O)-(CH2) q -CH(OH)-(CH2) q -or-C(=O)O-(CH2) q -CH(OH)-(CH2) q -;

[0156] Case (6): L3 and L4 are both -OC(=O)-P0-(CH2) q -or-C(=O)O-P0-C(=O)O-(CH2) q -, preferably both

[0157] In any of the above situations, the left end of L3 is connected to R1, and the left end of L4 is connected to R2.

[0158] 2.5. Other general formulas

[0159] In a specific embodiment of the present invention, the structure of the disulfide bond-containing cationic lipid is shown in any of the following general formulas:

[0160] In a specific embodiment of the present invention, the structure of the disulfide bond-containing cationic lipid is shown in formula (2) or (3):

[0161] In the general formula (3), G1 and G2 are both trivalent branched groups, the two L3 are the same, the two R1 are the same, the two L4 are the same, and the two R2 are the same;

[0162] In a more specific embodiment, the structure of the cationic lipid is as shown in formula (3), wherein G1 and G2 are each independently The * end is connected to X1 or X2, and the other two ends are connected to L3 or L4. g Any one selected from -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -OC(=O)O-, -OC(=O)NH-, -NHC(=O)O-;

[0163] Preferably, G1 and G2 are each independently selected from any one of the following structures:

[0164] 2.6. Specific structure example

[0165] In a specific embodiment of the present invention, the structure of the disulfide bond-containing cationic lipid is selected from any one of the following:

[0166] In a specific embodiment of the present invention, the structure of the disulfide bond-containing cationic lipid is selected from any one of the following:

[0167] 3. Preparation of disulfide-bonded cationic lipids

[0168] In the preparation method of the cationic lipids of the present invention, reactive groups are protected and deprotected as needed. When a reaction involves two or more reactive groups, to prevent a particular reactive group from affecting the desired reaction, the reactive group is typically protected, selectively allowing only the desired reactive group to react. The protected group remains stable during the desired reaction and can be deprotected as needed at any appropriate stage in the preparation process using conventional techniques in the art.

[0169] The reaction types involved in the preparation method of the cationic lipid of the present invention include coupling reactions. Based on the successful implementation of the present invention, the type of the coupling reaction is not particularly limited, as long as the reactive group can form a covalent linker through the reaction. The same preparation process may contain single-step or step-by-step coupling reactions, and preferably each coupling reaction step is independently any one of an alkylation reaction, a condensation reaction, an amidation reaction, an esterification reaction, a thioesterification reaction, a ring-opening reaction, a ring-closing condensation reaction, an addition reaction, a cycloaddition reaction, an α,β-unsaturated bond addition reaction, an alkynyl addition reaction, a Schiff base reaction combined with a reduction reaction, a click reaction, an azide-alkyne addition reaction, a 1,3-dipolar cycloaddition reaction, a Diels-Alder addition reaction, a thiol-yne reaction, a thiol-ene reaction, and a thiol-vinyl reaction. The reaction conditions of the coupling reaction are related to the type of covalent linker generated by the reaction, and existing public technologies can be used. The valence state of the covalent linker generated by the coupling reaction can be divalent or trivalent, preferably divalent. The coupling reaction can generate stable groups or degradable groups.

[0170] The disulfide bond-containing cationic lipids of the present invention can be prepared by any of the following methods, including but not limited to: Unless otherwise specified, the definitions of the parameters (e.g., X1, X2, L1, L2, etc.) in the following methods are the same as those described above.

[0171] Method 1: First couple the compound containing a disulfide bond with the compound containing X1 / X2, and then further couple the compound containing R1 / R2.

[0172] Step 1: The disulfide bond-containing bifunctionalized small molecule A is reacted with the bifunctionalized small molecule B-1 containing X1 and the bifunctionalized small molecule B-2 containing X2 to obtain the bifunctionalized intermediate C. When B-1 and B-2 are the same, compound A and B-1 can be reacted in one step to obtain compound C. G1 The same or different from each other, preferably the same. G1 and F G2 The divalent linking group obtained by the reaction and its adjacent two L from A and B-1 respectively m Together they constitute L1, FG1 and F G4 The divalent linking group obtained by the reaction and its adjacent two L from A and B-2 respectively m Together they constitute L2.

[0173] Step 2: The difunctionalized intermediate C is reacted with the monofunctionalized small molecules D-1 and D-2 containing R1 / R2 to obtain a cationic lipid containing a disulfide bond represented by the general formula (1). m Together they form L3 or L4, in which case the corresponding G1 or G2 is a connecting bond; or the trivalent connecting group generated by the reaction is connected to the L in C. m Together they form (L3)2G1 or (L4)2G2, where the corresponding G1 or G2 is a trivalent branched group. When D-1 and D-2 are the same, one molecule of compound C reacts with two molecules of D-1 to produce a cationic lipid of formula (2); or one molecule of compound C reacts with four molecules of D-1 to produce a cationic lipid of formula (3).

[0174] L m is a connecting bond or a divalent connecting group, any two L m They have the same or different structures, preferably a connecting bond, an alkylene group or 1-3 L m0 interrupted alkylene, said alkylene being substituted or unsubstituted, each L m0 are independently selected from any one of -O-, -S-, -C(=O), -C(=S)-, -C(=O)NH-, -NHC(=O)-, -C(=O)O- and -OC(=O)-, and any one of L m0 Not with any other L m0 Directly connected. G1 、F G2 、F G3 、F G4 、F G5 、F G6 、F G7 are all reactive groups or their slightly modified forms, and the selection of their specific types is subject to the smooth implementation of the present invention. G1 、F G2 、F G3 、F G4 、F G5 、F G6 、F G7Each is any one of -OH, -NH2, -COOH, active ester group, -F, -Cl, -Br, -I, -OMs, -OTs, anhydride group, epoxy group, -NCO, -NCS or a protected form thereof or an acid / base addition salt form. The protected form of the reactive group can be deprotected in any appropriate step to carry out the necessary subsequent reaction or to obtain the final product. In particular, F in B-1 G3 or F in B-2 G5 It can also be a hydrogen atom or an amino protecting group directly connected to the nitrogen atom of X1 or X2; the amino protecting group is preferably -Boc, -Cbz or Fmoc.

[0175] For example, exemplary compound A With exemplary compounds B-1 / B-2 The reaction gave exemplary compound C Furthermore, exemplary compounds D-1 / D-2 Reacting with the aforementioned exemplary compound C yields a disulfide bond-containing cationic lipid having the following structure:

[0176] The compound C obtained in the aforementioned step 1 can also be first subjected to a coupling reaction with other bifunctionalized small molecules to obtain another compound C having the same general formula but a different specific structure for the reaction in step 2.

[0177] Method 2: First couple the compound containing X1 / X2 with the compound containing R1 / R2, and then further couple the compound containing a disulfide bond.

[0178] The main difference between Method 2 and Method 1 is that in Method 2, Compound B-1 / B-2 is first reacted with Compound D-1 / D-2 to obtain Compound E-1 containing both R1 and X1 or Compound E-2 containing both R2 and X2. Further, Compound A reacts with Compound E-1 / E-2 to obtain a disulfide bond-containing cationic lipid represented by Formula (1). m 、F G1 、F G2 、F G3 、F G4 、F G5 、F G6 、F G7 The definition of and other details are the same as those in Method 1 and will not be repeated here.

[0179] Method 3: Cationic lipids with four tail chains are obtained through the coupling reaction of trifunctionalized small molecule compounds.

[0180] The compound C in method 1 is reacted with the trifunctionalized small molecule compound E to obtain a compound containing four reactive groups (2 F G9 and 2 Fs G10 ) intermediate compound F. Wherein compound E contains a trivalent branched core G0, and its reactive group F G8 and F of compound C G3 The resulting divalent linker reacts with two adjacent L groups from C and E. m and G0 together form the trivalent branched core G1; similarly, the trivalent branched core G2 is formed by the F of compound E. G8 and F of compound C G5 The reaction yields compound F. G9 、F G10 The reactive groups can be the same or different. Similar to step 2 of method 1, compound F is coupled with compound D-1 / D-2 to form a divalent linker with L in F. m Together they constitute L3 / L4 and give a cationic lipid represented by the general formula (3).

[0181] F G8 、F G9 、F G10 are all reactive groups or their slightly modified forms, and the selection of their specific types is subject to the smooth implementation of the present invention. G8 、F G9 、F G10 Each is any one of -OH, -NH2, -COOH or a protected form or acid / base addition salt form thereof. m 、F G3 、F G5 、F G6 、F G7 The definition of and other details are the same as those in Method 1 and will not be repeated here.

[0182] For example, exemplary compound C With exemplary compound E The reaction gave exemplary compound F Furthermore, the aforementioned exemplary compound F reacts with exemplary compound D-1 / D-2 (myristic acid) to obtain a compound of the general formula (3) having the following structure:

[0183] 4. Lipid compositions, lipid pharmaceutical compositions, lipid pharmaceutical composition preparations, and methods for preparing the same

[0184] One embodiment of the present invention:

[0185] A lipid composition characterized by containing any of the aforementioned disulfide bond-containing cationic lipids.

[0186] In a specific embodiment of the present invention, the aforementioned lipid composition further contains phospholipids; or, the aforementioned lipid composition further contains steroid lipids; or, the aforementioned lipid composition further contains PEGylated lipids; or, the aforementioned lipid composition further contains phospholipids and steroid lipids; or, the aforementioned lipid composition further contains phospholipids and PEGylated lipids; or, the aforementioned lipid composition further contains steroid lipids and PEGylated lipids; or, the aforementioned lipid composition further contains phospholipids, steroid lipids and PEGylated lipids; or, the aforementioned lipid composition further contains phospholipids, steroid lipids, PEGylated lipids and another cationic lipid; or, the aforementioned lipid composition further contains phospholipids, steroid lipids, PEGylated lipids and anionic lipids.

[0187] In a specific embodiment of the present invention, the phospholipids in the lipid composition are selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG) , palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE) and any combination thereof.

[0188] In a specific embodiment of the present invention, the steroid lipid in the lipid composition is selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and combinations thereof.

[0189] In a specific embodiment of the present invention, the PEGylated lipid in the lipid composition is selected from any one of a non-targeted PEGylated lipid, a targeted PEGylated lipid, and a combination thereof; preferably, the non-targeted PEGylated lipid is selected from polyethylene glycol-dipalmitoylphosphatidylcholine (PEG-DPPC), polyethylene glycol-dimyristyl glycerol (PEG-DMG), polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE), polyethylene glycol-dioleoylphosphatidylethanolamine (PEG-DOPE), polyethylene glycol-cholesterol (PEG-Chol), polyethylene glycol-diacylglycerol (PEG-DAG), polyethylene glycol-dialkyloxypropyl (PEG-DAP) A) and any one of combinations thereof; more preferably, the non-targeted PEGylated lipid is selected from any one of polyethylene glycol 500-dipalmitoylphosphatidylcholine, polyethylene glycol 2000-dipalmitoylphosphatidylcholine, polyethylene glycol 500-distearoylphosphatidylethanolamine, polyethylene glycol 2000-distearoylphosphatidylethanolamine, polyethylene glycol 500-dioleoylphosphatidylethanolamine, polyethylene glycol 2000-dioleoylphosphatidylethanolamine, polyethylene glycol 500-dimyristoylglycerol, polyethylene glycol 2000-dimyristoylglycerol (PEG2k-DMG) and any one of combinations thereof; preferably, the targeted PEGylated lipid is a PEGylated lipid modified with folic acid or N-acetylgalactosamine.

[0190] In a specific embodiment of the present invention, the other cationic lipid in the lipid composition is selected from 1,2-dioleoyl-3-trimethylammonium-propane (methylsulfate) (DOTAP), 1,2-dioctadecenyloxy-3-methylammonium propane chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 1,2-dioleyl-3-dimethylamino-propane (DODMA), 2 ,3-bis(tetradecanoyloxy)propyltrimethylammonium chloride (DMTAP), didecyldimethylammonium chloride (DDAC), didecyldimethylammonium bromide (DDAB), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-ammonium (DOBAQ), 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5- Diketone (cKK-E12), 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester (DLin-MC3-DMA), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholesterol Any one of base (EPC), ((4-hydroxybutyl) azadiyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino] octanoate (heptadecan-9-yl) ester (SM-102) and ((2-(2-hydroxyethoxy)ethyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (DHA-1).

[0191] In a specific embodiment of the present invention, the anionic lipid in the lipid composition is selected from any one of 1,2-dioleoyl-sn-glycero-3-phosphate sodium salt (18:1PA), 1,2-dimyristoyl-sn-glycero-3-phosphate sodium salt (14:0PA), bis(monooleoylglycerol) phosphate ammonium salt (18:1BMP) and cardiolipin (CL).

[0192] In a specific embodiment of the present invention, the molecular weight of the polyethylene glycol portion of any PEGylated lipid is preferably 0.5-2 kDa, more preferably 500 Da, 1000 Da or 2000 Da.

[0193] In a specific embodiment of the present invention, the PEGylated lipid in the lipid composition is a combination of a non-targeted PEGylated lipid and a targeted PEGylated lipid.

[0194] In a specific embodiment of the present invention, the molar percentage of PEGylated lipids in the lipid composition is 0.5-5% of the total lipids, preferably 1-3%, more preferably 1.5%, 1.6%, 1.7%, 1.8% or 1.9%; the molar percentage of cationic lipids in the total lipids is 30-65%, preferably 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50% or 55%; the molar percentage of phospholipids in the total lipids is 7.5-13%, preferably 8%, 9%, 10%, 11% or 12%; the molar percentage of steroid lipids in the total lipids is 35-50%, preferably 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%.

[0195] One embodiment of the present invention:

[0196] A lipid pharmaceutical composition comprises any of the aforementioned lipid compositions and a drug, wherein the drug is selected from any one of nucleic acid drugs, small molecule drugs, polypeptide drugs and protein drugs.

[0197] In a specific embodiment of the present invention, the aforementioned drug is a nucleic acid drug, selected from any one of DNA, RNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir and ribozyme; the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA and siRNA, preferably any one of mRNA, miRNA and siRNA.

[0198] In a specific embodiment of the present invention, the lipid pharmaceutical composition is used as a drug, and the drug is selected from any one of the following: a drug for treating cancer, an anti-infective agent, and a vaccine; the anti-infective agent is an antiparasitic agent, an antibiotic, an antifungal agent, or an antiviral agent.

[0199] In a specific embodiment of the present invention, the lipid pharmaceutical composition LNP pharmaceutical composition, LPP pharmaceutical composition or PNP pharmaceutical composition, is preferably an LNP pharmaceutical composition, more preferably an LNP-nucleic acid pharmaceutical composition, and most preferably an LNP-mRNA pharmaceutical composition. Wherein, "LNP pharmaceutical composition" is a pharmaceutical composition in the form of lipid nanoparticles (lipid nanoparticle), "LPP pharmaceutical composition" is a pharmaceutical composition in the form of lipid polyplexes (lipopolyplex), and "PNP pharmaceutical composition" is a pharmaceutical composition in the form of polypeptide nanoparticles (polypeptide nanoparticle); wherein, "LNP-nucleic acid pharmaceutical composition" is an LNP pharmaceutical composition loaded with nucleic acid, and "LNP-mRNA pharmaceutical composition" is an LNP pharmaceutical composition loaded with mRNA.

[0200] In a specific embodiment of the present invention, the drugs in the lipid pharmaceutical composition include but are not limited to doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracycline, nitrogen mustard, thiotepa, chlorambucil, razithromycin, melphalan, carmustine, lomustine, busulfan, dibromomannitol, mitomycin C, cis-dichlorodiamine platinum (II), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine, dibucaine, chlorpromazine, propranolol, dimerol, labetalol, clonidine, hydralazine, imipramine, amitriptyline, Doxepin, phenytoin, diphenhydramine, chlorpheniramine, promethazine, gentamicin, ciprofloxacin, cefoxitin, miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, netifine, amphotericin B, antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, sedatives, imaging agents, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, colchicine, daunorubicin, dihydroxyanthraquinone, mithramycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, puromycin, maytansine.

[0201] In a specific embodiment of the present invention, the drug in the lipid pharmaceutical composition is a nucleic acid drug, and the N / P ratio is (0.1-100):1, preferably (0.2-30):1, and more preferably (0.5-20):1.

[0202] One embodiment of the present invention:

[0203] A lipid pharmaceutical composition preparation, comprising any of the aforementioned lipid pharmaceutical compositions and a working solution; the working solution is a pharmaceutically acceptable diluent or excipient, preferably any one of deionized water, ultrapure water, phosphate buffer and normal saline, more preferably phosphate buffer or normal saline, most preferably normal saline.

[0204] In a specific embodiment of the present invention, the ratio of the lipid pharmaceutical composition to the working solution contained in the aforementioned lipid pharmaceutical composition preparation is not particularly limited. Preferably, the lipid pharmaceutical composition: working solution = 0.05-20 g: 100 mL, more preferably the lipid pharmaceutical composition: working solution = 0.1-10 g: 100 mL, and most preferably the lipid pharmaceutical composition: working solution = 0.2-5 g: 100 mL.

[0205] In a specific embodiment of the present invention, the preparation of the lipid pharmaceutical composition preparation comprises the following steps:

[0206] (1) equilibrating the lipid component in a diluent or excipient;

[0207] (2) adding the drug to the equilibrated mixture for compounding;

[0208] The equilibration time is not particularly limited, and is preferably 0.1 to 12 h, more preferably 0.2 to 6 h, and most preferably 0.5 to 3 h; the recombination time is not particularly limited, and is preferably 0.1 to 12 h, more preferably 0.2 to 5 h, and most preferably 0.5 to 2 h.

[0209] In a specific embodiment of the present invention, the preparation of the LNP-nucleic acid pharmaceutical composition comprises the following steps:

[0210] (1) dissolving the lipid component in an organic solvent to obtain an organic phase solution;

[0211] (2) adding the nucleic acid drug to a buffer solution to obtain an aqueous solution;

[0212] (3) mixing the organic phase solution and the aqueous phase solution to obtain an LNP-nucleic acid pharmaceutical composition, washing the mixture by ultrafiltration to remove the organic solvent and free molecules, and finally passing the mixture through a sterile filter for later use;

[0213] Among them, the organic solvent is preferably any one of methanol, ethanol, propanol, tert-butanol, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone, or a mixed solvent of any one or more thereof; the buffer is preferably a citrate buffer, and further, preferably, its concentration is 5-80 mM, pH is 2-6, more preferably a concentration is 10-50 mM, and pH is 3-5; the volume ratio of the organic phase solution to the aqueous phase solution is preferably 1:1-10, more preferably 1:2 or 1:3.

[0214] In a specific embodiment of the present invention, ultrasound, extrusion or microfluidics is used to control the particle size of lipid nanoparticles, and the particle size is 1 to 1000 nm, preferably 20 to 500 nm, more preferably 60 to 200 nm, and most preferably 60 to 150 nm. 5. Specific implementation methods

[0215] The preparation of disulfide bond-containing cationic lipids, lipid compositions, lipid pharmaceutical compositions, and the biological activity testing of LNP-nucleic acid pharmaceutical compositions are further described below in conjunction with some specific examples. The specific examples are provided to further illustrate the present invention and are not intended to limit the scope of the present invention.

[0216] Example 1: Preparation of L d Cationic lipids with ester groups

[0217] Example 1.1: Preparation of cationic lipid E1-1

[0218] In E1-1, L1 and L2 are both -(CH2)2-OC(=O)-CH2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -C(=O)O-(CH2)2-, and R1 and R2 are both tridecyl.

[0219] The preparation method is as follows:

[0220] Step a: Under argon atmosphere, a dichloromethane solution (30 mL) containing 2,2-dithiodiacetic acid (S1-1, 0.44 g, 2.4 mmol), N,N'-bis(2-hydroxyethyl)piperazine (S1-2, 2.09 g, 12.0 mmol), and 4-dimethylaminopyridine (DMAP, 0.12 g, 1.0 mmol) was placed in an ice bath. A dichloromethane solution (15 mL) of N,N'-dicyclohexylcarbodiimide (DCC, 1.09 g, 5.3 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to afford S1-3 (1.11 g).

[0221] Step b: Under an argon atmosphere, a dichloromethane solution (20 mL) containing S1-3 (0.99 g, 2.0 mmol), myristic acid (S1-4, 1.14 g, 5.0 mmol), and DMAP (0.12 g, 1.0 mmol) was placed in an ice bath, and a dichloromethane solution (15 mL) of DCC (1.13 g, 5.5 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain cationic lipid E1-1 (1.59 g). 1 H NMR(400MHz, CDCl3)δ:4.28(t,4H,-C(=O)OCH2-),4.20(t,4H,-C(=O)OCH2-),3.60( s,4H,-OC(=O)CH2S-),2.69-2.63(m,8H,-C(=O)OCH2CH2N<),2.56(s,16H,>N(CH2)2 N<),2.30(t,4H,-CH2CH2C(=O)O-),1.66-1.57(m,4H,-CH2CH2C(=O)O-),1.32-1.20 (m,40H,-CH2CH3,-CH2CH2CH2-),0.88(t,6H,-CH2CH3).MS(ESI):m / z=915.4([M+H] + ).

[0222] Example 1.2: Preparation of cationic lipid E1-2

[0223] In E1-2, L1 and L2 are both -CH2-OC(=O)-CH2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -C(=O)O-(CH2)3-, and R1 and R2 are both tridecyl.

[0224] The preparation method is as follows:

[0225] Step a: Under argon, a dichloromethane solution (30 mL) containing 2,2-dithiodiacetic acid (S1-1, 0.58 g, 3.2 mmol), a Boc derivative of 4-hydroxymethylpiperidine (S1-5, 1.72 g, 8.0 mmol), and DMAP (0.16 g, 1.3 mmol) was placed in an ice bath. A dichloromethane solution (20 mL) of DCC (1.45 g, 7.0 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was treated with a mixture of trifluoroacetic acid and dichloromethane (TFA / DCM) (1:1 v / v) to remove the Boc protecting group, followed by washing with purified water and extraction with dichloromethane. The extract was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to afford S1-6 (1.09 g).

[0226] Step b: Dissolve S1-6 (0.75 g, 2.0 mmol) and N,N-diisopropylethylamine (DIPEA, 0.77 g, 6.0 mmol) in 15 mL of dichloromethane. Add the TBS derivative of 3-bromo-1-propanol (S1-7, 1.11 g, 4.4 mmol) with stirring and allow to react overnight at room temperature. After completion, pour the reaction mixture into 30 mL of water and extract three times with dichloromethane (15 mL x 3). The organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Add 10 mL of anhydrous tetrahydrofuran to the residue, followed by a 1 M solution of tetrabutylammonium fluoride in tetrahydrofuran (TBAF / THF, 10 mL). Stir overnight to remove the TBS protection. After completion of the reaction, concentrate under reduced pressure. Dissolve the residue in 20 mL of dichloromethane, wash with saturated ammonium chloride solution, dry over anhydrous sodium sulfate, filter, and concentrate. The residue was purified by column chromatography to give S1-8 (0.76 g).

[0227] Step c: Under argon atmosphere, a dichloromethane solution (10 mL) containing S1-8 (0.59 g, 1.2 mmol), myristic acid (S1-4, 0.68 g, 3.0 mmol), and DMAP (0.07 g, 0.6 mmol) was placed in an ice bath, and a dichloromethane solution (10 mL) of DCC (0.68 g, 3.3 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain cationic lipid E1-2 (0.94 g). 1 H NMR(400MHz, CDCl3)δ:4.10(t,4H,-C(=O)OCH2CH2-),4.01(d,4H,>CHCH2OC(=O)-),3.58(s,4H,-OC(=O)CH2S-),2.93(d,4H,>NCH a H b CH2CH<),2.40(t,4H,-CH2N<),2.28(t,4H,-CH2CH2C(=O)O-),1.99-1.88(m,4H,>NCH a H b CH2CH<),1.87-1.76(m,6H,-C(=O)OCH2CH2CH2N<,>N(CH2)2CH<),1.73-1.67(m,4H,>NCH2CH a H b CH<),1.64-1.56(m,4H,>NCH2CH a H b CH<),1.39-1.19(m,44H,-CH2CH3,-CH2CH2CH2-),0.88(t,6H,-CH2CH3).MS(ESI):m / z=913.4([M+H] + ).

[0228] Example 1.3: Preparation of cationic lipid E1-3

[0229] In E1-3, L1 and L2 are both -CH2-OC(=O)-CH2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -C(=O)O-(CH2)4-CH(OH)-CH2-, and R1 and R2 are both nonyl groups.

[0230] The preparation method is as follows:

[0231] Epoxide S1-9 (2.2 mmol, 0.59 g, synthesized according to the method disclosed in WO2023126006A1) was dissolved in ethanol, and S1-6 (1.0 mmol, 0.38 g) was added. The mixture was stirred at 60°C for 10 hours. After completion of the reaction, dilution was added with dichloromethane and the mixture was washed with saturated sodium chloride solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E1-3 (0.81 g). 1 H NMR(400MHz, CDCl3)δ:4.06(t,4H,-C(=O)OCH2CH2-),4.03(d,4H,>CHCH2OC(=O)-),3.59(s,4H,-OC(=O)CH2S-),3.64-3.59(m,2H,>CHOH),2.67-2.45(m, 12H,-CH2N<),2.29(t,4H,-CH2CH2C(=O)O-),1.71-1.20(m,50H,>NCH2CH2CH<,-CH2CH3,-CH2CH2CH2-),0.87(t,6H,-CH2CH3).MS(ESI):m / z=917.4([M+H] + ).

[0232] Comparative Example 1.4: Preparation of comparative structure R1-1-ether

[0233] R1-1-ether is a comparative structure of E1-1. There is no degradable group between the polar head (piperazine group) containing the tertiary amine group and the disulfide bond (such as L in E1-1). d Instead of an ester group, a stable divalent ether group (-O-) is used.

[0234] The preparation method is as follows:

[0235] Step a: Dissolve the mesylate derivative of 2-hydroxyethyl disulfide (S2-1-OMs, 0.62 g, 2.0 mmol) in 40 mL of DMF, add S1-2 (1.74 g, 10.0 mmol) and K2CO3 (2.76 g, 20.0 mmol), and stir overnight at room temperature. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 40 mL of dichloromethane. After washing with 10% citric acid (20 mL x 2) and brine (20 mL x 2), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to afford S1-12 (0.81 g).

[0236] Step b: Under argon atmosphere, a dichloromethane solution (10 mL) containing S1-12 (0.56 g, 1.2 mmol), myristic acid (S1-4, 0.68 g, 3.0 mmol), and DMAP (0.07 g, 0.6 mmol) was placed in an ice bath, and a dichloromethane solution (10 mL) of DCC (0.68 g, 3.3 mmol) was slowly added dropwise. Subsequently, the reaction mixture was warmed to room temperature and stirred for 24 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the cationic lipid R1-1-ether (0.80 g). 1 H NMR(400MHz, CDCl3)δ:4.20(t,4H,-C(=O)OCH2-),3.75(t,4H,-OCH2CH2S-),3.6 2(t,4H,>NCH2CH2O-),2.90(m,4H,-OCH2CH2S-),2.70-2.44(m,24H,-CH2N<),2. 30(t,4H,-CH2CH2C(=O)O-),1.63-1.55(m,4H,-CH2CH2C(=O)O-),1.37-1.23(m, 40H,-CH2CH3,-CH2CH2CH2-),0.87(t,6H,-CH2CH3).MS(ESI):m / z=887.5([M+H] + ).

[0237] Comparative Example 1.5: Preparation of comparative structure R1-1-LB

[0238] R1-1-LB is a comparative structure of E1-1. There is no heteroatom group between the polar head (piperazine group) containing the tertiary amine group and the disulfide bond, and it can be regarded as L d The result is replaced by the join key.

[0239] The preparation method is as follows:

[0240] Step a: Dissolve the mesylate derivative of 2-hydroxyethyl disulfide (S2-1-OMs, 0.62 g, 2.0 mmol) in 20 mL of DMF. Add the TBS derivative of N-hydroxyethylpiperazine (S1-13, 1.22 g, 5.0 mmol) and K2CO3 (1.38 g, 10.0 mmol). Stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 20 mL of dichloromethane. Wash with 10% citric acid (10 mL x 2) and then brine (10 mL x 2). The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to afford S1-14 (0.53 g).

[0241] Step b: Under an argon atmosphere, a dichloromethane solution (10 mL) containing S1-14 (0.45 g, 1.2 mmol), myristic acid (S1-4, 0.68 g, 3.0 mmol), and DMAP (0.07 g, 0.6 mmol) was placed in an ice bath, and a dichloromethane solution (10 mL) of DCC (0.68 g, 3.3 mmol) was slowly added dropwise. Subsequently, the reaction mixture was warmed to room temperature and stirred for 24 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the cationic lipid R1-1-LB (0.79 g). 1 H NMR(400MHz, CDCl3)δ:4.18(t,4H,-C(=O)OCH2-),2.85-2.80(t,4H,>NCH2CH2S-),2.70-2.44(m,24H,-C(=O)OCH2CH2N<,>NCH2CH2N<,>NCH2CH2S-),2.32(t ,4H,-CH2CH2C(=O)O-),1.66-1.58(m,4H,-CH2CH2C(=O)O-),1.39-1.25(m,40 H,-CH2CH3,-CH2CH2CH2-),0.88(t,6H,-CH2CH3).MS(ESI):m / z=799.4([M+H] + ).

[0242] Example 2: Preparation of L d Cationic lipids containing oligopeptide divalent linkers

[0243] Example 2.1: Preparation of cationic lipid E2-1

[0244] In E2-1, L1 and L2 are both -(CH2)3-C(=O)-Gly-Phe-O-(CH2)2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -C(=O)O-(CH2)2-, and R1 and R2 are both tridecyl.

[0245] The preparation method is as follows:

[0246] Step a: Dissolve the Boc derivative of the phenylalanine-glycine dipeptide (S2-2, 4.83 g, 15.0 mmol) in 60 mL of anhydrous dichloromethane. Add NHS (2.59 g, 22.5 mmol) and then DCC (4.64 g, 22.5 mmol). Add DMAP (0.37 g, 3.0 mmol) to a solution of 2-hydroxyethyl disulfide (S2-1, 0.92 g, 6.0 mmol) in 10 mL of dichloromethane. Combine the two solutions and stir at room temperature for 24 hours. After completion of the reaction, remove the insoluble material by filtration and concentrate. The residue is then treated with a TFA / DCM mixture (1:1 v / v) to remove the Boc protecting group, washed with purified water, and extracted with dichloromethane. The extract is dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to yield S2-3 (2.66 g).

[0247] Step b: Dissolve 4-bromobutyric acid (S2-4, 1.67 g, 10.0 mmol) in 40 mL of anhydrous dichloromethane, add NHS (1.73 g, 15.0 mmol), and then add DCC (3.09 g, 15.0 mmol). Add DMAP (0.24 g, 2.0 mmol) to a solution of S2-3 (2.25 g, 4.0 mmol) in 30 mL of dichloromethane. Combine the two solutions and stir at room temperature for 24 hours. After completion of the reaction, filter to remove insoluble matter, concentrate, and purify the residue by column chromatography to afford S2-5 (3.00 g).

[0248] Step c: Dissolve S1-13 (1.83 g, 7.5 mmol) and DIPEA (1.45 g, 11.3 mmol) in 30 mL of dichloromethane. Add S2-5 (2.58 g, 3.0 mmol) with stirring and allow to react overnight at room temperature. After completion of the reaction, pour the reaction mixture into 60 mL of water and extract three times with dichloromethane (30 mL x 3). The organic phases are collected, combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is added to 20 mL of anhydrous tetrahydrofuran, followed by a 1 M TBAF / THF solution (20 mL). Stir overnight to remove the TBS protection. After completion of the reaction, concentrate under reduced pressure. The residue is dissolved in 40 mL of dichloromethane, washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to yield S2-6 (2.03 g).

[0249] Step d: Under an argon atmosphere, a dichloromethane solution (15 mL) containing S2-6 (1.15 g, 1.2 mmol), myristic acid (S1-4, 0.68 g, 3.0 mmol), and DMAP (0.07 g, 0.6 mmol) was placed in an ice bath, and a dichloromethane solution (10 mL) of DCC (0.68 g, 3.3 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain cationic lipid E2-1 (1.03 g). 1 H NMR(400MHz, CDCl3)δ:7.35-7.26(m,2H,Ar),7.25-7.19(m,8H,Ar),4.54-4.42(m,2H,Ar-CH2-CH< ),4.35(t,4H,-C(=O)OCH2CH2-S-),4.19(t,4H,-C(=O)OCH2CH2N<),3.74-3.70(m,2H,-C(=O)NHCH a H b C(=O)NH-),3.66-3.62(m,2H,-C(=O)NHCH a H b C(=O)NH-),3.15-2.73(m,8H,Ar-CH2-CH<,-C(=O)OCH2CH2S-),2.70-2.45(m,24H,>NC H2-),2.30(t,4H,-CH2CH2CH2C(=O)O-),2.18-2.13(m,4H,>NCH2CH2CH2C(=O)NH-),1. 75-1.71(m,4H,>NCH2CH2CH2C(=O)NH-),1.65-1.56(m,4H,-CH2CH2C(=O)O-),1.40-1. 24(m,40H,-CH2CH3,-CH2CH2CH2-),0.88(t,6H,-CH2CH3).MS(ESI):m / z=1379.7([M+H] + ).

[0250] Example 2.2: Preparation of cationic lipid E2-2

[0251] In E2-2, L1 and L2 are both -(CH2)3-C(=O)-Gly-Phe-O-(CH2)2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -C(=O)O-CH2-, and R1 and R2 are both tridecyl.

[0252] The preparation method is as follows:

[0253] Step a: Dissolve the TBS derivative of 4-hydroxymethylpiperidine (S2-7, 1.72 g, 7.5 mmol) and DIPEA (1.45 g, 11.3 mmol) in 30 mL of dichloromethane. Add S2-5 (2.58 g, 3.0 mmol) with stirring and allow to react overnight at room temperature. After completion of the reaction, pour the reaction mixture into 60 mL of water and extract three times with dichloromethane (30 mL x 3). The organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Add 20 mL of anhydrous tetrahydrofuran to the residue, followed by a 1 M TBAF / THF solution (20 mL). Stir overnight to remove the TBS protection. After completion of the reaction, concentrate under reduced pressure. The residue is dissolved in 40 mL of dichloromethane, washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to yield S2-0 (2.22 g).

[0254] Step b: Under an argon atmosphere, a dichloromethane solution (30 mL) containing S2-0 (1.86 g, 2.0 mmol), myristic acid (S1-4, 1.14 g, 5.0 mmol), and DMAP (0.12 g, 1.0 mmol) was placed in an ice bath, and a dichloromethane solution (15 mL) of DCC (1.13 g, 5.5 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain cationic lipid E2-2 (1.49 g). 1 H NMR(400MHz, CDCl3)δ:7.37-7.27(m,2H,Ar),7.26-7.20(m,8H,Ar),4.56-4.43(m,2H,Ar-CH2-CH<) ,4.34(t,4H,-C(=O)OCH2CH2-S-),3.98-3.95(d,4H,-C(=O)OCH2CH<),3.74-3.70(m,2H,-C(=O)NHCH a H b C(=O)NH-),3.66-3.62(m,2H,-C(=O)NHCH a H bC(=O)NH-),3.16-2.74(m,8H,Ar-CH2-CH<,-C(=O)OCH2CH2S-),2.71-2.44(m,12H,>N CH2-),2.29(t,4H,-CH2CH2CH2C(=O)O-),2.17-2.12(m,4H,>NCH2CH2CH2C(=O)NH-),1 .76-1.59(m,18H,>NCH2CH2CH2C(=O)NH-,>NCH2CH2CH<,-CH2CH2C(=O)O-),1.41-1.2 6(m,40H,-CH2CH3,-CH2CH2CH2-),0.89(t,6H,-CH2CH3).MS(ESI):m / z=1349.7([M+H] + ).

[0255] Example 3: Preparation of a plurality of L d Cationic lipids

[0256] In E3-1, L1 and L2 are both -(CH2)2-OC(=O)-(CH2)2-C(=O)O-(CH2)2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -OC(=O)-(CH2)7-, and R1 and R2 are both nonyl groups.

[0257] The preparation method is as follows:

[0258] Step a: Under argon, a solution of the tBu derivative containing succinic acid (S3-1, 2.61 g, 15.0 mmol), S2-1 (0.92 g, 6.0 mmol), and DMAP (0.37 g, 3.0 mmol) in dichloromethane (40 mL) was placed in an ice bath. A solution of DCC (3.71 g, 18.0 mmol) in dichloromethane (50 mL) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was treated with a TFA / DCM mixture (1:1 v / v) to remove the Boc protecting group, washed with purified water, and extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to afford S3-2 (1.88 g).

[0259] Step b: Under argon, a dichloromethane solution (30 mL) containing S3-2 (1.42 g, 4.0 mmol), a Boc derivative of N-hydroxyethylpiperazine (S3-5, 2.30 g, 10.0 mmol), and DMAP (0.20 g, 1.6 mmol) was placed in an ice bath. A dichloromethane solution (30 mL) of DCC (2.06 g, 10.0 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was treated with a TFA / DCM mixture (1:1 v / v) to remove the Boc protecting group, washed with purified water, and extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to afford S3-6 (1.96 g).

[0260] Step c: S3-6 (1.16 g, 2.0 mmol) and DIPEA (0.77 g, 6.0 mmol) were dissolved in 20 mL of dichloromethane. S3-4 (1.75 g, 5.0 mmol, obtained by esterification of 8-bromooctanoic acid and 1-nonanol) was added with stirring and allowed to react overnight at room temperature. After completion of the reaction, the reaction mixture was poured into 40 mL of water and extracted three times with dichloromethane (20 mL*3). The organic phases were collected, combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain cationic lipid E3-1 (1.63 g). 1 H NMR(400MHz, CDCl3)δ:4.36(t,4H,-C(=O)OCH2CH2S-),4.19(t,4H,>NCH2CH2OC(=O)-),4.08(t,4H,- CH2CH2CH2OC(=O)-),2.92-2.87(m,4H,-C(=O)OCH2CH2S-),2.73-2.44(m,32H,-CH2N<,-OC(=O)CH2CH 2C(=O)O-),2.28(t,4H,-CH2CH2CH2C(=O)O-),1.65-1.53(m,12H,-CH2CH2CH2N<,-CH2CH2OC(=O)CH2C H2-),1.40-1.25(m,36H,-CH2CH3,-CH2CH2CH2-),0.88(t,6H,-CH2CH3).MS(ESI):m / z=1115.5([M+H] + ).

[0261] Example 4: Preparation of L d Cationic lipids containing carbonate, carbamate or thiourea groups

[0262] Example 4.1: Preparation of cationic lipid E4-1

[0263] In E4-1, L1 and L2 are both -(CH2)3-OC(=O)O-(CH2)2-OC(=O)-CH2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -C(=O)O-(CH2)2-, and R1 and R2 are both tridecyl.

[0264] The preparation method is as follows:

[0265] Step a: Under nitrogen, compound S4-1 (3.65 g, 12.0 mmol, obtained by reacting p-nitrophenyl chloroformate and 3-bromo-1-propanol) was dissolved in DCM (50 mL). S4-2 (2.53 g, 14.4 mmol) was added with stirring at room temperature, followed by the slow dropwise addition of pyridine (1.45 mL, 18.0 mmol) and DMAP (0.29 g, 2.4 mmol). The reaction was stirred at room temperature for 16 h. After completion of the reaction, the mixture was washed with water and extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was added to 30 mL of anhydrous tetrahydrofuran, followed by a 1 M TBAF / THF solution (30 mL). The mixture was stirred overnight to remove the TBS protection. After completion of the reaction, the mixture was concentrated under reduced pressure. The residue was dissolved in 40 mL of dichloromethane, washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to give S4-3 (2.63 g).

[0266] Step b: Under argon atmosphere, a dichloromethane solution (30 mL) containing S4-3 (2.27 g, 10.0 mmol), S1-1 (0.73 g, 4.0 mmol), and DMAP (0.20 g, 1.6 mmol) was placed in an ice bath. A dichloromethane solution (20 mL) of DCC (1.81 g, 8.8 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to afford S4-4 (1.66 g).

[0267] Step c: Dissolve S1-13 (1.22 g, 5.0 mmol) and DIPEA (0.97 g, 7.5 mmol) in 15 mL of dichloromethane, add S4-4 (1.20 g, 2.0 mmol) with stirring, and react at room temperature overnight. After completion of the reaction, pour the reaction mixture into 30 mL of water and extract three times with dichloromethane (15 mL x 3). The organic phases are collected, combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to afford S4-5 (0.89 g).

[0268] Step d: Under an argon atmosphere, a dichloromethane solution (10 mL) containing S4-5 (0.70 g, 1.0 mmol), myristic acid (S1-4, 0.57 g, 2.5 mmol), and DMAP (0.06 g, 0.5 mmol) was placed in an ice bath, and a dichloromethane solution (10 mL) of DCC (0.57 g, 2.8 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain cationic lipid E4-1 (0.62 g). 1 H NMR(400MHz, CDCl3)δ:4.48-4.40(m,8H,-OC(=O)OCH2CH2OC(=O)-),4.20-4.16(m,8H,-C(=O)OCH2C H2N<,>NCH2CH2CH2OC(=O)O-),3.87-3.71(m,4H,-OC(=O)CH2S-),2.73-2.45(m,24H,-CH2N<),2.32( t,4H,-CH2CH2C(=O)O-),1.86-1.70(m,4H,>NCH2CH2CH2OC(=O)O-),1.67-1.58(m,4H,-CH2CH2C(=O) O-),1.40-1.24(m,40H,-CH2CH3,-CH2CH2CH2-),0.87(t,6H,-CH2CH3).MS(ESI):m / z=1119.5([M+H] + ).

[0269] Example 4.2: Preparation of cationic lipid E4-2

[0270] In E4-2, L1 and L2 are both -(CH2)2-OC(=O)O-(CH2)2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -OC(=O)-(CH2)7-, and R1 and R2 are both nonyl groups.

[0271] The preparation method is as follows:

[0272] Step a: S2-1 (0.92 g, 6.0 mmol) was dissolved in 30 mL of anhydrous dichloromethane and placed in an ice bath. Triethylamine (TEA, 1.67 mL, 12.0 mmol) and p-nitrobenzoyl chloride (S4-6, 1.82 g, 9.0 mmol) were added sequentially, and the mixture was allowed to warm to room temperature for 2 h. After completion of the reaction, the mixture was quenched with water, washed with purified water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to afford S4-7 (2.85 g).

[0273] Step b: Under nitrogen, S4-7 (1.94 g, 4.0 mmol) was dissolved in DCM (40 mL). S3-5 (2.30 g, 10.0 mmol) was added with stirring at room temperature, followed by the slow dropwise addition of pyridine (0.97 mL, 12.0 mmol) and DMAP (0.20 g, 1.6 mmol). The reaction was stirred at room temperature for 16 h. After completion of the reaction, the mixture was washed with water and extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was treated with a TFA / DCM mixture (1:1 v / v) to remove the Boc protecting group, washed with purified water, and extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to afford S4-8 (1.39 g).

[0274] Step c: S4-8 (0.93 g, 2.0 mmol) and DIPEA (0.77 g, 6.0 mmol) were dissolved in 20 mL of dichloromethane, and S3-4 (1.75 g, 5.0 mmol) was added with stirring. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the reaction mixture was poured into 40 mL of water and extracted three times with dichloromethane (20 mL*3). The organic phases were collected, combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain cationic lipid E4-2 (1.38 g). 1H NMR(400MHz, CDCl3)δ:4.43(t,4H,-OC(=O)OCH2CH2S-),4.27(t,4H,>NCH2CH2OC(=O)O-),4.09( t,4H,-CH2CH2CH2OC(=O)-),2.94-2.88(m,4H,-OC(=O)OCH2CH2S-),2.72-2.46(m,24H,-CH2N<) ,2.30(t,4H,-CH2CH2CH2C(=O)O-),1.68-1.55(m,12H,-CH2CH2CH2N<,-CH2CH2OC(=O)CH2CH2-) ,1.41-1.27(m,36H,-CH2CH3,-CH2CH2CH2-),0.89(t,6H,-CH2CH3).MS(ESI):m / z=1003.5([M+H] + ).

[0275] Example 4.3: Preparation of cationic lipid E4-3

[0276] In E4-3, L1 and L2 are both -(CH2)2-NHC(=O)O-(CH2)2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -OC(=O)-(CH2)7-, and R1 and R2 are both nonyl groups.

[0277] The preparation method is as follows:

[0278] Step a: Dissolve S2-1 (0.92 g, 6.0 mmol) in 50 mL of anhydrous dichloromethane. Add DIPEA (2.32 g, 18.0 mmol) and N,N'-succinimidyl carbonate (DSC, 3.84 g, 15.0 mmol) sequentially. Stir and react at room temperature overnight. After completion, concentrate under reduced pressure. The residue is dissolved in 50 mL of dichloromethane and washed with saturated sodium bicarbonate (20 mL x 3). The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to afford S4-9 (2.44 g).

[0279] Step b: Dissolve S4-9 (1.74 g, 4.0 mmol) in 30 mL of dichloromethane, add the Boc derivative of N-aminoethylpiperazine (S4-10, 2.29 g, 10.0 mmol) and TEA (2.78 mL, 20.0 mmol), and react at room temperature for 2 hours. After completion of the reaction, wash with saturated brine, and concentrate the organic phase under reduced pressure. The residue is treated with a TFA / DCM mixture (1:1 v / v) to remove the Boc protecting group, then wash with purified water and extract with dichloromethane. The extract is dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to afford S4-11 (1.44 g).

[0280] Step c: S4-11 (0.93 g, 2.0 mmol) and DIPEA (0.77 g, 6.0 mmol) were dissolved in 20 mL of dichloromethane, and S3-4 (1.75 g, 5.0 mmol) was added with stirring. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the reaction mixture was poured into 40 mL of water and extracted three times with dichloromethane (20 mL*3). The organic phases were collected, combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain cationic lipid E4-3 (1.23 g). 1 H NMR(400MHz, CDCl3)δ:4.32(t,4H,-NHC(=O)OCH2CH2S-),4.05(t,4H,-CH2CH2CH2OC(=O)-),3.32-3.25(m,4H, >NCH2CH2NHC(=O)O-),2.94(t,4H,-NHC(=O)OCH2CH2S-),2.69-2.47(m,20H,>N(CH2)2N<,>NCH2CH2NHC(=O)O- )2.45-2.40(m,4H,-CH2N<),2.29(t,4H,-CH2CH2CH2C(=O)O-),1.63-1.52(m,12H,-CH2CH2CH2N<,-CH2CH2OC( =O)CH2CH2-),1.33-1.25(m,36H,-CH2CH3,-CH2CH2CH2-),0.88(t,6H,-CH2CH3).MS(ESI):m / z=1001.5([M+H] + ).

[0281] Example 4.4: Preparation of cationic lipid E4-4

[0282] In E4-4, L1 and L2 are both -(CH2)2-NHC(=O)O-(CH2)2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -NHC(=O)O-(CH2)3-, and R1 and R2 are both dodecyl.

[0283] The preparation method is as follows:

[0284] Step a: Dissolve S4-11 (2.79 g, 6.0 mmol) and DIPEA (2.32 g, 18.0 mmol) in 50 mL of dichloromethane. Add S1-7 (3.80 g, 15.0 mmol) with stirring and allow to react overnight at room temperature. After completion of the reaction, pour the reaction mixture into 50 mL of water and extract three times with dichloromethane (50 mL x 3). The organic phases are collected, combined, washed with saturated brine, and concentrated under reduced pressure. Add 30 mL of anhydrous tetrahydrofuran to the residue, followed by a 1 M TBAF / THF solution (30 mL). Stir overnight to remove the TBS protection. After completion of the reaction, concentrate under reduced pressure. The residue is dissolved in 50 mL of dichloromethane, washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to yield S4-12 (2.41 g).

[0285] Step b: S4-12 (2.32 g, 4.0 mmol) was dissolved in 50 mL of anhydrous dichloromethane, and DIPEA (1.55 g, 12.0 mmol) and DSC (2.56 g, 10.0 mmol) were added sequentially. The mixture was stirred and reacted at room temperature overnight. After completion of the reaction, the mixture was washed with saturated sodium bicarbonate (20 mL*3). The organic phase was concentrated under reduced pressure, and the residue was dissolved in 50 mL of anhydrous dichloromethane. Dodecylamine (S4-13, 1.85 g, 10.0 mmol) and TEA (2.78 mL, 20.0 mmol) were added and reacted at room temperature for 2 hours. After completion of the reaction, the mixture was washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain cationic lipid E4-4 (2.54 g). 1H NMR(400MHz, CDCl3)δ:4.33(t,4H,-NHC(=O)OCH2CH2S-),4.25-2.20(m,4H,-NHC(=O)OCH2CH2CH2-),3.34 -3.29(m,4H,>NCH2CH2NHC(=O)O-),3.13-3.08(m,4H,-CH2CH2CH2NHC(=O)O-),2.93(t,4H,-NHC(=O)OCH2 CH2S-),2.71-2.45(m,24H,-CH2N<),1.74-1.58(m,4H,-OCH2CH2CH2N<),1.53-1.45(m,4H,-CH2CH2CH2NH C(=O)O-),1.37-1.23(m,36H,-CH2CH3,-CH2CH2CH2-),0.88(t,6H,-CH2CH3).MS(ESI):m / z=1003.5([M+H] + ).

[0286] Example 4.5: Preparation of cationic lipid E4-5

[0287] In E4-5, L1 and L2 are both -(CH2)2-NHC(=S)NH-(CH2)2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -NHC(=O)O-(CH2)3-, and R1 and R2 are both dodecyl.

[0288] The preparation method is as follows:

[0289] Step a: Dissolve S4-10 (2.75 g, 12.0 mmol) in 50 mL of tetrahydrofuran, add TEA (2.50 mL, 18.0 mmol), and add carbon disulfide (CS2, 1.19 g, 15.6 mmol) dropwise under ice-cooling. The reaction mixture is then warmed to room temperature. After stirring for 12 hours, DMAP (0.44 g, 3.6 mmol) is added. Di-tert-butyl dicarbonate ((Boc)2O, 3.40 g, 15.6 mmol) is added under ice-cooling, and the reaction is continued at room temperature for 3 hours. After completion of the reaction, the product is washed with saturated sodium chloride solution (20 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to afford S4-12 (2.35 g).

[0290] Step b: Under an ice bath, cystamine dihydrochloride (S4-13, 0.90 g, 4.0 mmol) was added to a dichloromethane solution (30 mL) containing S4-12 (2.17 g, 8.0 mmol), followed by the dropwise addition of a dichloromethane solution (20 mL) of TEA (1.11 mL, 8.0 mmol) over 15 min. The ice bath was removed and the reaction was allowed to proceed at room temperature for 5 hours. After completion of the reaction, the reaction mixture was washed sequentially with dilute hydrochloric acid (15 mL), water (15 mL), and brine (15 mL). The reaction mixture was concentrated under reduced pressure, the Boc protecting group was removed with a TFA / DCM mixture (1:1 v / v), and the mixture was washed with purified water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain S4-14 (1.27 g).

[0291] Step c: Dissolve S4-14 (0.99 g, 2.0 mmol) and DIPEA (0.77 g, 6.0 mmol) in 20 mL of dichloromethane. Add S4-15 (1.75 g, 5.0 mmol, obtained by esterification of undecanol and 6-bromohexanoic acid) with stirring and allow to react overnight at room temperature. After completion of the reaction, the reaction mixture was poured into 40 mL of water and extracted three times with dichloromethane (20 mL x 3). The organic phases were collected, combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain cationic lipid E4-5 (1.48 g). 1 H NMR(400MHz, CDCl3)δ:4.06(t,4H,-CH2OC(=O)-),3.93(q,4H,-NHC(=S)NHCH2CH2SS-),3.47-3 .43(m,4H,>NCH2CH2NHC(=S)NH-),2.96(t,4H,-NHC(=S)NHCH2CH2SS-),2.62-2.44(m,24H,>NC H2-),2.29(t,4H,-OC(=O)CH2-),1.66-1.53(m,12H,-CH2CH2CH2N<,-CH2CH2OC(=O)CH2CH2-), 1.33-1.17(m,36H,-CH2CH3,-CH2CH2CH2-),0.86(t,6H,-CH2CH3).MS(ESI):m / z=1031.5([M+H] + ).

[0292] Example 5: Preparation of cationic lipids with hydrophobic tails containing no heteroatom groups (E5-1)

[0293] In E5-1, L1 and L2 are both -(CH2)2-OC(=O)-(CH2)2-C(=O)O-(CH2)2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both connecting bonds, and R1 and R2 are both hexadecyl groups.

[0294] The preparation method is as follows:

[0295] S3-6 (1.16 g, 2.0 mmol) and DIPEA (0.77 g, 6.0 mmol) were dissolved in 20 mL of dichloromethane, and S5-1 (1.53 g, 5.0 mmol) was added with stirring. The mixture was allowed to react overnight at room temperature. After completion of the reaction, the reaction mixture was poured into 40 mL of water and extracted three times with dichloromethane (20 mL*3). The organic phases were collected, combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain cationic lipid E5-1 (1.52 g). 1 H NMR(400MHz, CDCl3)δ:4.37(t,4H,-C(=O)OCH2CH2S-),4.20(t,4H,>NCH2CH2OC(=O)-),2.94-2.88(m,4H,-C(=O)OCH2CH2S-),2.74-2.41(m,32H,-CH2N<,- OC(=O)CH2CH2C(=O)O-),1.64-1.53(m,4H,-CH2CH2CH2N<),1.40-1.24(m,52H,-CH2CH3,-CH2CH2CH2-),0.89(t,6H,-CH2CH3).MS(ESI):m / z=1027.5([M+H] + ).

[0296] Example 6: Preparation of cationic lipids with tail chains containing benzene ring structures

[0297] Example 6.1: Preparation of cationic lipid E6-1

[0298] In E6-1, L1 and L2 are both -(CH2)2-OC(=O)-CH2-, and X1 and X2 are both G1 and G2 are both connecting keys, L3 and L4 are R1 and R2 are both tridecyl.

[0299] The preparation method is as follows:

[0300] Step a: Under argon, a dichloromethane solution (50 mL) containing S1-3 (1.36 g, 6.0 mmol), a TBS derivative of p-hydroxyphenylacetic acid (S6-1, 2.73 g, 15.0 mmol), and DMAP (0.37 g, 3.0 mmol) was placed in an ice bath. A dichloromethane solution (50 mL) of DCC (3.71 g, 18.0 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was added to 30 mL of anhydrous tetrahydrofuran, followed by a 1 M TBAF / THF solution (30 mL). The mixture was stirred overnight to remove the TBS protection. After completion of the reaction, the residue was concentrated under reduced pressure, and the residue was dissolved in 50 mL of dichloromethane, washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to yield S6-2 (2.88 g).

[0301] Step b: Dissolve S6-2 (2.40 g, 4.0 mmol) and the anhydride compound S6-3 (5.27 g, 12.0 mmol, prepared using S1-4) in dichloromethane, add DMAP (0.20 g, 1.6 mmol), and react at room temperature for 9 hours. After completion of the reaction, the reaction solution was washed with 10% aqueous acetic acid and then deionized water, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain cationic lipid E6-1 (4.15 g). 1 H NMR(400MHz, CDCl3)δ:7.28(d,4H,Ar),7.02(d,4H,Ar),4.27(t,4H,-C(=O)OCH2-),4.21(t,4H ,-C(=O)OCH2-),3.60(d,8H,-OC(=O)CH2S-,Ar-CH2-C(=O)O-),2.68-2.59(m,8H,-C(=O)OCH2CH 2N<),2.59-2.36(m,20H,>N(CH2)2N<,-CH2CH2C(=O)O-),1.77-1.70(m,4H,-CH2CH2C(=O)O-), 1.39-1.13(m,40H,-CH2CH3,-CH2CH2CH2-),0.88(t,6H,-CH2CH3).MS(ESI):m / z=1183.5([M+H] + ).

[0302] Example 6.2: Preparation of cationic lipid E6-2

[0303] In E6-2, L1 and L2 are both -(CH2)2-OC(=O)-(CH2)2-C(=O)O-(CH2)2-, and X1 and X2 are both G1 and G2 are both connecting keys, L3 and L4 are R1 and R2 are both undecyl.

[0304] The preparation method is as follows:

[0305] Step a: Dissolve S3-6 (4.63 g, 8.0 mmol) and DIPEA (3.10 g, 24.0 mmol) in 60 mL of dichloromethane. Add S1-7 (5.06 g, 20.0 mmol) with stirring and allow to react overnight at room temperature. After completion of the reaction, pour the reaction mixture into 60 mL of water and extract three times with dichloromethane (60 mL x 3). The organic phases are collected, combined, and washed with saturated brine. The organic phase is concentrated under reduced pressure. The residue is added to 40 mL of anhydrous tetrahydrofuran, followed by a 1 M TBAF / THF solution (40 mL). Stir overnight to remove the TBS protection. After completion of the reaction, concentrate under reduced pressure. The residue is dissolved in 50 mL of dichloromethane, washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to yield S6-4 (3.70 g).

[0306] Step b: Under argon, a dichloromethane solution (30 mL) containing S6-4 (2.78 g, 4.0 mmol), a TBS derivative of 4-(hydroxymethyl)phenylacetic acid (S6-5, 2.80 g, 10.0 mmol), and DMAP (0.24 g, 2.0 mmol) was placed in an ice bath. A dichloromethane solution (30 mL) of DCC (2.47 g, 12.0 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was added to 20 mL of anhydrous tetrahydrofuran, followed by a 1 M TBAF / THF solution (20 mL). The mixture was stirred overnight to remove the TBS protection. After completion of the reaction, the residue was concentrated under reduced pressure, and the residue was dissolved in 40 mL of dichloromethane, washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to yield S6-6 (2.82 g).

[0307] Step c: Under argon atmosphere, a dichloromethane solution (30 mL) containing S6-6 (1.98 g, 2.0 mmol), lauric acid (S2-13, 1.00 g, 5.0 mmol), and DMAP (0.12 g, 1.0 mmol) was placed in an ice bath, and a dichloromethane solution (20 mL) of DCC (1.24 g, 6.0 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain cationic lipid E6-2 (1.65 g). 1 H NMR(400MHz, CDCl3)δ:7.28-7.24(m,4H,Ar),7.04-7.00(m,4H,Ar),5.09(s,4H,-C(=O)O-CH2-Ar-),4.34(t,4H,-C(=O)OCH 2CH2S-),4.18(t,4H,>NCH2CH2OC(=O)-),4.10(t,4H,-C(=O)OCH2CH2CH2N<),3.60(s,4H,-Ar-CH2-C(=O)O-),2.90-2.84(m, 4H,-C(=O)OCH2CH2S-),2.72-2.42(m,32H,-CH2N<,-OC(=O)CH2CH2C(=O)O-),2.29(t,4H,-CH2CH2CH2C(=O)O-),1.63-1.56( m,4H,-CH2CH2CH2C(=O)O-),1.43-1.22(m,36H,-CH2CH3,-CH2CH2CH2-),0.87(t,6H,-CH2CH3).MS(ESI):m / z=1355.6([M+H] + ).

[0308] Example 6.3: Preparation of cationic lipid E6-3

[0309] In E6-3, L1 and L2 are both -(CH2)2-OC(=O)-CH2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -OC(=O)-(CH2)2-C(=O)O-(CH2)2-, R1 and R2 are both

[0310] The preparation method is as follows:

[0311] Under an argon atmosphere, a dichloromethane solution (30 mL) containing S1-3 (0.99 g, 2.0 mmol), D-α-tocopheryl succinate (S6-7, 2.66 g, 5.0 mmol), and DMAP (0.12 g, 1.0 mmol) was placed in an ice bath, and a dichloromethane solution (15 mL) of DCC (1.13 g, 5.5 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 hours. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain cationic lipid E6-3 (2.41 g). 1 H NMR(400MHz, CDCl3)δ:4.21-4.17(m,8H,-C(=O)O-CH2-),3.86-3.72(m,4H,-OC(=O)CH2S-),2 .73-2.46(m,36H,-CH2N<,-OC(=O)CH2CH2C(=O)O-,-CH2CH2-Ar-),2.10-2.01(m,18H,-Ar-CH3 ),1.88-1.75(m,4H,-CH2CH2Ar-),1.61-1.03(m,48H,>CHCH2CH2CH2CH<,>CHCH2CH2CH2C(CH3) <,>C(CH3)O-),0.88-0.85(m,24H,(CH3)2CH-,-CH2C(CH3)CH2-).MS(ESI):m / z=1518.8([M+H] + ).

[0312] Example 7: Preparation of cationic lipids with double bonds in the tail chain

[0313] Example 7.1: Preparation of cationic lipid E7-1

[0314] In E7-1, L1 and L2 are both -(CH2)2-OC(=O)-CH2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -C(=O)O-(CH2)2-, and R1 and R2 are both heptadecan-8-enyl.

[0315] The preparation method is as follows:

[0316] Under an argon atmosphere, a dichloromethane solution (20 mL) containing S1-3 (0.99 g, 2.0 mmol), oleic acid (S7-1, 1.41 g, 5.0 mmol), and DMAP (0.12 g, 1.0 mmol) was placed in an ice bath, and a dichloromethane solution (15 mL) of DCC (1.24 g, 6.0 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 hours. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain cationic lipid E7-1 (1.41 g). 1 H NMR(400MHz, CDCl3)δ:5.38-5.31(m,4H,-CH2-CH=CH-CH2-),4.27(t,4H,-C(=O)OCH2-),4.19(t,4H,-C (=O)OCH2-),3.60(s,4H,-OC(=O)CH2S-),2.67-2.61(m,8H,-C(=O)OCH2CH2N<),2.54(s,16H,>N(CH2)2 N<),2.30(t,4H,-CH2CH2C(=O)O-),2.04-1.97(m,8H,-CH2-CH=CH-CH2-),1.64-1.58(m,4H,-CH2CH2C( =O)O-),1.33-1.25(m,40H,-CH2CH3,-CH2CH2CH2-),0.88(t,6H,-CH2CH3).MS(ESI):m / z=1023.5([M+H] + ).

[0317] Example 7.2: Preparation of cationic lipid E7-2

[0318] In E7-2, L1 and L2 are both -(CH2)2-OC(=O)-CH2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -C(=O)O-(CH2)2-, and R1 and R2 are both heptadeca-8,11-dienyl.

[0319] The preparation method is as follows:

[0320] Under an argon atmosphere, a dichloromethane solution (20 mL) containing S1-3 (0.99 g, 2.0 mmol), linoleic acid (S7-2, 1.40 g, 5.0 mmol), and DMAP (0.12 g, 1.0 mmol) was placed in an ice bath, and a dichloromethane solution (15 mL) of DCC (1.24 g, 6.0 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 hours. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain cationic lipid E7-2 (1.51 g). 1 H NMR(400MHz, CDCl3)δ:5.45-5.26(m,8H,-CH2-CH=CH-CH2-),4.26(t,4H,-C(=O)OCH2-),4.18(t,4H,-C( =O)OCH2-),3.62(s,4H,-OC(=O)CH2S-),2.79-2.73(m,4H,-CH=CH-CH2-CH=CH-),2.68-2.63(m,8H,-C(= O)OCH2CH2N<),2.54(s,16H,>N(CH2)2N<),2.29(t,4H,-CH2CH2C(=O)O-),2.03-1.99(m,8H,-CH2-CH=CH -CH2-),1.36-1.23(m,32H,-CH2CH3,-CH2CH2CH2-),0.87(t,6H,-CH2CH3).MS(ESI):m / z=1019.5([M+H] + ).

[0321] Example 7.3: Preparation of cationic lipid E7-3

[0322] In E7-3, L1 and L2 are both -(CH2)2-OC(=O)-CH2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -NHC(=O)O-(CH2)2-, and R1 and R2 are both octadec-9-enyl.

[0323] The preparation method is as follows:

[0324] S1-3 (0.99 g, 2.0 mmol) was dissolved in 20 mL of anhydrous dichloromethane, and DIPEA (1.55 g, 12.0 mmol) and DSC (1.23 g, 4.8 mmol) were added sequentially. The mixture was stirred and reacted at room temperature overnight. After completion, the reaction was washed with saturated sodium bicarbonate (10 mL x 3). The organic phase was concentrated under reduced pressure, and the residue was dissolved in 20 mL of anhydrous dichloromethane. Oleylamine (S7-3, 1.34 g, 5.0 mmol) and TEA (1.39 mL, 10.0 mmol) were added and reacted at room temperature for 2 hours. After completion, the reaction was washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain cationic lipid E7-3 (1.24 g). 1 H NMR(400MHz, CDCl3)δ:5.41-5.29(m,4H,-CH2-CH=CH-CH2-),4.31(t,4H,-NHC(=O)OCH2CH2N<),4. 20(t,4H,>NCH2CH2OC(=O)-),3.65(s,4H,-OC(=O)CH2S-),3.13-3.08(m,4H,-CH2NHC(=O)O-),2.72 -2.47(m,24H,-CH2N<),2.04-1.98(m,8H,-CH2-CH=CH-CH2-),1.64-1.57(m,4H,-CH2CH2NHC(=O)O -),1.38-1.25(m,44H,-CH2CH3,-CH2CH2CH2-),0.87(t,6H,-CH2CH3).MS(ESI):m / z=1081.6([M+H] + ).

[0325] Example 8: Preparation of cationic lipids containing branched hydrocarbon groups at the tail

[0326] Example 8.1: Preparation of cationic lipid E8-1

[0327] In E8-1, L1 and L2 are both -(CH2)2-OC(=O)-(CH2)2-C(=O)O-(CH2)2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -OC(=O)-(CH2)7-, and R1 and R2 are both 9-heptadecanyl.

[0328] The preparation method is as follows:

[0329] S3-6 (1.16 g, 2.0 mmol) and DIPEA (0.77 g, 6.0 mmol) were dissolved in 30 mL of dichloromethane. S8-1 (2.31 g, 5.0 mmol, obtained by esterification of 8-bromooctanoic acid and 9-heptadecanol) was added with stirring and allowed to react overnight at room temperature. After completion of the reaction, the reaction mixture was poured into 30 mL of water and extracted three times with dichloromethane (30 mL x 3). The organic phases were collected, combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain cationic lipid E8-1 (1.70 g). 1 H NMR(400MHz, CDCl3)δ:4.89-4.83(m,2H,>CH-),4.35(t,4H,-C(=O)OCH2CH2S-),4.18(t,4H,>N CH2CH2OC(=O)-),2.91-2.85(m,4H,-C(=O)OCH2CH2S-),2.76-2.44(m,32H,-CH2N<,-OC(=O)CH 2CH2C(=O)O-),2.29(t,4H,>CH-OC(=O)CH2-),1.68-1.48(m,12H,-CH2CH2CH2N<,>CH-CH2-),1 .42-1.24(m,64H,-CH2CH3,-CH2CH2CH2-),0.87(t,12H,-CH2CH3).MS(ESI):m / z=1339.8([M+H] + ).

[0330] Example 8.2: Preparation of cationic lipid E8-2

[0331] In E8-2, L1 and L2 are both -(CH2)2-OC(=O)-CH2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -NHC(=O)O-(CH2)2-, and R1 and R2 are both 2-octyldodecyl.

[0332] The preparation method is as follows:

[0333] S1-3 (0.99 g, 2.0 mmol) was dissolved in 20 mL of anhydrous dichloromethane, and DIPEA (1.55 g, 12.0 mmol) and DSC (1.23 g, 4.8 mmol) were added sequentially. The mixture was stirred and reacted at room temperature overnight. After completion of the reaction, the mixture was washed with saturated sodium bicarbonate (10 mL x 3). The organic phase was concentrated under reduced pressure, and the residue was dissolved in 20 mL of anhydrous dichloromethane. 2-Octyldodecylamine (S8-2, 1.49 g, 5.0 mmol) and TEA (1.39 mL, 10.0 mmol) were added and reacted at room temperature for 2 hours. After completion of the reaction, the mixture was washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain cationic lipid E8-2 (1.51 g). 1 H NMR(400MHz, CDCl3)δ:4.33(t,4H,-NHC(=O)OCH2CH2N<),4.21-4.16(m,4H,>NCH2CH2O C(=O)-),3.62(s,4H,-OC(=O)CH2S-),3.20-3.03(m,4H,>CH-CH2-NHC(=O)O-),2.70-2 .42(m,24H,-CH2N<),2.02-1.94(m,2H,>CH-),1.62-1.44(m,8H,-CH2CH<),1.39-1.23 (m,56H,-CH2CH3,-CH2CH2CH2-),0.86(t,12H,-CH2CH3).MS(ESI):m / z=1141.7([M+H] + ).

[0334] Example 9: Preparation of cationic lipids with trivalent branched cores G1 and G2

[0335] Example 9.1: Preparation of cationic lipid E9-1

[0336] In E9-1, L1 and L2 are both -(CH2)3-C(=O)O-(CH2)2-, and X1 and X2 are both G1 and G2 are L3 and L4 are both -OC(=O)-(CH2)7-, and R1 and R2 are both nonyl.

[0337] The preparation method is as follows:

[0338] Step a: Dissolve S3-4 (1.40 g, 4.0 mmol) in 20 mL of DMF, add S4-10 (0.92 g, 4.0 mmol) and K2CO3 (1.10 g, 8.0 mmol), and stir overnight at room temperature. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 20 mL of dichloromethane. The mixture was washed sequentially with 10% citric acid (10 mL x 2) and brine (10 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to afford S9-1 (2.32 g).

[0339] Step b: Dissolve S9-1 (2.00 g, 3.0 mmol) and DIPEA (0.58 g, 4.5 mmol) in 30 mL of dichloromethane. Add S9-2 (0.68 g, 1.5 mmol, prepared in step a of Example 9.2) with stirring and allow to react overnight at room temperature. After completion of the reaction, the reaction mixture was poured into 30 mL of water and extracted three times with dichloromethane (30 mL x 3). The organic phases were collected, combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to obtain cationic lipid E9-1 (1.23 g). 1 H NMR(400MHz, CDCl3)δ:4.34(t,4H,-C(=O)OCH2CH2S-),4.07(t,8H,-CH2CH2CH2OC(=O)- ),2.93-2.88(m,4H,-C(=O)OCH2CH2S-),2.70-2.44(m,36H,>NCH2-),2.32-2.28(m,12H, -CH2CH2CH2C(=O)O-),1.70-1.53(m,20H,-CH2CH2CH2N<,-CH2CH2CH2OC(=O)-),1.38-1. 22(m,80H,-CH2CH3,-CH2CH2CH2-),0.87(t,12H,-CH2CH3).MS(ESI):m / z=1622.1([M+H] + ).

[0340] Example 9.2: Preparation of cationic lipid E9-2

[0341] In E9-2, L1 and L2 are both -(CH2)3-C(=O)O-(CH2)2-, and X1 and X2 are both G1 and G2 are L3 and L4 are both -C(=O)O-(CH2)2-, and R1 and R2 are both tridecyl.

[0342] The preparation method is as follows:

[0343] Step a: Under argon atmosphere, a dichloromethane solution (50 mL) containing S2-1 (1.23 g, 8.0 mmol), S2-4 (3.34 g, 20.0 mmol), and DMAP (0.49 g, 4.0 mmol) was placed in an ice bath. A dichloromethane solution (50 mL) of DCC (4.53 g, 22.0 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to afford S9-2 (3.08 g).

[0344] Step b: Dissolve 3-piperazine-propionic acid (S9-3, 2.37 g, 15.0 mmol) and DIPEA (2.90 g, 22.5 mmol) in 30 mL of dichloromethane. Add S9-2 (2.71 g, 6.0 mmol) with stirring and allow to react overnight at room temperature. After completion of the reaction, pour the reaction mixture into 30 mL of water and extract three times with dichloromethane (30 mL x 3). The organic phases are collected, combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to yield S9-4 (2.69 g).

[0345] Step c: Dissolve S9-4 (2.43 g, 4.0 mmol) in 40 mL of anhydrous dichloromethane, add NHS (1.38 g, 12.0 mmol), and then add DCC (2.47 g, 12.0 mmol). Add DMAP (0.20 g, 1.6 mmol) to a solution of the TBS derivative of N,N-bis(2-hydroxyethyl)ethylenediamine (S9-5, 3.77 g, 10.0 mmol) dissolved in 40 mL of dichloromethane. Combine the two solutions and stir at room temperature for 24 hours. After completion, filter to remove insoluble matter and concentrate. Add the residue to 40 mL of anhydrous tetrahydrofuran, followed by a 1 M TBAF / THF solution (40 mL). Stir overnight to remove the TBS protection. After completion, concentrate under reduced pressure. Dissolve the residue in 50 mL of dichloromethane, wash with saturated ammonium chloride solution, dry over anhydrous sodium sulfate, filter, and concentrate. The residue was purified by column chromatography to give S9-6 (2.39 g).

[0346] Step d: Under an argon atmosphere, a dichloromethane solution (30 mL) containing S9-6 (1.73 g, 2.0 mmol), S1-4 (2.28 g, 10.0 mmol), and DMAP (0.24 g, 2.0 mmol) was placed in an ice bath, and a dichloromethane solution (30 mL) of DCC (2.47 g, 12.0 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain cationic lipid E9-2 (1.74 g). 1 H NMR(400MHz, CDCl3)δ:4.36(t,4H,-C(=O)OCH2CH2S-),4.22-4.17(t,8H,-CH2CH2CH2OC(=O)- ),3.31(q,4H,>NCH2CH2NHC(=O)-),2.90-2.85(m,4H,-C(=O)OCH2CH2S-),2.93-2.40(m,40H,> NCH2-),2.30-2.25(m,12H,-CH2CH2CH2C(=O)O-),1.70-1.63(m,4H,>NCH2CH2CH2C(=O)O-),1. 39-1.24(m,88H,-CH2CH3,-CH2CH2CH2-),0.86(t,12H,-CH2CH3).MS(ESI):m / z=1708.1([M+H] + ).

[0347] Example 9.3: Preparation of cationic lipid E9-3

[0348] In E9-3, L1 and L2 are both -CH2-OC(=O)-CH2-, and X1 and X2 are both G1 and G2 are L3 and L4 are both -C(=O)O-(CH2)2-, and R1 and R2 are both tridecyl.

[0349] The preparation method is as follows:

[0350] Step a: A triethanolamine derivative (S9-7, 4.56 g, 10.0 mmol) containing two TBS-protected hydroxyl groups and one hydroxyl group substituted with OMs was dissolved in 50 mL of dichloromethane. Potassium carbonate (2.21 g, 16.0 mmol) was added, followed by S1-6 (1.51 g, 4.0 mmol) and stirred overnight at room temperature. After completion of the reaction, the mixture was filtered, washed with water, and the organic phase was concentrated under reduced pressure. The residue was added to 40 mL of anhydrous tetrahydrofuran, followed by a 1 M TBAF / THF solution (40 mL), and stirred overnight to remove the TBS protection. After completion of the reaction, the mixture was concentrated under reduced pressure, and the residue was dissolved in 50 mL of dichloromethane, washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to afford S9-8 (2.14 g).

[0351] Step b: Under an argon atmosphere, a dichloromethane solution (30 mL) containing S9-8 (1.51 g, 2.0 mmol), S1-4 (2.28 g, 10.0 mmol), and DMAP (0.24 g, 2.0 mmol) was placed in an ice bath, and a dichloromethane solution (30 mL) of DCC (2.47 g, 12.0 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After the reaction was completed, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain cationic lipid E9-3 (1.42 g). 1 H NMR(400MHz, CDCl3)δ:4.20(t,8H,-C(=O)OCH2CH2-),3.98-3.93(m,4H,>CHCH2OC(=O)-) ,3.66(s,4H,-OC(=O)CH2S-),3.63-3.55(m,8H,-CH2OCH2-),2.69-2.46(m,24H,-CH2N<), 2.29(t,8H,-CH2CH2C(=O)O-),1.74-1.56(m,14H,-OCH2CH2CH2N<,>NCH2CH2CH<),1.40-1 .25(m,88H,-CH2CH3,-CH2CH2CH2-),0.88(t,12H,-CH2CH3).MS(ESI):m / z=1596.0([M+H] + ).

[0352] Example 10: Preparation of cationic lipids containing morpholine rings.

[0353] Example 10.1: Preparation of cationic lipid E10-1.

[0354] In E10-1, L1 and L2 are both -(CH2)3-C(=O)O-(CH2)2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -C(=O)NH-CH2-, and R1 and R2 are both tridecyl.

[0355] The preparation method is as follows:

[0356] Step a: Dissolve 2-aminomethyl-4-Boc-morpholine (S10-1, 2.16 g, 10.0 mmol) and DIPEA (1.94 g, 15.0 mmol) in 30 mL of dichloromethane. Add S9-2 (1.81 g, 4.0 mmol) with stirring and allow to react overnight at room temperature. After completion of the reaction, pour the reaction mixture into 30 mL of water and extract three times with dichloromethane (30 mL x 3). The organic phases are collected, combined, washed with saturated brine, and concentrated under reduced pressure. The residue is treated with a TFA / DCM mixture (1:1 v / v) to remove the Boc protecting group, then washed with purified water and extracted with dichloromethane. The extract is dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to yield S10-2 (1.71 g).

[0357] Step b: Dissolve S1-4 (1.14 g, 5.0 mmol) in 20 mL of anhydrous dichloromethane, add NHS (0.86 g, 7.5 mmol), and then add DCC (1.55 g, 7.5 mmol). Add DMAP (0.12 g, 1.0 mmol) to a solution of S10-2 (1.05 g, 2.0 mmol) in 15 mL of dichloromethane. Mix the two solutions and stir at room temperature for 24 hours. After completion of the reaction, filter to remove insoluble matter, concentrate, and purify the residue by column chromatography to obtain cationic lipid E10-1 (1.13 g). 1H NMR(400MHz, CDCl3)δ:4.35(t,4H,-C(=O)OCH2CH2S-),3.86-3.78(m,2H,-CH<),3.64-3.48(m,4H,>CH-OCH2-),3.43-3.22(m,4H ,-C(=O)NHCH2CH<),2.95-2.89(m,4H,-C(=O)OCH2CH2S-),2.77-2.45(m,8H,-OCH2CH2N<,>NCH2CH2CH2C(=O)O-),2.31(t,4H,-CH 2CH2CH2C(=O)O-),2.18-2.13(m,4H,-CH2C(=O)NH-),2.08-1.89(m,4H,>CHCH2N<),1.70-1.63(m,4H,>NCH2CH2CH2C(=O)O-),1.5 8-1.49(m,4H,-CH2CH2C(=O)NH-),1.33-1.21(m,40H,-CH2CH3,-CH2CH2CH2-),0.87(t,6H,-CH2CH3).MS(ESI):m / z=943.4([M+H] + ).

[0358] Example 10.2: Preparation of cationic lipid E10-2.

[0359] In E10-2, L1 and L2 are both -(CH2)3-C(=O)O-(CH2)2-, and X1 and X2 are both G1 and G2 are both connecting bonds, L3 and L4 are both -OC(=O)-(CH2)3-C(=O)NH-CH2-, and R1 and R2 are both dodecyl.

[0360] The preparation method is as follows:

[0361] Step a: Dissolve glutaric acid (S10-3, 2.64 g, 20.0 mmol) in 80 mL of anhydrous dichloromethane, add NHS (3.45 g, 30.0 mmol), and then add DCC (6.18 g, 30.0 mmol). Add DMAP (0.49 g, 4.0 mmol) to a solution of S10-2 (2.09 g, 4.0 mmol) in 30 mL of dichloromethane. Combine the two solutions and stir at room temperature for 24 hours. After completion of the reaction, filter to remove insoluble matter, concentrate, and purify the residue by column chromatography to afford S10-4 (2.20 g).

[0362] Step b: Under argon atmosphere, a dichloromethane solution (30 mL) containing S10-4 (1.50 g, 2.0 mmol), dodecanol (S10-5, 0.93 g, 5.0 mmol), and DMAP (0.10 g, 0.8 mmol) was placed in an ice bath, and a dichloromethane solution (30 mL) of DCC (1.03 g, 5.0 mmol) was slowly added dropwise. The reaction mixture was then warmed to room temperature and stirred for 24 h. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain cationic lipid E10-2 (1.37 g). 1 H NMR(400MHz, CDCl3)δ:4.33(t,4H,-C(=O)OCH2CH2S-),4.05(t,4H,-CH2CH2CH2OC(=O)-),3.85-3.76(m,2H,-CH<),3.65-3.48(m,4H,>CH- OCH2-),3.44-3.23(m,4H,-C(=O)NHCH2CH<),2.92-2.85(m,4H,-C(=O)OCH2CH2S-),2.75-2.46(m,8H,-OCH2CH2N<,>NCH2CH2CH2C(=O)O-) ,2.36-2.28(m,8H,-CH2CH2CH2C(=O)O-),2.20(t,4H,-CH2CH2CH2C(=O)NH-),2.10-1.89(m,4H,>CHCH2N<),1.76-1.61(m,8H,>NCH2CH2CH 2C(=O)O-,-OC(=O)CH2CH2CH2C(=O)NH-),1.38-1.23(m,40H,-CH2CH3,-CH2CH2CH2-),0.88(t,6H,-CH2CH3).MS(ESI):m / z=1087.5([M+H] + ).

[0363] Example 11: Preparation of LNP-mRNA Pharmaceutical Composition

[0364] In this example, an LNP-mRNA pharmaceutical composition containing Fluc-mRNA (LNP / Fluc-mRNA) was prepared. The phospholipids contained in the composition were all DSPC, the steroid lipids contained were all cholesterol, and the PEGylated lipids contained were all PEG2k-DMG. The only difference was the cationic lipid.

[0365] The preparation method of LNP / Fluc-mRNA is as follows:

[0366] Step (1): Pipette a certain amount of cationic lipid, DSPC, cholesterol and PEGylated lipid stock solution, dissolve the cationic lipid, DSPC, cholesterol and PEGylated lipid in ethanol at a molar ratio of 50:10:38:1.5 to obtain an ethanol phase solution (the lipid formula of each group is shown in Table 1 of Example 12, the cationic lipids used in the control groups A1, A2, A3 and A4 are R1-1-ether, R1-1-LB, R1-2-DE and R7-1-LB, respectively, and the cationic lipids in the experimental groups L1-1 to L9-3 are the disulfide bond-containing cationic lipids of the present invention);

[0367] Among them, R1-1-ether was synthesized by Comparative Example 1.4. Compared with E1-1, the difference is that the heteroatom group between the ionizable tertiary amine group and the disulfide bond of R1-1-ether is a stable ether group, which is different from the degradable ester group in E1-1;

[0368] Among them, R1-1-LB was synthesized by Comparative Example 1.5. Compared with E1-1, the difference is that there is no heteroatom group between the ionizable tertiary amine group and the disulfide bond of R1-1-LB;

[0369] R1-2-DE was synthesized according to the method described in CN114901360A. Compared with E1-2, R1-2-DE differs in that no heteroatom groups exist between the ionizable tertiary amine group and the disulfide bond, and all ester bonds in R1-2-DE are located in the hydrophobic tail chain. The structure of R1-2-DE is as follows:

[0370] R7-1-LB was synthesized according to the method described in CN107406396A. Compared with E7-1, R7-1-LB differs in that there is no heteroatom group between the ionizable tertiary amine group and the disulfide bond. The structure of R7-1-LB is as follows:

[0371] Step (2): Add Fluc-mRNA to 10-50 mM citrate buffer (pH = 4) to obtain an aqueous solution.

[0372] Step (3): The ethanol phase solution and the aqueous phase solution were mixed (1:3 v / v) to prepare LNP / Fluc-mRNA, and washed by DPBS ultrafiltration multiple times to remove ethanol and free molecules, and finally passed through a 0.2 μm sterile filter for use.

[0373] The above steps (1-3) were used, except that the molar ratio of each lipid was changed to cationic lipid: DSPC: cholesterol: PEGylated lipid = 48:9:42:1.5, where E1-1 was used as the cationic lipid. Other conditions remained unchanged to prepare LNP / Fluc-mRNA (L1-1-d).

[0374] Example 12: Biological activity test of lipid pharmaceutical composition

[0375] (1) Determination of nanoparticle size and nucleic acid complexing ability

[0376] Nucleic Acid Complexation Capacity Assay: Gel electrophoresis was used to investigate the nucleic acid complexation capacity of LNP / Fluc-mRNA. Weigh 0.8 g of agarose and dissolve it in 40 mL of TAE solution. Heat in a microwave oven to completely dissolve the agarose particles. Cool the mixture and add 5 μL of the nucleic acid dye GelGreen to the cooled agarose gel. The gel was then placed in a gel tank and allowed to air dry. LNP / Fluc-mRNA was mixed with 2 μL of loading buffer and added to the wells of the agarose gel. The electrophoresis voltage was set to 90 V and the electrophoresis was performed at room temperature for 10 minutes. Free Fluc-mRNA was essentially absent in all experimental and control groups, demonstrating that the disulfide-bond-containing cationic lipids of the present invention have excellent nucleic acid complexation capacity.

[0377] Encapsulation efficiency determination: LNP / Fluc-mRNA was ultracentrifuged (4°C, 60,000 rpm, 1 hour). The concentration of unencapsulated Fluc-mRNA in the supernatant was measured using a nucleic acid quantifier. The encapsulation efficiency of LNP for Fluc-mRNA was calculated, and the results are summarized in Table 1. The encapsulation efficiency in all experimental groups was above 86%, demonstrating that the LNPs of the present invention have a high encapsulation efficiency for nucleic acid drugs.

[0378] Particle size determination: According to the literature (Hassett et al., J. Controlled Release 2021, 335, 237-246), the particle size of the LNP preparation containing nucleic acid drugs can exert better efficacy when it is 60 to 150 nm. In this embodiment, the particle size of LNP / Fluc-mRNA was determined by dynamic light scattering (DLS). The measured LNP / Fluc-mRNA size uniformity is high, and its PDI is less than 0.3. The experimental results show that the LNP / Fluc-mRNA particle size prepared using the cationic lipid of the present invention is 66 nm to 97 nm, which is within the particle size range that can achieve better efficacy.

[0379] Table 1 Encapsulation efficiency and particle size determination

[0380] (2) Serum stability evaluation

[0381] The above-mentioned LNP / Fluc-mRNA (control groups A1 to A4, experimental groups L1-1 to L10-2 and L1-1-d) was added to a culture medium containing 10% fetal bovine serum (FBS), stirred at 37°C, and samples were taken regularly to measure the change in particle size of the LNP / Fluc-mRNA. The serum stability of the nucleic acid pharmaceutical preparation was analyzed by testing its particle size change. The experimental results showed that within 7 days, the particle size of the control group changed by 2 to 15%, and the particle size of the experimental group changed by 3 to 8%. These results show that the present invention, by appropriately introducing more degradable groups into the disulfide-bonded cationic lipid, does not significantly affect the serum stability of the prepared LNP-nucleic acid pharmaceutical composition.

[0382] (3) Cytotoxicity evaluation

[0383] Prepare DMEM high glucose complete medium (containing 10% FBS). Prepare samples (L1-1 to L10-2 and L1-1-d) with complete medium to 0.1, 0.15, 0.2, 0.25, and 0.3 μg / 100 μL working solutions and store for later use. Take 293T cells in the logarithmic growth phase and add 7×10 3 / well, 100 μL / well were inoculated into 96-well plates. Three replicates were set up for both the control group and the sample group. After incubation in a 5% CO2, 37°C constant temperature incubator for 24 hours, the original culture medium was retained, 100 μL / well of complete culture medium was added to the control group, and 100 μL / well of working solution was added to the sample group. After further incubation for 24 hours, culture medium containing 10% CCK-8 was added at 100 μL / well and incubated in a 5% CO2, 37°C constant temperature incubator for 2 hours. The absorbance value at 450 nm was detected by a microplate reader. The relative viability of the cells was calculated according to the following formula:

[0384] Relative activity % = (absorbance value of sample group - background absorbance value) / (absorbance value of control group - background absorbance value) × 100%, wherein the background absorbance value is the absorbance when only CCK-8 reagent and culture medium are added.

[0385] The experimental results showed that the lipid drug nanoparticles prepared using the cationic lipid of the present invention did not produce significant cytotoxicity under five concentration gradients, and the cell survival rate was greater than 95%. Specifically, the results of sample group L7-1 are shown in Figure 8.

[0386] (4) Evaluation of in vitro transfection effect

[0387] Designing to introduce additional degradable groups at appropriate positions in the disulfide-containing cationic lipid structure, especially in the part between the ionizable tertiary amine group and the disulfide bond, can significantly improve the delivery efficiency of LNP for nucleic acid drugs and achieve more efficient transfection.

[0388] In order to investigate the mRNA transfection efficiency of each group of LNP / Fluc-mRNA compositions prepared in Example 11 of the present invention at the cellular level, Luciferase bioluminescence was used for testing. The LNP / Fluc-mRNA composition preparation was dissolved in culture medium to prepare the required dose. HeLa cells were used as a cell model, and 100 μL / well of the cell suspension was inoculated into a 96-well plate with a black-edged transparent bottom at a seeding density of 6000 cells / well. After inoculation, the cells were incubated in a cell culture incubator for 24 hours and then administered at a dose of 0.2 μg mRNA per well. A1 to A4 were set as positive control groups, and L1-1 to L10-2 were set as experimental groups. A blank control group was also set up, and the corresponding dose of free Fluc-mRNA was added. After 24 hours of transfection, the old culture medium was removed and replaced with a new culture medium containing D-luciferin sodium (1.5 mg / mL) substrate. After incubation for 5 minutes, bioluminescence was detected using a microplate reader. The stronger the fluorescence, the more Fluc-mRNA was transported into the cytoplasm and translated into the corresponding fluorescent protein. The results showed that the transfection effect of most experimental groups was better than that of the control group. In particular, when the cationic lipid structure was similar, the transfection effect of the experimental group was significantly better than that of the positive control group.

[0389] Table 2 Cell transfection test results

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

[0391] With the dosage of 10 μ g / only, 6-8 week old female BALB / c mice are delivered lipid nanoparticle L7-1 by tail vein injection, and small animal in vivo fluorescence imaging is carried out respectively after administration 6, 12 and 24 hours. After the last time point imaging, mice are euthanized, and major organs heart, liver, spleen, lung, kidney are imaged. 10-15min before imaging, the D-luciferin sodium (15mg / mL) of 0.2mL is injected intraperitoneally. Experimental result (Fig. 9) shows that the lipid drug nanoparticles prepared by cationic lipid of the present invention can realize efficient nucleic acid drug delivery in vivo, and the LNP-mRNA pharmaceutical composition delivered into the body is mainly distributed in liver and spleen.

Claims

1. A disulfide bond-containing cationic lipid, characterized in that The structure is shown in the general formula (1): or a salt, tautomer, stereoisomer, isotope-substituted product or solvate thereof; Among them, -SS- is a disulfide bond; L1 and L2 are each independently -(CH2) t -L d -(CH2) t -or-(CH2) t -L d -(CH2) t -L d -(CH2) t -, wherein each t is independently an integer from 1 to 4; L d is a degradable divalent linking group. Any two L d have the same or different structures; X1 and X2 are independently Among them, R d2 , R d3 are each independently a hydrogen atom or an optionally substituted C 1-6 Alkyl, and R d2 , R d3 The number of is independently 1, 2, 3 or 4; s is 1, 2 or 3; n is 1 or 2; G1 and G2 are each independently a connecting bond or a trivalent branching group; When G1 is a connecting bond, k1 is 1; when G1 is a trivalent branching group, k1 is 2; When G2 is a connecting bond, k1 is 1; when G2 is a trivalent branching group, k2 is 2; L3, L4 each appear independently selected from a linker, -(CH2) q -, Z, P0, and any combination thereof, wherein q is an integer of 1 to 10, Z is a divalent linking group containing a heteroatom, and P0 is p is independently 0 or 1 each time it occurs; R1 and R2 are each independently an optionally substituted C 5-30 Hydrocarbon or C 5-30 Hydrocarbon derivative residues.

2. The disulfide bond-containing cationic lipid according to claim 1, characterized in that L d Selected from -OC(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)NH-, -NHC(=O)O-, -NHC(=S)O-, -NHC(=O)NH-, -NHC(=S)NH-, -L x -(AA) m -L x -Any one; among which, -(AA) m - is a divalent oligopeptide linker, m is 2 or 3; L x Each occurrence is independently -C(=O), -NH- or -O-, and forms an amide bond or an ester bond with the adjacent AA; the m AAs are each independently an amino acid residue or an amino acid derivative residue, and the amino acid is selected from any one of glycine, alanine, β-alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, ornithine and citrulline.

3. The disulfide bond-containing cationic lipid according to claim 2, characterized in that L1 and L2 are each independently -(CH2) t -OC(=O)-(CH2) t -、-(CH2) t -OC(=O)O-(CH2) t -、-(CH2) t -NHC(=O)O-(CH2) t -、-(CH2) t -NHC(=S)O-(CH2) t -、-(CH2) t -NHC(=O)NH-(CH2) t -、-(CH2) t -NHC(=S)NH-(CH2) t -、-(CH2) t -OC(=O)-(CH2) t -C(=O)O-(CH2) t -、-(CH2) t -OC(=O)O-(CH2) t -OC(=O)-(CH2) t -、-(CH2) t -L x -(AA) m -L x -(CH2) t -, and any of its connecting ends is connected to a disulfide bond; preferably, L1 and L2 are each independently -CH2-OC(=O)-CH2-, -(CH2)2-OC(=O)-CH2-, -(CH2)3-OC(=O)-CH2-, -(CH2)3-C(=O)O-(CH2)2-, -(CH2)2-OC(=O)O-(CH2)2-, -(CH2)2-NHC(=O)O-(CH2)2-, -(CH2)2-NHC(=O)O-(CH2)2- =S)NH-(CH2)2-, -(CH2)2-OC(=O)-(CH2)2-C(=O)O-(CH2)2-, -(CH2)3-OC(=O)O-(CH2)2-OC(=O)-CH2-, -(CH2)3-C(=O)-Gly-Phe-O-(CH2)2-, -(CH2)3-C(=O)-(Gly)3-O-(CH2)2-; wherein Gly is a glycine residue and Phe is a phenylalanine residue.

4. The disulfide bond-containing cationic lipid according to claim 1, characterized in that R d2 , R d3 each independently a hydrogen atom; Preferably, X1 and X2 are each independently Any one of.

5. The disulfide bond-containing cationic lipid according to claim 1, characterized in that The structure of the cationic lipid is shown in any of the following general formulas:

6. The disulfide bond-containing cationic lipid according to claim 1, characterized in that R1 and R2 are the same or different and are independently C 5-30 Aliphatic hydrocarbon group, C 5-30 Aliphatic hydrocarbon derivative residues, C 7-30 Aromatic hydrocarbon or C 7-30 Aromatic hydrocarbon derivative residue; preferably, R1, R2 are each independently selected from R L , R B , R r and R A More preferably, R1 and R2 are each independently R L , R B or R A ; R L C is a linear structure 5-30 Aliphatic hydrocarbon group, containing 0-4 carbon-carbon double bonds or carbon-carbon triple bonds; preferably, R L for Any one or any substituted form of; wherein, p R Each occurrence is independently an integer from 1 to 15; the substituted form contains 1 to 4 substituents; each occurrence of the substituent is independently a linear or branched C 1-4 An alkyl group, a hydroxyl group or a halogen atom, preferably a methyl group or a hydroxyl group; More preferably, R L Any of the following structures: R B C for branched structure 5-30 Aliphatic or C 5-30 Aliphatic hydrocarbon derivative residue, selected from any of the following structures: Preferably, R B Any of the following structures: R r C containing ring 5-30 Aliphatic hydrocarbon group, preferably R A C 7-30 Aromatic hydrocarbon or C 7-30 Aromatic hydrocarbon derivative residue, preferably 7. The disulfide bond-containing cationic lipid according to claim 1, characterized in that L3 and L4 are each independently a linking bond, -Z-(CH2) q -、-Z-(CH2) q -Z-(CH2) q -、-Z-P0-(CH2) q -、-Z-P0-Z-(CH2) q -or-P0-(CH2) q -, and the left end is connected to R1 or R2; wherein the structures of any two Zs are the same or different.

8. The disulfide bond-containing cationic lipid according to claim 7, characterized in that Z at each occurrence is independently selected from -O-, -S-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -NR c -、-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 -、-NR c C(=O)S-、-C(R c )=N-NR c -、-NR c -N=C(R c )-, -CH(OH)-, R c Each occurrence is independently H or methyl; preferably, each occurrence of Z is independently selected from any one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -OC(=O)O-, -OC(=O)NH-, -NHC(=O)O-, and -CH(OH)-.

9. The disulfide bond-containing cationic lipid according to claim 7, characterized in that P0 is 10. The disulfide bond-containing cationic lipid according to claim 1, characterized in that L3 and L4 are the same and belong to any of the following situations: Case (1): L3 and L4 are both connecting bonds; Case (2): L3 and L4 are both -C(=O)O-(CH2) q -or-OC(=O)-(CH2) q -; Case (3): L3 and L4 are both -C(=O)NH-(CH2) q -or-NHC(=O)-(CH2) q -; Case (4): L3 and L4 are both -OC(=O)NH-(CH2) q -or-NHC(=O)O-(CH2) q -; Case (5): Both L3 and L4 are -OC(=O)-(CH2) q -C(=O)O-(CH2) q -、-OC(=O)-(CH2) q -OC(=O)-(CH2) q -、-C(=O)O-(CH2) q -C(=O)O-(CH2) q -、-C(=O)O-(CH2) q -OC(=O)-(CH2) q -、-OC(=O)-(CH2) q -C(=O)NH-(CH2) q -、-OC(=O)-(CH2) q -NHC(=O)-(CH2) q -、-C(=O)O-(CH2) q -C(=O)NH-(CH2) q -、-C(=O)O-(CH2) q -NHC(=O)-(CH2) q -、-OC(=O)-(CH2) q -CH(OH)-(CH2) q -、-C(=O)O-(CH2) q -CH(OH)-(CH2) q -、-NHC(=O)-(CH2) q -CH(OH)-(CH2) q - or -C(=O)NH-(CH2) q -CH(OH)-(CH2) q -; Case (6): L3 and L4 are both -OC(=O)-P0-(CH2) q -or-C(=O)O-P0-C(=O)O-(CH2) q -, preferably both In any of the above situations, the left end of L3 is connected to R1, and the left end of L4 is connected to R2.

11. The disulfide bond-containing cationic lipid according to claim 1, characterized in that The structure of the cationic lipid is shown in general formula (2):

12. The disulfide bond-containing cationic lipid according to claim 1, characterized in that The structure of the cationic lipid is shown in general formula (3): Among them, two L3 are the same, two R1 are the same, two L4 are the same, two R2 are the same; G1 and G2 are each independently The * end is connected to X1 or X2, and the other two ends are connected to L3 or L4. g Any one selected from -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -OC(=O)O-, -OC(=O)NH-, -NHC(=O)O-; Preferably, G1 and G2 are each independently selected from any of the following structures:

13. The disulfide bond-containing cationic lipid according to claim 1, characterized in that The structure of the cationic lipid is selected from any one of the following:

14. A lipid composition, characterized in that A cationic lipid containing a disulfide bond according to any one of claims 1 to 13.

15. The lipid composition according to claim 14, characterized in that The lipid composition further comprises phospholipids; alternatively, the lipid composition further comprises steroid lipids; alternatively, the lipid composition further comprises PEGylated lipids; alternatively, the lipid composition further comprises phospholipids and steroid lipids; alternatively, the lipid composition further comprises phospholipids and PEGylated lipids; alternatively, the lipid composition further comprises steroid lipids and PEGylated lipids; alternatively, the lipid composition further comprises phospholipids, steroid lipids and PEGylated lipids; alternatively, the lipid composition further comprises phospholipids, steroid lipids and PEGylated lipids; alternatively, the lipid composition further comprises phospholipids, steroid lipids, PEGylated lipids and another cationic lipid; alternatively, the lipid composition further comprises phospholipids, steroid lipids, PEGylated lipids and anionic lipids.

16. The lipid composition according to claim 15, characterized in that The phospholipids are selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecanoyl-sn-glycero-3-phosphocholine, sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn- Glycerol-3-phosphoethanolamine, 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycerol-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycerol-3-phospho-rac-(1-glycerol) sodium salt, dioleoylphosphatidyl silk any one of amino acid, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine and combinations thereof.

17. The lipid composition according to claim 15, characterized in that The steroid lipid is selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and combinations thereof.

18. The lipid composition according to claim 15, characterized in that The PEGylated lipid is selected from any one of non-targeted PEGylated lipids, targeted PEGylated lipids and combinations thereof; preferably, the non-targeted PEGylated lipid is selected from any one of polyethylene glycol-dipalmitoylphosphatidylcholine, polyethylene glycol-dimyristylglycerol, polyethylene glycol-distearoylphosphatidylethanolamine, polyethylene glycol-dioleoylphosphatidylethanolamine, polyethylene glycol-cholesterol, polyethylene glycol-diacylglycerol, polyethylene glycol-dialkoxypropyl and combinations thereof; more preferably, the non-targeted PEGylated lipid is selected from any one of polyethylene glycol 5 00-dipalmitoylphosphatidylcholine, polyethylene glycol 2000-dipalmitoylphosphatidylcholine, polyethylene glycol 500-distearoylphosphatidylethanolamine, polyethylene glycol 2000-distearoylphosphatidylethanolamine, polyethylene glycol 500-dioleoylphosphatidylethanolamine, polyethylene glycol 2000-dioleoylphosphatidylethanolamine, polyethylene glycol 500-dimyristoylglycerol, polyethylene glycol 2000-dimyristoylglycerol and any one of their combinations; preferably, the targeted PEGylated lipid is a PEGylated lipid modified with folic acid or N-acetylgalactosamine.

19. The lipid composition according to claim 15, characterized in that The other cationic lipid is selected from 1,2-dioleoyl-3-trimethylammonium-propane (methyl sulfate), 1,2-dioctadecenyloxy-3-methylammonium propane chloride, 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride, 1,2-dioleyl-3-dimethylamino-propane, 2,3-di(tetradecanoyloxy)propyltrimethylazonium chloride, didecyldimethylammonium chloride, didecyldimethylammonium bromide, N,N-dioleyl-N,N-dimethylammonium chloride, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-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)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol), 4-(N,N-dimethylamino)butyric acid (dilinoleyl)methyl ester, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 8-[(2-hydroxyethyl)(6-oxo-6-(undecanyloxy)hexyl)amino]octanoic acid (heptadecan-9-yl) ester, and ((2-(2-hydroxyethoxy)ethyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

20. The lipid composition according to claim 15, characterized in that The anionic lipid is selected from any one of 1,2-dioleoyl-sn-glycero-3-phosphate sodium salt, 1,2-dimyristoyl-sn-glycero-3-phosphate sodium salt, bis(monooleylglycerol)phosphate ammonium salt and cardiolipin.

21. The lipid composition according to claim 15, characterized in that The molar percentage of PEGylated lipids to total lipids is 0.5-5%, preferably 1-3%, more preferably 1.5%, 1.6%, 1.7%, 1.8% or 1.9%; The molar percentage of cationic lipids to total lipids is 30-65%, preferably 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50% or 55%; The molar percentage of phospholipids to total lipids is 7.5-13%, preferably 8%, 9%, 10%, 11% or 12%; The molar percentage of steroid lipids to total lipids is 35-50%, preferably 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%.

22. A lipid pharmaceutical composition, characterized in that A lipid composition and a drug according to any one of claims 14 to 21, wherein the drug is selected from any one of nucleic acid drugs, small molecule drugs, polypeptide drugs and protein drugs.

23. The lipid pharmaceutical composition according to claim 22, characterized in that The drug is a nucleic acid drug, selected from any one of DNA, RNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir and ribozyme; the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA and siRNA, preferably any one of mRNA, miRNA and siRNA.

24. The lipid pharmaceutical composition according to claim 22, characterized in that The lipid pharmaceutical composition is used as a medicine, and the medicine is selected from any one of the following: a medicine for treating cancer, an anti-infective agent, and a vaccine; the anti-infective agent is an anti-parasitic agent, an antibiotic, an antifungal agent, or an antiviral agent.

25. The lipid pharmaceutical composition according to claim 22, characterized in that The lipid pharmaceutical composition is an LNP pharmaceutical composition, an LPP pharmaceutical composition or a PNP pharmaceutical composition, preferably an LNP pharmaceutical composition, more preferably an LNP-nucleic acid pharmaceutical composition, and most preferably an LNP-mRNA pharmaceutical composition.

26. A lipid pharmaceutical composition preparation, characterized in that: A lipid pharmaceutical composition and a working solution comprising any one of claims 22-25; the working solution is a pharmaceutically acceptable diluent or excipient, preferably any one of deionized water, ultrapure water, phosphate buffer and normal saline, more preferably phosphate buffer or normal saline, most preferably normal saline.

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