Cationic lipid containing disulfide bond and ionizable side group and application thereof

Abstract

The invention discloses a disulfide bond-containing cationic lipid as shown in a formula (1), and in the formula (1),-S-S-is a disulfide bond; l1, L2, L3, L4, L5, L6, L7, L8, L9 and L10 are each independently a linking bond or a divalent linking group; g1 and G2 are trivalent linking groups and are respectively and independently gt; cH-or gt; n <->; m1, M2, B1, B2, B3 and B4 are each independently a linking bond or a C1-12 alkylene group; m3 and M4 are each independently a connecting bond or a C1-6 alkylene group; r1 and R2 are respectively and independently C1-30 alkyl or C1-30 hydrocarbon derivative residues; and Y1 and Y2 are each independently an aza-alkyl group containing 0-1 cyclic structure. The cationic lipid structure disclosed by the invention is constructed through the bifunctional disulfide and small molecules without disulfide bonds, and the raw materials are easy to obtain. The lipid pharmaceutical composition prepared by adopting the cationic lipid disclosed by the invention obtains an excellent effect in a biological activity test, especially in the aspect of a nucleic acid drug transfection effect.

Description

Cationic lipid containing disulfide bonds and ionizable side groups 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 at present. The lipid formula of LNPs can be composed of one or more lipids selected from cationic lipids, neutral lipids, steroid lipids, and pegylated lipids. As a key component in the LNP-nucleic acid drug composition preparation, cationic lipids can be compounded with negatively charged nucleic acid drugs through electrostatic interactions, thereby achieving the encapsulation of nucleic acid drugs. Cationic lipids include cationizable lipids and permanent cationic lipids. Among them, cationizable lipids can be electrically neutral under physiological conditions to avoid excessive interference with biological membranes in the systemic circulation, so the toxicity they may produce is far less than that of 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 endosomal escape. 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 drug delivery efficiency of LNPs. Whether LNPs can fully release drugs within cells is one of the key factors affecting delivery efficiency. The concentration of glutathione (GSH) within cells is significantly higher than outside cells, which is also one of the main factors causing the reducing environment within 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 within cells to achieve destabilization of the LNP structure, thereby promoting the release of encapsulated substances. Cationic lipids with these characteristics disclosed in CN107406396A, CN103930398A and EP4130282A1 improve the expression efficiency of nucleic acid drugs within cells. However, for LNPs that enter cells through endocytosis, 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] Conventional cationic lipids containing disulfide bonds are not completely satisfactory when used for the delivery of nucleic acid drugs. To solve this problem, it is necessary to make some substantial improvements to the specific structure of 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, the structure of which is shown in general formula (1):

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

[0009] in,

[0010] -SS- is a disulfide bond;

[0011] L1, L2, L3, and L4 are each independently a linking bond, -(CH2) h -、-Z-、-Z(CH2) h - and -(CH2) j Z(CH2) k - any one; wherein h, j, k are each an integer from 1 to 6, and the sum of j and k is an integer from 2 to 6;

[0012] L5, L6, L7, L8, L9, L 10 Each is independently a linking bond or -Z-;

[0013] Z is independently selected at each occurrence from any one of -O-, -C(=O)-, -NR'-, -OC(=O)-, -C(=O)O-, -NR'C(=O)-, -C(=O)NR'-, -OC(=O)O-, -NR'C(=O)O-, -OC(=O)NR'-, -NR'C(=O)NR'- and -NR'C(=S)NR'-, wherein R' is a hydrogen atom or a linear, branched or cyclic C containing 0-2 nitrogen atoms. 1-12 alkyl;

[0014] G1 and G2 are trivalent linking groups, each independently >CH- or >N-;

[0015] M1, M2, B1, B2, B3, B4 are each independently a connecting bond or C 1-12 alkylene;

[0016] M3 and M4 are each independently a connecting bond or C 1-6 alkylene;

[0017] When G1 is >CH-, M1 and M3 are not simultaneously connected bonds;

[0018] When G2 is >CH-, M2 and M4 are not simultaneously connected bonds;

[0019] When B1 is a connecting bond, at least one of L5 and L7 is a connecting bond; when B2 is a connecting bond, at least one of L6 and L8 is a connecting bond; when B3 is a connecting bond, at least one of L7 and L9 is a connecting bond; when B4 is a connecting bond, at least one of L8 and L9 is a connecting bond. 10 At least one of them is a connecting key;

[0020] R1 and R2 are each independently C 1-30 Hydrocarbon or C 1-30 hydrocarbon derivative residues;

[0021] Y1 and Y2 are each independently a cyclic or acyclic azaalkyl group;

[0022] The alkyl group, alkylene group, hydrocarbon group, hydrocarbon derivative residue, and azaalkyl group are each independently substituted or unsubstituted.

[0023] The present invention also provides another embodiment:

[0024] A lipid composition comprises a disulfide bond-containing cationic lipid having a structure as shown in formula (1).

[0025] The present invention also provides another embodiment:

[0026] 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).

[0027] The present invention also provides another embodiment:

[0028] A lipid pharmaceutical composition preparation contains the aforementioned lipid pharmaceutical composition and a pharmaceutically acceptable diluent or excipient.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The cationic lipid structure of the present invention can be regarded as two lipid segments covalently linked by a disulfide-containing linker, each of which has a cationizable polar head and a hydrophobic tail chain. Therefore, the cleavage of the disulfide bond not only causes a change in the lipid structure and promotes LNP destabilization, but also obtains a product that is also a cationic lipid and retains the ability to destroy the endosomal membrane structure, thereby effectively promoting the release of the loaded drug. The cationic lipid structure of the present invention contains a disulfide bond and two cationizable side groups; in particular, unlike the structure constructed with a cystine backbone in the prior art, the cationic lipid structure of the present invention is constructed by a difunctionalized disulfide and a small molecule without a disulfide bond, and the raw materials are easily available. The lipid pharmaceutical composition prepared using the cationic lipid of the present invention has achieved excellent results in biological activity tests, especially in terms of nucleic acid drug transfection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 shows the cationic lipid A-a1b2 1 H NMR spectrum.

[0032] Figure 2 shows the cationic lipid A-a16b12 1 H NMR spectrum.

[0033] Figure 3 shows the cationic lipid D-a3e2 1 H NMR spectrum.

[0034] Figure 4 shows the cationic lipid E-c7d1 1 H NMR spectrum.

[0035] Figure 5 shows the cationic lipid F-d6e1 1 H NMR spectrum.

[0036] FIG6 shows the high performance liquid chromatography (HPLC) test results of cationic lipid E-c7d1.

[0037] FIG7 is a mass spectrum (MS) of cationic lipid E-c7d1.

[0038] FIG8 shows the cytotoxicity test results of the LNP-mRNA pharmaceutical composition containing E-c7d1 prepared in Example 12.

[0039] FIG9 shows the imaging results of mice after injection of the LNP-mRNA pharmaceutical composition containing E-c7d1 prepared in Example 12.

[0040] Detailed Description of the Invention

[0041] 1. Terminology

[0042] 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.

[0043] 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."

[0044] “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.

[0045] “Independently” applies not only to objects with different names, but also to objects with the same name appearing in different positions, meaning that the specific selection of one object is not restricted by another object.

[0046] When at least two items are listed, the "combination" of the listed items refers to the combination of any two or more of the aforementioned items, or the combination of any two or more of the same item; in the combination, the number of any item can be one or more, and the specific forms of multiple items with the same name can be the same or different from each other. For the combination of linkers, it is assumed that the linkers are connected to each other to form a new linker. The combination of a linker and any linker is still the linker itself. For example, the combination of an alkylene group and -O- can be -CH2CH2O-, -CH2OCH2CH2-, -CH2OCH2CH2O- or other combinations. For example, -SS- can be regarded as a combination of two -S-.

[0047] 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.

[0048] Groups with a valence greater than or equal to 2 are collectively referred to as "linking groups" and by default contain at least one atom. The word "group" in a divalent linking group can be replaced with "bond" without changing the 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-).

[0049] Unless otherwise specified, there are no particular limitations on the end of a linking group used to connect to other groups. For example, when -C(=O)NH- is used as a divalent linking group between group A and group B, either end (left or right) of -C(=O)NH- can be used to connect to A or B, i.e., both AC(=O)NH-B and A-NHC(=O)-B are optional.

[0050] Numerical intervals can be represented by dashes, wavy lines, or "to / to", such as 1-6, 1-6, and 1 to / to 6, which all represent the group consisting of all values ​​between 1 and 6 (including endpoints). Unless otherwise specified, the numerical values ​​include but are not limited to integers, non-integers, percentages, fractions, etc. Unless otherwise specified, the numerical interval representing the number of groups is composed of integers by default, for example, -(CH2) 1-4 - represents a group consisting of -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, and express The group composed of.

[0051] 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, "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 type and number of atoms in the substituent are not particularly limited.

[0052] 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.

[0053] 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 ).

[0054] 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.

[0055] 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.

[0056] "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.

[0057] "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.

[0058] "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.

[0059] 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.

[0060] Relative to a compound, a group formed after losing some atoms or groups is also called a "residue".

[0061] 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 1-10 Alkyl; 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.

[0062] "Optionally substituted" means that the group or compound being described may be substituted or unsubstituted.

[0063] "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.

[0064] "Hydrocarbons" refer to compounds containing only carbon and hydrogen atoms. Hydrocarbons without benzene or other aromatic rings are collectively referred to as "aliphatic hydrocarbons," while hydrocarbons containing benzene 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.

[0065] "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 a monovalent hydrocarbon 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.

[0066] "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.

[0067] "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.

[0068] "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.

[0069] "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.

[0070] "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 ring-containing structures and are optionally substituted.

[0071] The "benzene ring skeleton" refers to a divalent linking group formed by the loss of two hydrogen atoms from a benzene ring. Does not include phenyl as a substituent or terminal group

[0072] "Heteroalkyl" refers to an alkyl group with at least one heteroatom in its backbone, and its structure can be linear, branched, or contain one or more cyclic structures. Depending on the heteroatom, heteroalkyl groups can be divided into oxaalkyl, azaalkyl, etc. For example, "azaalkyl" is an alkyl group with at least one nitrogen atom in its backbone, including but not limited to -NHCH2CH3, -N(CH3)2, etc. "Functional group" also refers to "functional group," including but not limited to reactive groups, protected reactive groups, precursors of reactive groups, etc. A polyfunctional compound contains at least two functional groups, such as a polyol, which refers to a compound containing at least two hydroxyl groups, and a polythiol, which refers to a compound containing at least two sulfhydryl groups. It should be noted that a polyfunctional compound can contain more than one functional group. For example, tris(hydroxymethyl)aminomethane is a triol that also contains an amino group, and citric acid is a tricarboxylic acid that also contains a hydroxyl group.

[0073] "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.

[0074] "Deprotection" refers to the process of converting a protected form into an unprotected form.

[0075] "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.

[0076] "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" refers to a group 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.

[0077] "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), Fmoc (9-fluorenylmethyleneoxycarbonyl), or Cbz (benzyloxycarbonyl). 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). The removal of Cbz is preferably performed by catalytic hydrogenolysis.

[0078] "Lipids" are a broad group of organic compounds, including fats, waxes, sterols, fat-soluble vitamins, 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, also known as "lipidoids," include phospholipids, sphingolipids, glycolipids, steroids, sterols, and lipoproteins. The derived lipids, including simple lipid derivatives and complex lipid derivatives, have the general properties of lipids.

[0079] "Lipid nanoparticles" or "LNPs" (lipid nanoparticles) refer to nanoscale (e.g., 1 nm to 1000 nm) particles containing one or more lipids. LNPs can further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules).

[0080] 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).

[0081] "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.

[0082] "Neutral lipids" refer to lipids, including phospholipids, that exist in an overall electrically neutral nonionic or zwitterionic form at a selected pH.

[0083] "Steroid lipids" refer to fused ring systems consisting of three cyclohexanes and one cyclopentane. The main characteristic lipids.

[0084] "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.

[0085] "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.

[0086] "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.

[0087] "RNA" refers to naturally occurring or non-naturally occurring ribonucleic acids. 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. RNA can have a nucleotide sequence that encodes a specific polypeptide, such as mRNA that, upon translation, can produce the encoded polypeptide.

[0088] "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.

[0089] "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.

[0090] "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.

[0091] 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.

[0092] "Antagomirs," also known as "anti-miRs," are a class of chemically engineered oligonucleotides designed to silence endogenous miRNAs.

[0093] 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.

[0094] "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.

[0095] 2. Cationic lipids containing disulfide bonds and their preparation methods

[0096] 2.1. Cationic lipids containing disulfide bonds

[0097] One embodiment of the present invention:

[0098] A cationic lipid containing a disulfide bond, the structure of which is shown in general formula (1):

[0099] or a salt, tautomer, stereoisomer, isotope-substituted product or solvate thereof;

[0100] in,

[0101] -SS- is a disulfide bond;

[0102] L1, L2, L3, and L4 are each independently a linking bond, -(CH2) h -、-Z-、-Z(CH2) h - and -(CH2) j Z(CH2) k - any one; wherein h, j, k are each an integer from 1 to 6, and the sum of j and k is an integer from 2 to 6;

[0103] L5, L6, L7, L8, L9, L 10 Each is independently a linking bond or -Z-;

[0104] Z is independently selected at each occurrence from any one of -O-, -C(=O)-, -NR'-, -OC(=O)-, -C(=O)O-, -NR'C(=O)-, -C(=O)NR'-, -OC(=O)O-, -NR'C(=O)O-, -OC(=O)NR'-, -NR'C(=O)NR'- and -NR'C(=S)NR'-, wherein R' is a hydrogen atom or a linear, branched or cyclic C containing 0-2 nitrogen atoms. 1-12 alkyl;

[0105] G1 and G2 are trivalent linking groups, each independently >CH- or >N-;

[0106] M1, M2, B1, B2, B3, B4 are each independently a connecting bond or C 1-12 alkylene;

[0107] M3 and M4 are each independently a connecting bond or C 1-6 alkylene;

[0108] When G1 is >CH-, M1 and M3 are not simultaneously connected bonds;

[0109] When G2 is >CH-, M2 and M4 are not simultaneously connected bonds;

[0110] When B1 is a connecting bond, at least one of L5 and L7 is a connecting bond; when B2 is a connecting bond, at least one of L6 and L8 is a connecting bond; when B3 is a connecting bond, at least one of L7 and L9 is a connecting bond; when B4 is a connecting bond, at least one of L8 and L9 is a connecting bond. 10 At least one of them is a connecting key;

[0111] R1 and R2 are each independently C 1-30 Hydrocarbon or C 1-30 hydrocarbon derivative residues;

[0112] Y1 and Y2 are each independently a cyclic or acyclic azaalkyl group;

[0113] The alkyl group, alkylene group, hydrocarbon group, hydrocarbon derivative residue, and azaalkyl group are each independently substituted or unsubstituted.

[0114] In a specific embodiment of the present invention, L1 and L2 are the same or different and are independently selected from a connecting bond, -(CH2) h -、-Z(CH2) h -* and -(CH2) j Z(CH2) k-*; wherein the * end of L1 and L2 is connected to a disulfide bond; preferably, Z in L1 and L2 is any one of -O-, -C(=O)-, -C(=O)NR'-, -NR'C(=O)-, -NR'C(=O)NR'- and -NR'C(=S)NR'-, and each R' in L1 and L2 is independently a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group or a cyclopropyl group; more preferably, L1 and L2 are each independently selected from any one of a connecting bond, -CH2CH2-, -NR'C(=O)CH2-*, -C(=O)NR'CH2CH2-* and -NR'C(=S)NR'CH2CH2-*, and each R' in L1 and L2 is preferably -H.

[0115] In a specific embodiment of the present invention, L3 and L4 are the same or different and are independently selected from a connecting bond, -(CH2) h -、Z、-Z(CH2) h -*, -(CH2) h Z-* and -(CH2) j Z(CH2) k -*; wherein the * end of L3 and L4 is connected to Y1 and Y2 respectively; preferably, Z in L3 and L4 is any one of -O-, -C(=O)-, -C(=O)NR'-, -NR'C(=O)-, -NR'C(=O)NR'- and -NR'C(=S)NR'-, and each R' in L3 and L4 is independently a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, -CH2N(CH3)2, -CH2N(CH2CH3)2, -CH2CH2N(CH3)2 or -CH2CH2N(CH2CH3)2; more preferably, L3 and L4 are each independently selected from a connecting bond, -C(=O)-, -C(=O)(CH2) h -*, -C(=O)NR'-*, -C(=O)NR'(CH2) h -*, -NR'C(=O)-* and -NR'C(=O)(CH2) h -*, and h in L3 and L4 is preferably 2.

[0116] In a specific embodiment of the present invention, Y1 and Y2 are the same or different and are independently selected from and any of its substituted forms; wherein X1 is N or CH; X2 is NR d , CH or O; R d Linear or branched C 1-5 Alkyl; when X1 is CH, X2 is NR d; The substitution form of Y1 and Y2 is a form in which 1 to 2 hydrogen atoms at any position are substituted by hydroxyl groups; preferably, Y1 and Y2 are each independently selected from any one of the following:

[0117] In a specific embodiment of the present invention, the structure of the cationic lipid is shown in formula (2-1), (2-2), (2-3) or (2-4):

[0118] More specifically, the cationic lipid is selected from any one of the following situations:

[0119] Case 1: The structure of the cationic lipid is shown in the general formula (2-1), wherein M3 and M4 are each independently a connecting bond or C 1-6 Alkylene, L3 and L4 are connecting bonds; or, M3 and M4 are connecting bonds, L3 and L4 are each independently -C(=O)- or -C(=O)(CH2) h -*;

[0120] Case 2: The structure of the cationic lipid is shown in the general formula (2-2), wherein M3 and M4 are each independently a connecting bond or C 1-6 Alkylene, L3, L4 are each independently -C(=O)NR'-* or -C(=O)NR'(CH2) h -*, and R' in L3 and L4 is preferably -H, -CH3 or -CH2CH2N(CH2CH3)2;

[0121] Case 3: The structure of the cationic lipid is as shown in the general formula (2-3), wherein M3 and M4 are each independently a connecting bond, and L3 and L4 are each independently -NR'C(=O)-* or -NR'C(=O)(CH2) h -*, and R' in L3 and L4 is preferably -H;

[0122] Case 4: The structure of the cationic lipid is shown in the general formula (2-4), wherein M3 and M4 are each independently a connecting bond, and L3 and L4 are each independently -C(=O)NR'-* or -C(=O)NR'(CH2) h -*, and R' in L3 and L4 is preferably -H, -CH3 or -CH2CH2N(CH2CH3)2;

[0123] In either case, the * ends of L3 and L4 are connected to Y1 and Y2, respectively.

[0124] In a specific embodiment of the present invention, M1 and M2 are both connecting bonds; or, one of M1 and M2 is a connecting bond and the other is C 1-12Alkylene or its substituted form; or, M1, M2 are each independently C 1-12 Alkylene or its substituted form, its structure is the same or different; wherein the substituted form of M1 and M2 is C 1-12 One or more hydrogen atoms at any position of the alkylene group are independently substituted by an aliphatic hydrocarbon group, an aromatic group, a heterocyclic group, a halogen group or a hydroxyl group, preferably C 1-12 The form in which one hydrogen atom in the alkylene group is replaced by one hydroxyl group is more preferably wherein t is an integer selected from 0-10, and the * ends of M1 and M2 are connected to G1 and G2, respectively.

[0125] In a specific embodiment of the present invention, B1 and B2 are the same or different, B3 and B4 are the same or different; B1, B2, B3, and B4 are each independently a connecting bond, C 1-8 Alkylene or its substituted form, wherein the substituted form of B1, B2, B3, B4 is C 1-8 A form in which one hydrogen atom of an alkylene group is substituted by one benzene ring-containing substituent; the benzene ring-containing substituent is preferably a benzyl group.

[0126] In a specific embodiment of the present invention, L5 and L6 are the same or different, L7 and L8 are the same or different, L9 and L 10 Same or different; L5, L6, L7, L8, L9, L 10 are each independently selected from any one of a connecting bond, -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NR'C(=O)-, -C(=O)NR'-, -OC(=O)O-, -NR'C(=O)O- and -OC(=O)NR'-, and L5, L6, L7, L8, L9, L 10 R' is a hydrogen atom or a linear, branched or ring-containing C 1-12 Alkyl, preferably hydrogen, methyl, ethyl, propyl, isopropyl or cyclopropyl; preferably, at least one of L5, L7, L9 is not -OC(=O)- or -C(=O)O-, and / or L6, L8, L 10 At least one of them is not -OC(=O)- or -C(=O)O-; More preferably, L5, L6, L7, L8, L9, L 10 Each is independently selected from any one of a linking bond, -C(=O)-, -OC(=O)-, -C(=O)O-, -C(=O)NH- and -NHC(=O)-.

[0127] In a specific embodiment of the present invention,

[0128] R1 and R2 are the same or different and are independently C 5-30Aliphatic 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 Any one of, and containing 0-4 carbon-carbon double bonds and / or carbon-carbon triple bonds;

[0129] R L For linear C 5-30 aliphatic hydrocarbon group or a substituted form thereof; said R L The substitution form is linear C 5-30 The form in which 1-4 hydrogen atoms of the aliphatic hydrocarbon group are replaced by 1-4 substituents, each substituent being independently hydroxyl, C 1-6 alkyl, phenyl or benzyl; preferably, R L Any of the following structures:

[0130] R B For branched C 5-30 Aliphatic hydrocarbon group or C containing 1-2 oxygen atoms 5-30 Aliphatic hydrocarbon derivative residue; preferably, R B Any of the following structures:

[0131] R C C containing a ring 5-30 Aliphatic hydrocarbon group; preferably, R C Any of the following structures:

[0132] R A C 7-30 Aromatic hydrocarbon or C 7-30 Aromatic hydrocarbon derivative residue, preferably

[0133] In a specific embodiment of the present invention, the structure of the cationic lipid is shown in formula (3-1), (3-2), (3-3), (3-4), (3-5) or (3-6):

[0134] wherein each Z is independently preferably any one of -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -OC(=O)NH-, and -NHC(=O)O-;

[0135] Preferably, the structure of the cationic lipid is as shown in the general formula (3-1) or any one of the following general formulas (3-2-1), (3-2-2), (3-4-1), (3-4-2), (3-4-3), (3-4-4), (3-4-5), (3-4-6), (3-6-1), and (3-6-2):

[0136] Wherein, G1 and G2 in the general formulas (3-2-1), (3-2-2), (3-4-3), (3-4-4), (3-4-5), and (3-4-6) are the same and are >CH- or >N-; G1 and G2 in the general formulas (3-4-1), (3-4-2), (3-6-1), and (3-6-2) are both >N-;

[0137] Wherein, M1 and M2 in the general formula (3-2-1), (3-2-2), (3-4-3), (3-4-4), (3-4-5), and (3-4-6) are each independently -(CH2) j -or j is an integer selected from 1-12, t is an integer selected from 0-10, the * ends of M1 and M2 are connected to G1 and G2 respectively; B1 and B2 in the general formulas (3-4-1), (3-4-2), (3-4-3), (3-4-4), (3-4-5), (3-4-6), (3-6-1), and (3-6-2) are each independently -(CH2) k -, k is an integer selected from 1-12.

[0138] In a specific embodiment of the present invention, the structure of the cationic lipid is selected from any one of the following:

[0139] 2.2. Lipid Preparation Method

[0140] The raw materials used to synthesize disulfide-bonded cationic lipids include, but are not limited to, compounds a1-a22, b1-b34, c1-c9, d1-d8, e1-e12, f1-f12, g1-g6, h1-h7, and k1-k9, whose structures are as follows:

[0141] Compounds a1 to a22:

[0142] Compounds b1 to b34:

[0143] The structures containing ester bonds, carbonate bonds or ether bonds in compounds b1 to b34 can be synthesized with reference to the methods disclosed in WO2023126006A1 and CN117126071A.

[0144] Compounds c1-c9:

[0145] Among them, C3 is synthesized by esterification reaction of 1-hexadecanol and glutaric acid; C4 is synthesized by esterification reaction and amidation reaction using 1-hexadecanol, phenylalanine and glutaric acid as raw materials; C6 is synthesized by esterification reaction of 9-heptadecanol and adipic acid.

[0146] Compounds d1-d8:

[0147] Compounds e1-e12:

[0148] Compounds f1-f12:

[0149] Compounds g1-g6:

[0150] Compounds h1-h7:

[0151] Compounds k1-k9:

[0152] Among them, k7~k9 were synthesized with reference to the method disclosed in WO2023126006A1.

[0153] The cationic lipid of the present invention can be obtained by any one of the following general preparation methods 1-9:

[0154] General preparation method 1:

[0155] Step 1: The difunctionalized disulfide ds-LG contains two leaving groups LG1, which reacts with amino-containing compounds H2N-M3-L3-Y1 and / or H2N-M4-L4-Y2 to obtain an intermediate IM1 containing two secondary amino groups; wherein the two LG1s are leaving groups and are preferably mesylate groups (-OMs) or p-toluenesulfonate groups (-OTs); preferably, the compounds H2N-M3-L3-Y1 and H2N-M4-L4-Y2 are each independently any one of a1 to a22;

[0156] Step 2: The secondary amine group of the intermediate IM1 reacts with the leaving group LG2 in the compound T1-LG2 and / or T2-LG2 to obtain a compound of formula (1), wherein G1 and G2 are both >N-; wherein T1 and T2 are hydrophobic tail chains, and LG2 in T1-LG2 and T2-LG2 are each independently -F, -Cl, -Br, -I, -OMs or -OTs; preferably, the compounds T1-LG2 and T2-LG2 are each independently any one of b1 to b34;

[0157] The definitions of other parameters are the same as those in general formula (1).

[0158] General preparation method 2:

[0159] Step 1: The difunctionalized disulfide ds-LG contains two leaving groups LG1, which reacts with amino-containing compounds T1-NH2 and / or T2-NH2 to obtain an intermediate IM2 containing two secondary amino groups; wherein the two LG1s are leaving groups and are preferably -OMs or -OTs, and T1 and T2 are hydrophobic tail chains; preferably, the compounds T1-NH2 and T2-NH2 are each independently any one of h1 to h7;

[0160] Step 2: The secondary amine group of the intermediate IM2 reacts with the leaving group LG2 in the compound LG2-M3-L3-Y1 and / or LG2-M4-L4-Y2 to obtain a compound of formula (1), wherein G1 and G2 are both >N-; wherein the two LG2 are each independently -F, -Cl, -Br, -I, -OMs or -OTs; preferably, the compounds LG2-M3-L3-Y1 and LG2-M4-L4-Y2 are each independently any one of g1 to g6;

[0161] The definitions of other parameters are the same as those in general formula (1).

[0162] General preparation method 3:

[0163] Step 1: The difunctionalized disulfide ds-LG contains two leaving groups LG1, and reacts with amino-containing compounds H2N-M3-L3-Y1 and / or H2N-M4-L4-Y2 to obtain an intermediate IM1 containing two secondary amino groups; wherein the two LG1s are leaving groups and are preferably -OMs or -OTs; preferably, the compounds H2N-M3-L3-Y1 and H2N-M4-L4-Y2 are each independently any one of a1 to a22;

[0164] Step 2: The secondary amine group of the intermediate IM1 reacts with the carboxyl group of the compound T1-COOH and / or T2-COOH to obtain a compound of formula (1), wherein G1 and G2 are both >N-, M1 and M2 are both connecting bonds, and L5 and L6 are both -C(=O)-; wherein T1 and T2 are hydrophobic tail chains; preferably, the compounds T1-COOH and T2-COOH are each independently any one of c1 to c9;

[0165] The definitions of other parameters are the same as those in general formula (1).

[0166] General preparation method 4:

[0167] Step 1: A trifunctionalized compound Tri-1 contains a protected carboxyl group, a protected amino group, and a naked carboxyl group, wherein PG1 is a carboxyl protecting group, PG2 is an amino protecting group, and L0' is a linker or an alkylene group; the naked carboxyl group of the compound Tri-1 undergoes an esterification reaction with the hydroxyl group of the compound T1-OH, followed by a deprotection reaction to remove the PG2 protecting group to obtain the intermediate IM3; preferably, one of PG1 and PG2 is a tert-butyloxycarbonyl (Boc) group and the other is a benzyloxycarbonyl (Cbz) group; wherein T1 is a hydrophobic tail chain; preferably, the compound T1-OH is selected from any one of e1 to e12;

[0168] Step 2: The exposed amino group of the intermediate IM3 reacts with a disulfide containing two carboxyl groups, ds-COOH, followed by a deprotection reaction to remove the PG1 protecting group to obtain the intermediate IM4; wherein the two L0s in ds-COOH are independently alkylene groups; the two M1s in IM4 are the same, and the two T1s are the same;

[0169] Step 3: The two exposed carboxyl groups of the intermediate IM4 react with amino-containing compounds H2N-L"-Y1 and / or H2N-L"-Y2, wherein L" is a connecting bond or C 1-6 Alkylene; reaction to obtain a compound of formula (1), wherein G1, G2 are both> CH-, L1, L2 are both -L0'NHC(=O)L0-* and are selected from -NHC(=O)(CH2) h -* or -(CH2) j NHC(=O)(CH2) k -*, the * ends of L1 and L2 are connected to disulfide bonds, and L3 and L4 are each independently -C(=O)O-* or -C(=O)O(CH2) h -* and its * end is connected to Y1 and Y2 respectively, M1 and M2 are the same, L5 and L6 are both -OC(=O)-* and its * end is connected to M1 and M2 respectively, B1 and B2 are the same, L7 and L8 are the same, B3 and B4 are the same, L9 and L10 The same, R1 and R2 are the same; preferably, the compounds H2N-L"-Y1 and H2N-L"-Y2 are each independently any one of a1 to a22;

[0170] The definitions of other parameters are the same as those in general formula (1).

[0171] General preparation method 5:

[0172] Step 1: The difunctionalized disulfide ds-LG contains two leaving groups LG1, which reacts with amino-containing compounds PG3-O-M1-NH2 and / or PG3-O-M2-NH2 to obtain an intermediate IM5 containing two secondary amino groups; wherein the two LG1 are leaving groups and are preferably -OMs or -OTs, and PG3 is a hydroxyl protecting group and is preferably tert-butyldimethylsilyl (-TBS);

[0173] Step 2: The two secondary amine groups of the intermediate IM5 react with the carboxyl groups of the compounds HOOC-L"-Y1 and / or HOOC-L"-Y2, and then a deprotection reaction is performed to remove the PG3 protecting group to obtain the intermediate IM6; wherein L" is a connecting bond or C 1-6 Alkylene; preferably, the compounds HOOC-L"-Y1 and HOOC-L"-Y2 are each independently any one of d1 to d8;

[0174] Step 3: The two exposed hydroxyl groups of the intermediate IM6 react with the carboxyl groups of the compound T1-COOH and / or T2-COOH to obtain a compound of formula (1), wherein G1 and G2 are both >N-, M3 and M4 are both connecting bonds, and L3 and L4 are both -C(=O)L"-* and are each independently selected from -C(=O)- or -C(=O)(CH2) h -*, the * ends of L3 and L4 are connected to Y1 and Y2, respectively, L5 and L6 are both -C(=O)O-* and their * ends are connected to M1 and M2, respectively; wherein T1 and T2 are hydrophobic tail chains; preferably, the compounds T1-COOH and T2-COOH are each independently any one of c1 to c9;

[0175] The definitions of other parameters are the same as those in general formula (1).

[0176] General preparation method 6:

[0177] Step 1: A disulfide containing two amino groups, ds-NH2, reacts with the exposed carboxyl group in the trifunctional compound Tri-2, wherein Tri-2 also contains a protected carboxyl group and a protected amino group, wherein PG1 is a carboxyl protecting group, PG2 is an amino protecting group, L0' is a linker or an alkylene group, and the two L0's are each independently an alkylene group; a deprotection reaction is then performed to remove the PG1 protecting group to obtain the intermediate IM7; preferably, one of PG1 and PG2 is a tert-butyloxycarbonyl (Boc) group and the other is a benzyloxycarbonyl (Cbz) group;

[0178] Step 2: The two exposed carboxyl groups of the intermediate IM7 are subjected to an esterification reaction with the hydroxyl groups of the compounds T1-OH and / or T2-OH, followed by a deprotection reaction to remove the protecting group of PG2 to obtain the intermediate IM8; wherein T1 and T2 are hydrophobic tail chains; preferably, the compounds T1-OH and T2-OH are each independently any one of e1 to e12;

[0179] Step 3: The two exposed amino groups of intermediate IM8 react with the carboxyl groups of compounds HOOC-L"-Y1 and / or HOOC-L"-Y2, wherein L" is a connecting bond or C 1-6 Alkylene; reaction to obtain a compound of formula (1), wherein G1, G2 are both> CH-, L1, L2 are both -LO'C(=O)NHL0-* and are selected from -C(=O)NH(CH2) h -* or -(CH2) j C(=O)NH(CH2) k -*, the * ends of L1 and L2 are connected to disulfide bonds, and L3 and L4 are each independently -NHC(=O)-* or -NHC(=O)(CH2) h -* and its * end is connected to Y1 and Y2 respectively, L5 and L6 are both -OC(=O)-* and its * end is connected to M1 and M2 respectively; preferably, the compounds HOOC-L"-Y1 and HOOC-L"-Y2 are each independently any one of d1 to d8;

[0180] The definitions of other parameters are the same as those in general formula (1).

[0181] General preparation method 7:

[0182] Step 1: A disulfide containing two amino groups, ds-NH2, reacts with the leaving group LG2 of compound T1-LG2 and / or T2-LG2 to obtain an intermediate IM9 containing two secondary amino groups; wherein the two L0s are each independently an alkylene group, and T1 and T2 are hydrophobic tail chains; LG2 in T1-LG2 and T2-LG2 is each independently -F, -Cl, -Br, -I, -OMs or -OTs; preferably, compounds T1-LG2 and T2-LG2 are each independently any one of b1 to b34;

[0183] Step 2: The secondary amine group of the intermediate IM9 reacts with the leaving group LG2 in the compound LG2-M3-L3-Y1 and / or LG2-M4-L4-Y2 to obtain a compound of formula (1), wherein G1 and G2 are both >N-, L1 and L2 are both L0 and each independently is C 1-6 Alkylene; wherein LG2 in the compounds LG2-M3-L3-Y1 and LG2-M4-L4-Y2 are each independently -F, -Cl, -Br, -I, -OMs or -OTs; preferably, the compounds LG2-M3-L3-Y1 and LG2-M4-L4-Y2 are each independently any one of g1 to g6;

[0184] The definitions of other parameters are the same as those in general formula (1).

[0185] General preparation method 8:

[0186] Step 1: A disulfide containing two amino groups, ds-NH2, reacts with the leaving group LG2 in the compound LG2-M3-L3-Y1 and / or LG2-M4-L4-Y2 to obtain an intermediate IM10 containing two secondary amino groups; wherein the two L0s are each independently an alkylene group, and the LG2s in the compounds LG2-M3-L3-Y1 and LG2-M4-L4-Y2 are each independently -F, -Cl, -Br, -I, -OMs or -OTs; preferably, the compounds LG2-M3-L3-Y1 and LG2-M4-L4-Y2 are each independently any one of g1 to g6;

[0187] Step 2: The two secondary amine groups of the intermediate IM10 react with the carboxyl groups of the compound T1-COOH and / or T2-COOH to obtain a compound of formula (1), wherein G1 and G2 are both >N-, L1 and L2 are both L0 and are independently C 1-6 Alkylene, M1 and M2 are both connecting bonds, L5 and L6 are both -C(=O)-; wherein T1 and T2 are hydrophobic tail chains; preferably, the compounds T1-COOH and T2-COOH are each independently any one of c1 to c9;

[0188] The definitions of other parameters are the same as those in general formula (1).

[0189] General preparation method 9:

[0190] Step 1: The difunctionalized disulfide ds-LG contains two leaving groups LG1, and reacts with amino-containing compounds H2N-M3-L3-Y1 and / or H2N-M4-L4-Y2 to obtain an intermediate IM1 containing two secondary amino groups; wherein the two LG1s are leaving groups and are preferably -OMs or -OTs; preferably, the compounds H2N-M3-L3-Y1 and H2N-M4-L4-Y2 are each independently any one of a1 to a22;

[0191] Step 2: The two secondary amine groups of intermediate IM1 react with and / or Reaction to obtain a compound of formula (1), wherein G1 and G2 are both >N-, M1 and M2 are both -OH substituted alkylene; wherein T1 and T2 are hydrophobic tail chains; preferably, Each independently represents any one of k1 to k9;

[0192] The definitions of other parameters are the same as those in general formula (1).

[0193] General preparation method 10:

[0194] Step 1: The trifunctionalized compound Tri-3 contains a protected carboxyl group, a protected amino group, and a naked carboxyl group, wherein PG1 is a carboxyl protecting group, PG2 is an amino protecting group, and L0' is a connecting bond or an alkylene group; the naked carboxyl group of the compound Tri-3 reacts with the amino-containing compound H2N-L"-Y1 and / or H2N-L"-Y2, and then a deprotection reaction is performed to remove the PG2 protecting group to obtain the intermediate IM11; wherein L" is a connecting bond or C 1-6 Alkylene; preferably, the compounds H2N-L"-Y1 and H2N-L"-Y2 are each independently any one of a1 to a22; preferably, one of PG1 and PG2 is Boc and the other is Cbz;

[0195] Step 2: converting the exposed amino group of intermediate IM11 into a -N=C=Q group, wherein Q is an oxygen atom or a sulfur atom, to obtain intermediate IM12;

[0196] Step 3: The disulfide ds-NH2 containing two amino groups reacts with the isocyanate group (-NCO) or isothiocyanate group (-NCS) in the intermediate IM12, and then a deprotection reaction is performed to remove the PG1 protecting group to obtain the intermediate IM13; wherein the two L0 are each independently an alkylene group; wherein M1 and M2 are the same, M3 and M4 are the same, and L3 and L4 are both -C(=O)NH-L"-* and are selected from -C(=O)NH(CH2) h -*, the * ends of L3 and L4 are connected to Y1 and Y2 respectively;

[0197] Step 4: The two carboxyl groups of the intermediate IM13 react with T1-OH and / or T2-OH, or the two carboxyl groups of the intermediate IM13 react with T1-NH2 and / or T2-NH2, wherein T1 and T2 are hydrophobic tail chains; the reaction yields a compound of formula (1); wherein G1 and G2 are both >CH-, L1 and L2 are both -LO'NHC(=Q)NHL0-* and are selected from -NHC(=Q)NH(CH2) h -* or -(CH2) j NHC(=Q)NH(CH2) k -*, Q is an oxygen atom or a sulfur atom, the * ends of L1 and L2 are connected to a disulfide bond; wherein, L5 and L6 are both -OC(=O)-* and their * ends are connected to M1 and M2 respectively, B1 and B2 are the same, L7 and L8 are the same, B3 and B4 are the same, L9 and L 10 The same, R1 and R2 are the same; preferably, T1-OH and T2-OH are each independently any one of e1 to e12, and T1-NH2 and T2-NH2 are each independently any one of h1 to h7;

[0198] The definitions of other parameters are the same as those in general formula (1).

[0199] In any of the aforementioned general preparation methods, functional groups not participating in the reaction may be optionally protected and deprotected at any appropriate stage.

[0200] 3. Lipid compositions, lipid pharmaceutical compositions, lipid pharmaceutical composition preparations, and methods for preparing the same

[0201] One embodiment of the present invention:

[0202] A lipid composition comprising any one of the aforementioned disulfide bond-containing cationic lipids.

[0203] In a specific embodiment of the present invention, the lipid composition further contains phospholipids; alternatively, the lipid composition further contains steroid lipids; alternatively, the lipid composition further contains PEGylated lipids; alternatively, the lipid composition further contains phospholipids and steroid lipids; alternatively, the lipid composition further contains phospholipids and PEGylated lipids; alternatively, the lipid composition further contains steroid lipids and PEGylated lipids; alternatively, the lipid composition further contains phospholipids, steroid lipids and PEGylated lipids; alternatively, the lipid composition further contains phospholipids, steroid lipids, PEGylated lipids and another cationic lipid; alternatively, the lipid composition further contains phospholipids, steroid lipids, PEGylated lipids and anionic lipids.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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).

[0208] 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).

[0209] 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.

[0210] 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.

[0211] In a specific embodiment of the present invention, the molar percentage of PEGylated lipids in the lipid composition is 0.5-5%, preferably 1-3%, more preferably 1.5%, 1.6%, 1.7%, 1.8% or 1.9% of the total lipids; the molar percentage of cationic lipids in the lipid composition is 30-65%, preferably 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50% or 55% of the total lipids; the molar percentage of phospholipids in the lipid composition is 7.5-13%, preferably 8%, 9%, 10%, 11% or 12% of the total lipids; the molar percentage of steroid lipids in the lipid composition is 35-50%, preferably 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50% of the total lipids.

[0212] One embodiment of the present invention:

[0213] 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.

[0214] In a specific embodiment of the present invention, the drug in the lipid pharmaceutical composition 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.

[0215] 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.

[0216] In a specific embodiment of the present invention, 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 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.

[0217] 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.

[0218] 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.

[0219] One embodiment of the present invention:

[0220] 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, and most preferably normal saline.

[0221] 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.

[0222] In a specific embodiment of the present invention, the preparation of the lipid pharmaceutical composition preparation comprises the following steps:

[0223] (1) equilibrating the lipid component in a diluent or excipient;

[0224] (2) adding the drug to the equilibrated mixture for compounding;

[0225] 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.

[0226] In a specific embodiment of the present invention, the preparation of the LNP-nucleic acid pharmaceutical composition comprises the following steps:

[0227] (1) dissolving the lipid component in an organic solvent to obtain an organic phase solution;

[0228] (2) adding the nucleic acid drug to a buffer solution to obtain an aqueous solution;

[0229] (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;

[0230] 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.

[0231] 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. 4. Specific Implementation Methods

[0232] The preparation of disulfide bond-containing cationic lipids, lipid compositions, lipid pharmaceutical compositions, and the biological activity test of LNP-nucleic acid pharmaceutical compositions are further described below in conjunction with some specific examples. The intermediates and final products prepared in the present invention can be purified by purification methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, thin film dialysis, supercritical extraction, column chromatography, etc. The structures of the following examples are purified by nuclear magnetic resonance spectroscopy ( 1 The specific examples are provided to further illustrate the present invention, but are not intended to limit the scope of protection of the present invention.

[0233] Example 1: Synthesis of Cationic Lipids - Series A

[0234] A-a1b2 complies with the general formula (2-1) and the general formula (3-2), wherein L1 and L2 are ethylene, M3 and M4 are ethylene, L3 and L4 are connecting bonds, and Y1 and Y2 are M1 and M2 are heptylene, L5 and L6 are -OC(=O)-, and R1 and R2 are nonyl.

[0235] The preparation method is as follows:

[0236] Step 1: Dissolve N,N-dimethylethylenediamine (a1, 12.0 mmol, 1.06 g, 2.2 eq) and N,N-diisopropylethylamine (DIPEA, 16.4 mmol, 2.12 g, 3.0 eq) in dichloromethane. Add the methanesulfonate derivative of bis(2-hydroxyethyl) disulfide (S1-1, 5.5 mmol, 1.69 g, 1.0 eq) with stirring. Allow to react overnight at room temperature. After completion of the reaction, pour the reaction mixture into water and extract three times with dichloromethane. The organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to afford S1-2 (1.49 g, 93%).

[0237] Step 2: Dissolve S1-2 (4.0 mmol, 1.18 g, 1.0 eq) and DIPEA (12.0 mmol, 1.55 g, 3.0 eq) in dichloromethane. Add 8-bromooctanoic acid nonyl ester (b2, 8.8 mmol, 3.07 g, 2.2 eq) with stirring and allow to react overnight at room temperature. After completion of the reaction, pour the reaction mixture into water and extract three times with dichloromethane. The organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to yield cationic lipid A-a1b2 (2.79 g, 84%). 1 H NMR(400MHz, CDCl3)δ:4.05(t,4H,-CH2OC(=O)-),2.83-2.71(m,8H,>N(CH2)2N(CH3)2),2.69(t, 4H,>NCH2CH2SS-),2.46(t,4H,-CH2CH2CH2N<),2.38(t,4H,>NCH2CH2SS-),2.29(t,4H,-OC(=O)CH 2-),2.24(s,12H,-N(CH3)2),1.67-1.56(m,8H,-CH2CH2OC(=O)-),1.48-1.39(m,4H,-CH2CH2CH2N <),1.35-1.24(m,36H,-CH2CH3,-CH2CH2CH2-),0.88(t,6H,-CH2CH3).MS(ESI):m / z=831.5([M+H] + ).

[0238] Referring to the synthetic route of A-a1b2, a1 was replaced by one of a1 to a19, and / or b2 was replaced by one of b1 to b34, and other conditions remained unchanged to synthesize the cationic lipids in Table 1.

[0239] Table 1 Structure and NMR data of series A cationic lipids

[0240] Example 2: Synthesis of Cationic Lipids - Series B

[0241] B-g2h1 complies with the general formula (2-1) and the general formula (3-1), wherein L1 and L2 are ethylene, M3 and M4 are ethylene, L3 and L4 are connecting bonds, and Y1 and Y2 are R1 and R2 are octadec-9-enyl.

[0242] The preparation method is as follows:

[0243] Step 1: Dissolve oleylamine (h1, 12.0 mmol, 3.21 g, 2.2 eq) and DIPEA (16.4 mmol, 2.12 g, 3.0 eq) in dichloromethane. Add S1-1 (5.5 mmol, 1.69 g, 1.0 eq) with stirring and allow to react overnight at room temperature. After completion of the reaction, pour the reaction mixture into water and extract three times with dichloromethane. The organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to afford S2-1 (3.05 g, 86%).

[0244] Step 2: Dissolve S2-1 (4.0 mmol, 2.61 g, 1.0 eq) and DIPEA (20.0 mmol, 2.59 g, 5.0 eq) in dichloromethane. Add 1-(2-chloroethyl)piperidine hydrochloride (g2, 8.8 mmol, 1.62 g, 2.2 eq) with stirring and allow to react overnight at room temperature. After completion of the reaction, pour the reaction mixture into water and extract three times with dichloromethane. 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 cationic lipid B-g2h1 (3.06 g, 87%). 1 H NMR(400MHz, CDCl3)δ:5.37-5.13(m,4H,-CH2-CH=CH-CH2-),2.87-2.82(t,4H,>NCH2CH2SS-),2.74-2.39(m,24H,-CH2CH2CH2N<,>NCH2CH2N<,>N CH2CH2SS-),2.02-1.97(m,8H,-CH2-CH=CH-CH2-),1.58-1.21(m,60H,-CH2CH3,-CH2CH2CH2-),0.86(t,6H,-CH2CH3).MS(ESI):m / z=875.6([M+H] + ).

[0245] Referring to the synthetic route of B-g2h1, h1 was replaced by one of h1 to h7, and / or g2 was replaced by one of b1 to b34. Other conditions remained unchanged to synthesize the cationic lipids in Table 2.

[0246] Table 2 Structure and NMR data of series B cationic lipids

[0247] Example 3: Synthesis of Cationic Lipids - Series C

[0248] C-a1c4 complies with the general formula (2-1) and the general formula (3-6), wherein L1 and L2 are ethylene, M3 and M4 are ethylene, L3 and L4 are connecting bonds, Y1 and Y2 are, M1 and M2 are connecting bonds, L5 and L6 are -C(=O)-, B1 and B2 are propylene, L7 and L8 are -NHC(=O)-, B3 and B4 are methylene substituted with benzyl, L9 and L 10 It is -OC(=O)-, and R1 and R2 are hexadecyl.

[0249] The preparation method is as follows:

[0250] Step 1: Same as step 1 of Example 1.

[0251] Step 2: Compound c4 (4.0 mmol, 2.01 g, 1.0 eq) was dissolved in anhydrous dichloromethane. N-hydroxysuccinimide (NHS, 6.0 mmol, 0.69 g, 1.5 eq) was added, followed by N,N'-dicyclohexylcarbodiimide (DCC, 6.0 mmol, 1.24 g, 1.5 eq). 4-Dimethylaminopyridine (DMAP, 0.8 mmol, 0.10 g, 0.2 eq) was added to a dichloromethane solution containing S1-2 (1.6 mmol, 0.47 g, 0.4 eq). The two solutions were mixed and stirred at room temperature for 24 hours. After completion of the reaction, the insoluble material was removed by filtration and concentrated. The residue was purified by column chromatography to yield cationic lipid C-a1c4 (1.80 g, 89%). 1 H NMR(400MHz, CDCl3)δ:7.32-7.24(m,2H,Ph),7.22-7.16(m,8H,Ph),4.84-4.76(m,2H,Ph-CH2-CH<),4.05(t,4H,-CH2OC(=O)- ),3.58-3.45(m,8H,-C(=O)N(CH2-)CH2-),3.12-2.96(m,4H,Ph-CH2-),2.80-2.55(m,8H,>NCH2CH2SS-,>NCH2CH2N(CH3)2),2. 41(t,4H,-NHC(=O)CH2CH2CH2C(=O)N<),2.27(t,4H,-NHC(=O)CH2CH2CH2C(=O)N<),2.25(s,12H,-N(CH3)2),1.97-1.93(m,4H, -NHC(=O)CH2CH2CH2C(=O)N<),1.67-1.22(m,56H,-CH2CH3,-CH2CH2CH2-),0.86(t,6H,-CH2CH3).MS(ESI):m / z=1265.7([M+H] + ).

[0252] Referring to the synthetic route of C-a1c4, a1 was replaced by one of a1 to a19, and / or c4 was replaced by one of c1 to c9, and other conditions remained unchanged to synthesize the cationic lipids in Table 3.

[0253] Table 3 Structure and NMR data of series C cationic lipids

[0254] Example 4: Synthesis of Cationic Lipids - Series D

[0255] D-a3e2 conforms to the general formula (2-2) and the general formula (3-2), wherein L1 and L2 are -NHC(=O)CH2-, M3 and M4 are ethylene, L3 and L4 are -C(=O)NHCH2CH2-, Y1 and Y2 are, M1 and M2 are connecting bonds, L5 and L6 are -OC(=O)-, and R1 and R2 are hexadecyl.

[0256] The preparation method is as follows:

[0257] Step 1: Under argon, a glutamic acid derivative (S4-1, 8.0 mmol, 2.70 g, 1.0 eq) containing one benzyl (Bn)-protected carboxyl group and one tert-butyloxycarbonyl (Boc)-protected amino group, hexadecanol (e2, 9.6 mmol, 2.33 g, 1.2 eq), and DMAP (1.6 mmol, 0.20 g, 0.2 eq) were added sequentially to a flask containing dichloromethane. The mixture was stirred under an ice bath, and a dichloromethane solution of DCC (12.0 mmol, 2.48 g, 1.5 eq) was slowly added dropwise. The reaction mixture was then allowed to warm to room temperature and stirred for 24 hours. After completion of the reaction, the precipitate was removed by filtration. Trifluoroacetic acid (TFA) was added to the filtrate to remove the Boc protecting group. After completion of the reaction, the mixture was 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 give S4-2 (2.00 g, 90%).

[0258] Step 2: Dissolve 2,2-dithiodiacetic acid (S4-3, 3.0 mmol, 0.55 g, 1.0 eq) in anhydrous dichloromethane. Add NHS (9.0 mmol, 1.04 g, 3.0 eq) and then DCC (9.0 mmol, 1.86 g, 3.0 eq). Add DMAP (1.2 mmol, 0.15 g, 0.4 eq) to a dichloromethane solution of S4-2 (6.6 mmol, 1.84 g, 2.2 eq). Combine the two solutions and stir at room temperature for 24 hours. After completion, filter to remove insoluble matter and concentrate. Dissolve the crude product in methanol, add Pd / C catalyst, and bubble hydrogen gas at room temperature for 14 hours to remove the Bn protecting group. Remove the catalyst by filtration through celite, evaporate the methanol, and purify the residue by column chromatography to yield S4-4 (2.24 g, 84%).

[0259] Step 3: Dissolve S4-4 (2.0 mmol, 1.78 g, 1.0 eq) in anhydrous dichloromethane, add NHS (6.0 mmol, 0.69 g, 3.0 eq), and then add DCC (6.0 mmol, 1.24 g, 3.0 eq). Add DMAP (0.8 mmol, 0.10 g, 0.4 eq) to a dichloromethane solution of 1-(2-aminoethyl)piperidine (a3, 4.4 mmol, 0.56 g, 2.2 eq). 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 purified by column chromatography to yield the cationic lipid D-a3e2 (1.94 g, 87%). 1 H NMR(400MHz, CDCl3)δ:4.63-4.53(m,2H,-OC(=O)CH<),4.12(t,4H,-CH2OC(=O)-),3.65-3.49(m,4H,-NHC(=O)CH2SS-),3.46-3.2 5(m,4H,-C(=O)NHCH2CH2N<),2.60-2.29(m,16H,-C(=O)NHCH2CH2N<,-CH2N(CH2-)CH2-,-CH2CH2C(=O)NH-),2.28-2.20(m,2H,-CH a H b CH2C(=O)NH-),2.09-2.00(m,2H,-CH a H b CH2C(=O)NH-),1.63-1.23(m,68H,-CH2CH3,-CH2CH2CH2-),0.88(t,6H,-CH2CH3).MS(ESI):m / z=1109.6([M+H] + ).

[0260] Referring to the synthetic route of D-a3e2, e2 was replaced by one of e1 to e12, and / or a3 was replaced by one of a1 to a19 or f1 to f12, and / or the glutamic acid derivative was replaced by an aspartic acid derivative (S6-1), and other conditions remained unchanged to synthesize the cationic lipids in Table 4.

[0261] Table 4 Structure and NMR data of series D cationic lipids

[0262] Example 5: Synthesis of Cationic Lipids - Series E

[0263] E-c7d1 conforms to the general formula (2-1) and the general formula (3-2), wherein L1 and L2 are -CH2CH2-, M3 and M4 are connecting bonds, L3 and L4 are -C(=O)CH2CH2-, Y1 and Y2 are, M1 and M2 are ethylene, L5 and L6 are -C(=O)O-, and R1 and R2 are heptadeca-8,11-dienyl.

[0264] The preparation method is as follows:

[0265] Step 1: Ethanolamine (S5-1, 10.0 mmol, 1.75 g, 2.2 eq) containing one tert-butyldimethylsilyl (TBS)-protected hydroxyl group and DIPEA (13.6 mmol, 1.76 g, 3.0 eq) were dissolved in dichloromethane. S1-1 (4.5 mmol, 1.41 g, 1.0 eq) was added with stirring and allowed to react overnight at room temperature. After completion of the reaction, the reaction mixture was poured into water and extracted three times with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to afford S5-2 (2.04 g, 96%).

[0266] Step 2: Dissolve 3-(4-phenyl-piperazin-1-yl)-propionic acid (d1, 8.0 mmol, 1.38 g, 1.0 eq) in anhydrous dichloromethane. Add NHS (12.0 mmol, 1.38 g, 1.5 eq) and DCC (12.0 mmol, 2.48 g, 1.5 eq). Add DMAP (1.6 mmol, 0.20 g, 0.2 eq) to a dichloromethane solution of S5-2 (3.2 mmol, 1.50 g, 0.4 eq). 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 treated with tetrabutylammonium fluoride in tetrahydrofuran (1 M TBAF / THF) to remove the TBS protecting group. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was dissolved in dichloromethane, washed with saturated ammonium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain S5-3 (1.59 g, 90%).

[0267] Step 3: Under argon, linoleic acid (c7, 4.0 mmol, 1.12 g, 2.2 eq), S5-3 (1.8 mmol, 1.00 g, 1.0 eq), and DMAP (0.7 mmol, 0.09 g, 0.4 eq) were added sequentially to a flask containing dichloromethane. The mixture was stirred under an ice bath, and a dichloromethane solution of DCC (5.5 mmol, 1.13 g, 3.0 eq) was slowly added dropwise. The reaction mixture was then allowed to warm 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 yield cationic lipid E-c7d1 (1.63 g, 83%). 1H NMR(400MHz, CDCl3)δ:5.45-5.23(m,8H,-CH2-CH=CH-CH2-),4.24-4.13(m,4H,-C(=O)OCH2CH2N<),3.73-3.54(m,8H,-C(=O)OC H2CH2N<,>NC(=O)CH2CH2N<),2.90-2.81(m,4H,>NCH2CH2SS-),2.81-2.71(m,8H,-CH=CH-CH2-CH=CH-,>NC(=O)CH2CH2N<),2.70 -2.34(m,20H,>NCH2CH2SS-,-CH2N(CH2-)CH2-,-CH2N(CH3)CH2-,),2.34-2.27(m,10H,>NCH3,-CH2C(=O)O-),2.08-2.01(m,8H, -CH2-CH=CH-CH2-CH=CH-CH2-),1.39-1.26(m,32H,-CH2CH3,-CH2CH2CH2-),0.89(t,6H,-CH2CH3).MS(ESI):m / z=1073.6([M+H] + ).

[0268] Referring to the synthetic route of E-c7d1, d1 was replaced by one of d1 to d8, and / or c7 was replaced by one of c1 to c9. Other conditions remained unchanged to synthesize the cationic lipids in Table 5.

[0269] Table 5 Structure and NMR data of series E cationic lipids

[0270] Example 6: Synthesis of Cationic Lipids - Series F

[0271] F-d6e1 conforms to the general formula (2-3) and the general formula (3-2), wherein L1 and L2 are -C(=O)NHCH2CH2-, M3 and M4 are connecting bonds, L3 and L4 are -NHC(=O)CH2CH2-, Y1 and Y2 are, M1 and M2 are methylene, L5 and L6 are -OC(=O)-, and R1 and R2 are dodecyl.

[0272] The preparation method is as follows:

[0273] Step 1: Dissolve an aspartic acid derivative (S6-1, 10.0 mmol, 3.23 g, 1.0 eq) containing one benzyl-protected carboxyl group and one tert-butyloxycarbonyl-protected amino group in anhydrous dichloromethane. Add NHS (15.0 mmol, 1.73 g, 1.5 eq) and DCC (15.0 mmol, 3.09 g, 1.5 eq). Add DMAP (2.0 mmol, 0.24 g, 0.2 eq) and DIPEA (10.0 mmol, 1.29 g, 1.0 eq) to a dichloromethane solution containing cystamine dihydrochloride (S7-1, 5.0 mmol, 1.13 g, 0.5 eq). Combine the two solutions and stir at room temperature for 24 hours. After completion, filter to remove insoluble matter and concentrate. Dissolve the crude product in methanol, add Pd / C catalyst, and bubble hydrogen gas at room temperature for 14 hours to remove the Bn protecting group. The catalyst was removed by filtration using celite, the methanol was evaporated, and the residue was purified by column chromatography to give S6-2 (2.51 g, 86%).

[0274] Step 2: Under an argon atmosphere, S6-2 (4.0 mmol, 2.33 g, 1.0 eq), dodecanol (e1, 8.8 mmol, 1.64 g, 2.2 eq), and DMAP (1.6 mmol, 0.20 g, 0.4 eq) were added sequentially to a flask containing dichloromethane. The mixture was stirred under an ice bath, and a dichloromethane solution of DCC (12.0 mmol, 2.48 g, 3.0 eq) was slowly added dropwise. The reaction mixture was then allowed to warm to room temperature and stirred for 24 h. After completion of the reaction, the precipitate was removed by filtration. TFA was added to the filtrate to remove the Boc protecting group. After completion of the reaction, the mixture was 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 yield S6-3 (2.60 g, 90%).

[0275] Step 3: Dissolve 1-piperidinepropionic acid (d6, 8.0 mmol, 1.26 g, 1.0 eq) in anhydrous dichloromethane, add NHS (12.0 mmol, 1.38 g, 1.5 eq), and then add DCC (12.0 mmol, 2.48 g, 1.5 eq). Add DMAP (1.6 mmol, 0.20 g, 0.2 eq) to a dichloromethane solution of S6-3 (3.2 mmol, 2.30 g, 0.4 eq). 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 purified by column chromatography to yield cationic lipid F-d6e1 (2.51 g, 79%). 1H NMR(400MHz, CDCl3)δ:4.88-4.74(m,2H,>CH-),4.06(t,4H,-CH2OC(=O)-),3.64-3.45(m,4H,-C( =O)NHCH2CH2SS-),2.89-2.74(m,4H,-OC(=O)CH2CH<),2.65-2.29(m,20H,-NHC(=O)CH2-,-CH2N( CH2-)CH2-,-C(=O)NHCH2CH2SS-),1.67-1.57(m,12H,>NCH2(CH2)3CH2-),1.52-1.44(m,4H,-CH2 CH2OC(=O)-),1.32-1.22(m,36H,-(CH2)9CH3),0.88(t,6H,-CH2CH3).MS(ESI):m / z=997.4([M+H] + ).

[0276] Referring to the synthetic route of F-d6e1, d6 was replaced by one of d1 to d8, and / or e1 was replaced by one of e1 to e12, and / or the aspartic acid derivative was replaced by a glutamic acid derivative (S4-1). Other conditions remained unchanged to synthesize the cationic lipids in Table 6.

[0277] Table 6 Structure and NMR data of series F cationic lipids

[0278] Example 7: Synthesis of Cationic Lipids - Series G

[0279] G-b2g5 conforms to the general formula (2-1) and the general formula (3-2), wherein L1 and L2 are -CH2CH2-, M3 and M4 are ethylene, L3 and L4 are connecting bonds, Y1 and Y2 are, M1 and M2 are heptylene, L5 and L6 are -OC(=O)-, and R1 and R2 are nonyl.

[0280] The preparation method is as follows:

[0281] Step 1: Dissolve cystamine dihydrochloride (S7-1, 4.0 mmol, 0.90 g, 1.0 eq) and DIPEA (20.0 mmol, 2.59 g, 5.0 eq) in dichloromethane. Add b2 (8.0 mmol, 2.79 g, 2.0 eq) with stirring and allow to react overnight at room temperature. After completion of the reaction, pour the reaction mixture into water and extract three times with dichloromethane. The organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to yield S7-2 (2.36 g, 86%).

[0282] Step 2: Dissolve S7-2 (2.0 mmol, 1.38 g, 1.0 eq) and DIPEA (6.0 mmol, 0.78 g, 3.0 eq) in dichloromethane. Add g5 (4.4 mmol, 0.72 g, 2.2 eq) with stirring and allow to react overnight at room temperature. After completion of the reaction, pour the reaction mixture into water and extract three times with dichloromethane. 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 the cationic lipid G-b2g5 (1.48 g, 78%). 1 H NMR(400MHz, CDCl3)δ:4.05(t,4H,-CH2OC(=O)-),2.83-2.78(t,4H,>NCH2CH2SS-),2.69-2.40(m,32H,-CH2N<,-CH2SS-),2.33(s,6 H,>NCH3),2.30(t,4H,-OC(=O)CH2-),1.68-1.23(m,48H,-CH2CH3,-CH2CH2CH2-),0.86(t,6H,-CH2CH3).MS(ESI):m / z=941.5([M+H] + ).

[0283] Referring to the synthetic route of G-b2g5, b2 was replaced by one of b1 to b34, and / or g5 was replaced by one of g1 to g6. Other conditions remained unchanged to synthesize the cationic lipids in Table 7.

[0284] Table 7 Structure and NMR data of series G cationic lipids

[0285] Example 8: Synthesis of Cationic Lipids - Series H

[0286] H-c8g4 conforms to the general formula (2-1) and the general formula (3-2), wherein L1 and L2 are -CH2CH2-, M3 and M4 are ethylene, L3 and L4 are connecting bonds, Y1 and Y2 are, M1 and M2 are connecting bonds, L5 and L6 are -C(=O)-, and R1 and R2 are heptadeca-8-enyl.

[0287] The preparation method is as follows:

[0288] Step 1: Dissolve cystamine dihydrochloride (S7-1, 6.0 mmol, 1.35 g, 1.0 eq) and DIPEA (30.0 mmol, 3.88 g, 5.0 eq) in dichloromethane. Add 4-(2-chloroethyl)morpholine (g4, 12.0 mmol, 1.80 g, 2.0 eq) with stirring and allow to react overnight at room temperature. After completion of the reaction, pour the reaction mixture into water and extract three times with dichloromethane. The organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to afford S8-1 (1.83 g, 80%).

[0289] Step 2: Dissolve oleic acid (c8, 6.0 mmol, 1.69 g, 1.0 eq) in anhydrous dichloromethane, add NHS (9.0 mmol, 1.04 g, 1.5 eq), and then add DCC (9.0 mmol, 1.86 g, 1.5 eq). Add DMAP (1.2 mmol, 0.45 g, 0.2 eq) to a dichloromethane solution of S8-1 (2.4 mmol, 1.73 g, 0.4 eq). 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 purified by column chromatography to yield the cationic lipid H-c8g4 (1.88 g, 86%). 1 H NMR(400MHz, CDCl3)δ:5.34-5.14(m,4H,>CH=CH<),3.65-3.45(m,16H,-C(=O)N(CH 2-)CH2-,-CH2OCH2-),2.77-2.56(m,4H,-CH2SS-),2.45-2.37(m,12H,-CH2N(CH2- )CH2-),2.25(t,4H,-CH2C(=O)N<),2.03-1.99(m,8H,-CH2CH=CHCH2-),1.44-1.21 (m,44H,-CH2CH3,-CH2CH2CH2-),0.86(t,6H,-CH2CH3).MS(ESI):m / z=907.5([M+H] + ).

[0290] Referring to the synthetic route of H-c8g4, g4 was replaced by one of g1 to g6, and / or c8 was replaced by one of c1 to c9. Other conditions remained unchanged to synthesize the cationic lipids in Table 8.

[0291] Table 8 Structure and NMR data of series H cationic lipids

[0292] Example 9: Synthesis of Cationic Lipids - Series K

[0293] K-a1k7 conforms to the general formula (2-1) and the general formula (3-2), wherein L1 and L2 are -CH2CH2-, M3 and M4 are ethylene, L3 and L4 are connecting bonds, Y1 and Y2 are, M1 and M2 are hexamethylene substituted with -OH, L5 and L6 are -OC(=O)-, and R1 and R2 are 9-heptadecanyl.

[0294] The preparation method is as follows:

[0295] Step 1: Same as step 1 of Example 1.

[0296] Step 2: Dissolve the epoxy compound k7 (2.2 mmol, 0.84 g, 2.2 eq) in ethanol, add S1-2 (1.0 mmol, 0.29 g, 1.0 eq), and stir at 60°C for 10 hours. After completion of the reaction, dilute with dichloromethane and wash with saturated sodium chloride solution. The organic phase is separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to yield the cationic lipid K-a1k7 (0.82 g, 77%). 1 H NMR(400MHz, CDCl3)δ:4.87-4.82(m,2H,>CHOC(=O)-),3.63-3.57(m,2H,>CHOH), 2.82-2.78(t,4H,>NCH2CH2SS-),2.69-2.40(m,16H,>CHCH2N<,>NCH2CH2N(CH3)2, >NCH2CH2SS-),2.30(t,4H,-OC(=O)CH2-),2.23(s,12H,-N(CH3)2),1.70-1.23(m, 68H,-CH2CH3,-CH2CH2CH2-),0.87(t,12H,-CH2CH3).MS(ESI):m / z=1059.7([M+H] + ).

[0297] Referring to the synthetic route of K-a1k7, a1 was replaced with one of a1 to a19, and / or k7 was replaced with one of k1 to k9. Other conditions remained unchanged to synthesize the cationic lipids in Table 9.

[0298] Table 9 Structure and NMR data of a series of K cationic lipids

[0299] Example 10: Synthesis of Cationic Lipids - Series L

[0300] L-a16e1 conforms to the general formula (2-4) and the general formula (3-2), wherein L1 and L2 are -NHC(=S)NHCH2CH2-, M3 and M4 are connecting bonds, L3 and L4 are -C(=O)NHCH2CH2-, Y1 and Y2 are, M1 and M2 are methylene, L5 and L6 are -OC(=O)-, and R1 and R2 are dodecyl.

[0301] The preparation method is as follows:

[0302] Step 1: Dissolve S6-1 (8.0 mmol, 2.59 g, 1.0 eq) in anhydrous dichloromethane, add NHS (24.0 mmol, 2.76 g, 3.0 eq), and then add DCC (24.0 mmol, 4.95 g, 3.0 eq). Add DMAP (3.2 mmol, 0.39 g, 0.4 eq) to a dichloromethane solution of 4-methyl-1-piperazineethylamine (a16, 17.6 mmol, 2.52 g, 2.2 eq). Combine the two solutions and stir at room temperature for 24 hours. After completion of the reaction, filter to remove insoluble matter and concentrate. Remove the Boc protecting group using a mixture of trifluoroacetic acid / dichloromethane (TFA / DCM) (1:1 v / v) and wash with purified water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to give S10-1 (2.34 g, 84%).

[0303] Step 2: Dissolve S10-1 (6.0 mmol, 2.09 g, 1.0 eq) in tetrahydrofuran, add triethylamine (TEA, 9.0 mmol, 1.25 mL, 1.5 eq), and add carbon disulfide (CS2, 7.8 mmol, 0.59 g, 1.3 eq) dropwise in an ice bath. The reaction mixture is then warmed to room temperature. After stirring for 12 hours, DMAP (1.8 mmol, 0.22 g, 0.3 eq) is added. Di-tert-butyl dicarbonate ((Boc)2O, 7.8 mmol, 1.70 g, 1.3 eq) is added in an ice bath, and stirring is continued at room temperature for 3 hours. After completion of the reaction, the mixture is washed three times with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue is purified by column chromatography to afford S10-2 (2.06 g, 88%).

[0304] Step 3: Under an ice bath, S7-1 (2.0 mmol, 0.45 g, 1.0 eq) was added to a dichloromethane solution containing S10-2 (4.0 mmol, 1.56 g, 2.0 eq). TEA (4.0 mmol, 0.40 g, 2.0 eq) in dichloromethane was then added dropwise over 15 min. The ice bath was removed and the mixture was allowed to react at room temperature for 5 h. After completion of the reaction, the mixture was washed sequentially with dilute hydrochloric acid, water, and saturated brine. The reaction mixture was concentrated under reduced pressure, and the crude product was dissolved in methanol. Pd / C catalyst was added, and hydrogen was bubbled through the reaction at room temperature for 14 h to remove the Bn protecting group. The catalyst was removed by filtration through celite, the methanol was evaporated, and the residue was purified by column chromatography to afford S10-3 (1.12 g, 74%).

[0305] Step 4: Under an argon atmosphere, S10-3 (1.0 mmol, 0.75 g, 1.0 eq), e1 (2.2 mmol, 0.41 g, 2.2 eq), and DMAP (0.4 mmol, 0.05 g, 0.4 eq) were added sequentially to a flask containing dichloromethane. The mixture was stirred under an ice bath, and a dichloromethane solution of DCC (3.0 mmol, 0.62 g, 3.0 eq) was slowly added dropwise. The reaction mixture was then allowed to warm 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 yield the cationic lipid L-a16e1 (0.73 g, 67%). 1 H NMR(400MHz, CDCl3)δ:4.08(t,4H,-CH2OC(=O)-),3.93(q,4H,-NHC(=S)NHCH2CH2SS-),3.55-3.4 9(m,2H,>CH-),3.34-3.28(m,4H,-C(=O)NHCH2CH2N<),2.94(t,4H,-NHC(=S)NHCH2CH2S-),2.63-2 .39(m,24H,>NCH2-,-OC(=O)CH2CH<),2.34(s,6H,>NCH3),1.65-1.58(m,4H,-CH2CH2CH2OC(=O)- ),1.35-1.17(m,36H,-CH2CH3,-CH2CH2CH2-),0.86(t,6H,-CH2CH3).MS(ESI):m / z=1089.5([M+H] + ).

[0306] Referring to the synthetic route of L-a16e1, a1 was replaced with one of a1 to a19 or f1 to f12, and / or e1 was replaced with one of e1 to e12, and other conditions remained unchanged to synthesize the cationic lipids in Table 10.

[0307] Table 10 Structure and NMR data of series L cationic lipids

[0308] Example 11: Lipids containing a cystine backbone (comparative lipids)

[0309] The structure of the comparative lipid XE is closest to that of the present invention's E-c7d1, having the same polar head and hydrocarbon tail chain with the same number of carbon-carbon double bonds, except that the disulfide bonds of XE are contained in the cystine backbone.

[0310] The preparation method of XE is as follows:

[0311] Step 1: Dissolve L-cystine (S11-1, 4.0 mmol, 2.03 g, 1.0 eq) containing two Cbz protecting groups in anhydrous dichloromethane. Add NHS (12.0 mmol, 1.38 g, 3.0 eq) and DCC (12.0 mmol, 2.48 g, 3.0 eq). Add DMAP (1.6 mmol, 0.20 g, 0.4 eq) to a dichloromethane solution of 1-(2-aminoethyl)pyrrolidine (a2, 8.8 mmol, 1.26 g, 2.2 eq). Combine the two solutions and stir at room temperature for 24 hours. After completion, filter to remove insoluble matter and concentrate. Dissolve the crude product in methanol, add Pd / C catalyst, and bubble hydrogen gas at room temperature for 14 hours to remove the Cbz protecting groups. The catalyst was removed by filtration using celite, the methanol was evaporated, and the residue was purified by column chromatography to give S11-2 (1.59 g, 81%).

[0312] Step 2: Dissolve linoleic acid (c7, 4.0 mmol, 1.12 g, 1.0 eq) in anhydrous dichloromethane, add NHS (6.0 mmol, 0.69 g, 1.5 eq), and then add DCC (6.0 mmol, 1.24 g, 1.5 eq). Add DMAP (0.8 mmol, 0.10 g, 0.2 eq) to a dichloromethane solution of S11-2 (1.6 mmol, 0.79 g, 0.4 eq). 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 purified by column chromatography to yield comparative lipid XE (1.37 g, 84%). 1 H NMR(400MHz, CDCl3)δ:5.45-5.30(m,8H,-CH2-CH=CH-CH2-),4.88-4.80(m,2H,>CH-),3.43-3.38(m,2H,-CH a H bSS-),3.32-3.27(m,4H,-C(=O)NHCH2CH2N<),3.12-3.06(m,2H,-CH a H b SS-),2.80-2.72(m,4H,-CH=CH-CH2-CH=CH-),2.66-2.38(m,20H,-CH2N(CH2-)CH2-,-CH2N(CH3)CH2-).2.35(s,6H,>NCH3),2.27(t,4H,-CH2C(=O)NH -),2.02-1.98(m,8H,-CH2-CH=CH-CH2-CH=CH-CH2-),1.47-1.22(m,32H,-CH2CH3,-CH2CH2CH2-),0.86(t,6H,-CH2CH3).MS(ESI):m / z=1015.6([M+H] + ).

[0313] Example 12: Preparation of LNP-nucleic acid pharmaceutical composition

[0314] In this example, a LNP-nucleic acid pharmaceutical composition (LNP / Fluc-mRNA) containing Fluc-mRNA was prepared. The phospholipids contained were all DSPC, the steroid lipids contained were all cholesterol, and the PEGylated lipids contained were all PEG2k-DMG, except for the cationic lipids. Among them, Fluc-mRNA can express luciferase protein and emit bioluminescence in the presence of a luciferin substrate. The experimental group used the cationic lipids prepared in Examples 1 to 10, and the control group used the comparative example lipid prepared in Example 11 containing a cystine backbone lipid (XE).

[0315] The preparation method of LNP / Fluc-mRNA is as follows:

[0316] Step (1): a certain amount of cationic lipid, DSPC, cholesterol and PEGylated lipid stock solution is pipetted, and the cationic lipid, DSPC, cholesterol and PEGylated lipid are dissolved in ethanol at a molar ratio of 50:10:38:1.5 to obtain an ethanol phase solution;

[0317] Step (2): Add Fluc-mRNA to 10-50 mM citrate buffer (pH = 4) to obtain an aqueous solution.

[0318] 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.

[0319] Example 13: Biological activity test of lipid pharmaceutical composition

[0320] (1) Determination of nanoparticle size and nucleic acid complexing ability

[0321] 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.

[0322] Encapsulation efficiency was determined by ultracentrifuging the LNP / Fluc-mRNA mixture (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 the LNP for Fluc-mRNA was calculated. The encapsulation efficiency was above 80% in all experimental groups, demonstrating the high encapsulation efficiency of the LNP for nucleic acid drugs.

[0323] 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 61 nm to 89 nm, which is within the particle size range that can achieve better efficacy.

[0324] Table 1 Encapsulation efficiency and particle size determination

[0325] (2) Serum stability evaluation

[0326] LNP / Fluc-mRNA was added to a culture medium containing 10% fetal bovine serum (FBS) and stirred at 37°C. Samples were taken periodically to measure changes in LNP / Fluc-mRNA particle size. The experimental results showed that over 7 days, the particle size of the experimental group varied between 0% and 8%, while the particle size of the control group varied by 7%. These results demonstrate that the disulfide-bond-containing cationic lipids of the present invention exhibit excellent serum stability.

[0327] (3) Cytotoxicity evaluation

[0328] Prepare DMEM high glucose complete medium (containing 10% FBS). Prepare samples (groups 1 to 10 in Table 1) 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 culture them at 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:

[0329] 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.

[0330] 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 the sample group containing E-c7d1 are shown in Figure 8.

[0331] (4) Transfection effect evaluation

[0332] In order to investigate the mRNA transfection efficiency of each group of LNP / Fluc-mRNA compositions prepared in the present invention at the cellular level, Luciferase bioluminescence was used for testing. The LNP / Fluc-mRNA composition preparation prepared in Example 12 was dissolved in culture medium and prepared into 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 ug mRNA per well. XE-containing LNP / Fluc-mRNA was set as a positive control group, and the cationic lipid-containing LNP / Fluc-mRNA of Examples 1 to 10 was used as an experimental group. A blank control group was also set, and a corresponding dose of free Fluc-mRNA was added. 24 hours after transfection, the old culture medium was removed and replaced with fresh culture medium containing D-luciferin sodium (1.5 mg / mL) substrate. After incubation for 5 minutes, bioluminescence was measured using a microplate reader. Stronger fluorescence indicates more Fluc-mRNA transported into the cytoplasm and translated into the corresponding fluorescent protein. The relative fluorescence value of the blank control group was set to 1. The calculated values ​​showed that all experimental groups had good in vitro transfection efficacy. In particular, the transfection efficiency of all experimental groups was better than that of the positive control group.

[0333] Table 2 Cell transfection test results

[0334] (5) Evaluation of in vivo transfection effect

[0335] The LNP / Fluc-mRNA containing E-c7d1 was delivered to 6-8 week-old female BALB / c mice by tail vein injection at a dosage of 10 μg / , and small animal in vivo fluorescence imaging was performed respectively after administration for 6, 12, and 24 hours. After the last time point imaging, mice were euthanized, and major organs, heart, liver, spleen, lung, and kidney were imaged. 10-15 min before imaging, 0.2 mL of D-luciferin sodium (15 mg / mL) was injected intraperitoneally. Experimental result (Fig. 9) shows that the lipid drug nanoparticles prepared by the 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 the liver and spleen.

Claims

1. A cationic lipid containing a disulfide bond, the structure of which is shown in the general formula (1): or a salt, tautomer, stereoisomer, isotope-substituted product or solvate thereof; in, -SS- is a disulfide bond; L1, L2, L3, and L4 are each independently a linking bond, -(CH2) h -、-Z-、-Z(CH2) h - and -(CH2) j Z(CH2) k - any one; wherein h, j, k are each an integer from 1 to 6, and the sum of j and k is an integer from 2 to 6; L5, L6, L7, L8, L9, L 10 Each is independently a linking bond or -Z-; Z is independently selected at each occurrence from any one of -O-, -C(=O)-, -NR'-, -OC(=O)-, -C(=O)O-, -NR'C(=O)-, -C(=O)NR'-, -OC(=O)O-, -NR'C(=O)O-, -OC(=O)NR'-, -NR'C(=O)NR'- and -NR'C(=S)NR'-, wherein R' is a hydrogen atom or a linear, branched or cyclic C containing 0-2 nitrogen atoms. 1-12 alkyl; G1 and G2 are trivalent linking groups, each independently >CH- or >N-; M1, M2, B1, B2, B3, B4 are each independently a connecting bond or C 1-12 alkylene; M3 and M4 are each independently a connecting bond or C 1-6 alkylene; When G1 is >CH-, M1 and M3 are not simultaneously connected bonds; When G2 is >CH-, M2 and M4 are not simultaneously connected bonds; When B1 is a connecting bond, at least one of L5 and L7 is a connecting bond; when B2 is a connecting bond, at least one of L6 and L8 is a connecting bond; when B3 is a connecting bond, at least one of L7 and L9 is a connecting bond; when B4 is a connecting bond, at least one of L8 and L9 is a connecting bond. 10 At least one of them is a connecting key; R1 and R2 are each independently C 1-30 Hydrocarbon or C 1-30 hydrocarbon derivative residues; Y1 and Y2 are each independently a cyclic or acyclic azaalkyl group; The alkyl group, alkylene group, hydrocarbon group, hydrocarbon derivative residue, and azaalkyl group are each independently substituted or unsubstituted.

2. The disulfide bond-containing cationic lipid according to claim 1, wherein L1, L2 are the same or different, and are independently selected from a connecting bond, -(CH2) h -、-Z(CH2) h -* and -(CH2) j Z(CH2) k -Any of *; wherein the * ends of L1 and L2 are connected with a disulfide bond; Preferably, Z in L1 and L2 is any one of -O-, -C(=O)-, -C(=O)NR'-, -NR'C(=O)-, -NR'C(=O)NR'- and -NR'C(=S)NR'-, and each R' in L1 and L2 is independently a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group or a cyclopropyl group; More preferably, L1 and L2 are each independently selected from any one of a linker, -CH2CH2-, -NR'C(=O)CH2-*, -C(=O)NR'CH2CH2-* and -NR'C(=S)NR'CH2CH2-*, and each R' in L1 and L2 is preferably -H.

3. The disulfide bond-containing cationic lipid according to claim 1, wherein L3, L4 are the same or different, and are independently selected from a connecting bond, -(CH2) h -、-Z-、-Z(CH2) h -*, -(CH2) h Z-* and -(CH2) j Z(CH2) k -*; wherein the * ends of L3 and L4 are connected to Y1 and Y2 respectively; Preferably, Z in L3 and L4 is any one of -O-, -C(=O)-, -C(=O)NR'-, -NR'C(=O)-, -NR'C(=O)NR'- and -NR'C(=S)NR'-, and each R' in L3 and L4 is independently a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, -CH2N(CH3)2, -CH2N(CH2CH3)2, -CH2CH2N(CH3)2 or -CH2CH2N(CH2CH3)2; More preferably, L3 and L4 are each independently selected from a linker, -C(=O)-, -C(=O)(CH2) h -*, -C(=O)NR'-*, -C(=O)NR'(CH2) h -*, -NR'C(=O)-* and -NR'C(=O)(CH2) h -*, and h in L3 and L4 is preferably 2.

4. The disulfide bond-containing cationic lipid according to claim 1, wherein Y1 and Y2 are the same or different and are independently selected from and any of its substituted forms; wherein X1 is N or CH; X2 is NR d , CH or O; R d Linear or branched C 1-5 Alkyl; when X1 is CH, X2 is NR d The substitution form of Y1 and Y2 is a form in which 1 to 2 hydrogen atoms at any position are substituted by hydroxyl groups; Preferably, Y1 and Y2 are each independently selected from any one of the following:

5. The disulfide bond-containing cationic lipid according to any one of claims 3-4, characterized in that The structure of the cationic lipid is shown in general formula (2-1), (2-2), (2-3) or (2-4):

6. The disulfide bond-containing cationic lipid according to claim 5, characterized in that The cationic lipid is selected from any one of the following situations: Case 1: The structure of the cationic lipid is shown in the general formula (2-1), wherein M3 and M4 are each independently a connecting bond or C 1-6 Alkylene, L3 and L4 are connecting bonds; or, M3 and M4 are connecting bonds, L3 and L4 are each independently -C(=O)- or -C(=O)(CH2) h -*; Case 2: The structure of the cationic lipid is shown in the general formula (2-2), wherein M3 and M4 are each independently a connecting bond or C 1-6 Alkylene, L3, L4 are each independently -C(=O)NR'-* or -C(=O)NR'(CH2) h -*, and R' in L3 and L4 is preferably -H, -CH3 or -CH2CH2N(CH2CH3)2; Case 3: The structure of the cationic lipid is as shown in the general formula (2-3), wherein M3 and M4 are each independently a connecting bond, and L3 and L4 are each independently -NR'C(=O)-* or -NR'C(=O)(CH2) h -*, and R' in L3 and L4 is preferably -H; Case 4: The structure of the cationic lipid is shown in the general formula (2-4), wherein M3 and M4 are each independently a connecting bond, and L3 and L4 are each independently -C(=O)NR'-* or -C(=O)NR'(CH2) h -*, and R' in L3 and L4 is preferably -H, -CH3 or -CH2CH2N(CH2CH3)2; In either case, the * ends of L3 and L4 are connected to Y1 and Y2, respectively.

7. The disulfide bond-containing cationic lipid according to claim 1, characterized in that M1 and M2 are both connecting keys; or, One of M1 and M2 is a connecting key and the other is C 1-12 Alkylene or a substituted form thereof; or, M1 and M2 are each independently C 1-12 Alkylene or substituted forms thereof, which may be structurally the same or different; Wherein, the substitution form of M1 and M2 is C 1-12 One or more hydrogen atoms at any position of the alkylene group are independently substituted by an aliphatic hydrocarbon group, an aromatic group, a heterocyclic group, a halogen group or a hydroxyl group, preferably C 1-12 The form in which one hydrogen atom in the alkylene group is replaced by one hydroxyl group is more preferably wherein t is an integer selected from 0-10, and the * ends of M1 and M2 are connected to G1 and G2, respectively.

8. The disulfide bond-containing cationic lipid according to claim 1, wherein B1, B2 are the same or different, B3, B4 are the same or different; B1, B2, B3, B4 are each independently a connecting bond, C 1-8 Alkylene or its substituted form, wherein the substituted form of B1, B2, B3, B4 is C 1-8 A form in which one hydrogen atom of an alkylene group is substituted by one benzene ring-containing substituent; the benzene ring-containing substituent is preferably a benzyl group.

9. The disulfide bond-containing cationic lipid according to claim 1, wherein L5, L6 are the same or different, L7, L8 are the same or different, L9, L 10 Same or different; L5, L6, L7, L8, L9, L 10 are each independently selected from any one of a connecting bond, -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NR'C(=O)-, -C(=O)NR'-, -OC(=O)O-, -NR'C(=O)O- and -OC(=O)NR'-, and L5, L6, L7, L8, L9, L 10 R' is a hydrogen atom or a linear, branched or ring-containing C 1-12 an alkyl group, preferably a hydrogen atom, a methyl group, an ethyl group, a propyl group, an isopropyl group or a cyclopropyl group; Preferably, at least one of L5, L7, and L9 is not -OC(=O)- or -C(=O)O-, and / or L6, L8, L 10 At least one of them is not -OC(=O)- or -C(=O)O-; More preferably, L5, L6, L7, L8, L9, L 10 Each is independently selected from any one of a linking bond, -C(=O)-, -OC(=O)-, -C(=O)O-, -C(=O)NH- and -NHC(=O)-.

10. 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 Any one of, and containing 0-4 carbon-carbon double bonds and / or carbon-carbon triple bonds; R L For linear C 5-30 aliphatic hydrocarbon group or a substituted form thereof; said R L The substitution form is linear C 5-30 The form in which 1-4 hydrogen atoms of the aliphatic hydrocarbon group are replaced by 1-4 substituents, each substituent being independently hydroxyl, C 1-6 alkyl, phenyl or benzyl; preferably, R L Any of the following structures: R B For branched C 5-30 Aliphatic hydrocarbon group or C containing 1-2 oxygen atoms 5-30 Aliphatic hydrocarbon derivative residue; preferably, R B Any of the following structures: R C C containing a ring 5-30 Aliphatic hydrocarbon group; preferably, R C Any of the following structures: R A C 7-30 Aromatic hydrocarbon or C 7-30 Aromatic hydrocarbon derivative residue, preferably 11. The disulfide bond-containing cationic lipid according to any one of claims 7 to 10, characterized in that The structure of the cationic lipid is shown in general formula (3-1), (3-2), (3-3), (3-4), (3-5) or (3-6): wherein each Z is independently preferably any one of -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -OC(=O)NH-, and -NHC(=O)O-; Preferably, the structure of the cationic lipid is as shown in the general formula (3-1) or any one of the following general formulas (3-2-1), (3-2-2), (3-4-1), (3-4-2), (3-4-3), (3-4-4), (3-4-5), (3-4-6), (3-6-1), and (3-6-2): Wherein, G1 and G2 in the general formulas (3-2-1), (3-2-2), (3-4-3), (3-4-4), (3-4-5), and (3-4-6) are the same and are >CH- or >N-; G1 and G2 in the general formulas (3-4-1), (3-4-2), (3-6-1), and (3-6-2) are both >N-; Wherein, M1 and M2 in the general formula (3-2-1), (3-2-2), (3-4-3), (3-4-4), (3-4-5), and (3-4-6) are each independently -(CH2) j -or j is an integer selected from 1-12, t is an integer selected from 0-10, the * ends of M1 and M2 are connected to G1 and G2 respectively; B1 and B2 in the general formulas (3-4-1), (3-4-2), (3-4-3), (3-4-4), (3-4-5), (3-4-6), (3-6-1), and (3-6-2) are each independently -(CH2) k -, k is an integer selected from 1-12.

12. The disulfide bond-containing cationic lipid according to claim 1, wherein The structure of the cationic lipid is selected from any one of the following:

13. A lipid composition, characterized in that Contains the disulfide bond-containing cationic lipid according to any one of claims 1 to 12.

14. The lipid composition according to claim 13, characterized in that The lipid composition further comprises phospholipids; or, The lipid composition further comprises a steroid lipid; or, The lipid composition further comprises a PEGylated lipid; or The lipid composition further comprises phospholipids and steroid lipids; or, The lipid composition further comprises phospholipids and PEGylated lipids; or, The lipid composition further comprises a steroid lipid and a PEGylated lipid; or The lipid composition further comprises phospholipids, steroid lipids and PEGylated lipids; or, The lipid composition further comprises a phospholipid, a steroid lipid, a pegylated lipid and another cationic lipid; or, The lipid composition also contains phospholipids, steroid lipids, PEGylated lipids and anionic lipids.

15. The lipid composition according to claim 14, characterized in that The phospholipid is 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-diamidonoyl-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, dioleoylphosphatidylserine 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.

16. The lipid composition according to claim 14, 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.

17. The lipid composition according to claim 14, 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-distearylphosphatidylethanolamine, 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 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.

18. The lipid composition according to claim 14, 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)propan-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-(undecyloxy)hexyl)amino]octanoate (heptadecan-9-yl), and ((2-(2-hydroxyethoxy)ethyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

19. The lipid composition according to claim 14, characterized in that The anionic lipid is selected from any one of 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt, 1,2-dimyristoyl-sn-glycerol-3-phosphate sodium salt, bis(monooleoylglycerol)phosphate ammonium salt and cardiolipin.

20. The lipid composition according to claim 14, 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%.

21. A lipid pharmaceutical composition, characterized in that Contains the lipid composition and drug according to any one of claims 13 to 20, wherein the drug is selected from any one of nucleic acid drugs, small molecule drugs, polypeptide drugs and protein drugs.

22. The lipid pharmaceutical composition according to claim 21, 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.

23. The lipid pharmaceutical composition according to claim 21, characterized in that The lipid pharmaceutical composition is used as a medicine, and the medicine 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.

24. The lipid pharmaceutical composition according to claim 21, 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.

25. A lipid pharmaceutical composition preparation, characterized in that: Containing the lipid pharmaceutical composition and working solution according to any one of claims 21-24; 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.

Images