Cationic lipid with functional groups on side chains and its application

Abstract

The present invention discloses a novel cationic lipid and its pegylated derivative, as well as cationic liposomes, cationic liposome nucleic acid drug compositions and cationic liposome nucleic acid drug composition preparations containing the same. The present invention provides an ionizable lipid compound of the structure shown in formula (1), which is less ionized or electrically neutral at physiological pH, ionized to a greater extent under acidic conditions, has lower systemic circulation toxicity and higher endosomal escape ability; the polar head of the compound has an ionizable tertiary amine group, the side chain may carry a functional group, and the tail chain may contain an easily degradable connecting bond; the cationic liposome containing the compound has higher nucleic acid drug complexing ability, serum stability, no obvious cytotoxicity, and high transfection efficiency. Compared with traditional liposomes, the cationic liposomes provided by the present invention have higher nucleic acid delivery and transfection efficiency, and can be used as a new carrier functional reagent in nucleic acid drugs and vaccines.

Description

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 cationic lipid with a side chain containing a functional group, as well as a lipid composition comprising the cationic lipid, a lipid pharmaceutical composition, and preparations and applications thereof. Background Art

[0002] Liposomes are widely used to deliver nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, peptide drugs, or protein drugs. With the U.S. Food and Drug Administration (FDA) urgently approving two messenger RNA (mRNA) vaccines for the prevention of the novel coronavirus SARS-CoV-2 in response to COVID-19, mRNA-loaded lipid nanoparticles (LNPs) have become a popular delivery technology. In addition to negatively charged mRNA, LNPs also contain four components: ionizable cationic lipids, neutral co-lipids, sterol lipids, and PEGylated lipids. Cationic lipids interact with negatively charged mRNA through electrostatics. Neutral co-lipids, typically phospholipids, prevent lipid oxidation, attach ligands to the liposome surface, or reduce lipid particle aggregation. Sterol lipids have strong membrane fusogenicity, promoting mRNA intracellular uptake and cytosolic entry. PEGylated lipids, located on the surface of lipid nanoparticles, improve their hydrophilicity, prevent rapid clearance by the immune system, prevent particle aggregation, and increase stability. Among the four lipids used to prepare LNP, the most critical one is the ionizable cationic lipid, which is not ionized or weakly ionized under physiological conditions and is basically electrically neutral, avoiding excessive interference with biological membranes in the systemic circulation, thereby reducing toxicity. After being taken up by cells, it can ionize under the acidic conditions of the endosomal cavity and carry partial positive charge, increasing membrane permeability. At the same time, the liposomes are degraded or reorganized, promoting the escape of mRNA from the endosome and releasing the mRNA into the cytoplasm for further translation, thereby completing the delivery and transfection of the mRNA molecules.

[0003] Many LNP drug formulations have been approved for marketing and are used in medical fields such as anticancer, antifungal, analgesia, and vaccines. Traditional LNP delivery systems have significant limitations, such as short blood circulation time, poor in vivo stability, poor drug complexation ability, low efficacy, and low transfection efficiency for nucleic acid drugs. Therefore, whether to introduce functional groups into the polar head of the cationic lipid, how to introduce functional groups, and whether the cationic lipid works synergistically with other lipids are important entry points for solving these problems, especially with respect to nanoparticle stability and nucleic acid drug transfection efficiency.

[0004] In order to solve the above problems, it is necessary to develop a new cationic lipid and its derivatives. Summary of the Invention

[0005] The present invention provides novel cationic lipids, as well as lipid compositions, lipid pharmaceutical compositions, and formulations thereof comprising the cationic lipids, for use in the field of drug delivery. The cationic lipids of the present invention have a cationizable nitrogen-branched polar head, particularly a side chain containing a functional group. The lipid compositions prepared using the cationic lipids of the present invention have a high nucleic acid drug complexing capacity and good in vivo stability, enabling nucleic acid drugs to fully exert their efficacy without causing significant cytotoxicity, thereby improving immune or therapeutic effects.

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

[0007] One embodiment of the present invention:

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

[0009]

[0010] Wherein, N is a nitrogen branching center;

[0011] L1 and L2 are each independently a connecting bond or a divalent connecting group;

[0012] L3 is a trivalent linking group, and together with R3 forms a divalent linking group -L3(R3)- with the side chain being R3;

[0013] B1 and B2 are each independently a connecting bond or C 1-30 alkylene;

[0014] R1 and R2 are each independently C 2-30 Aliphatic hydrocarbon or C containing 1-2 O 2-30 Aliphatic hydrocarbon derivative residues;

[0015] R3 is -OR d 、-NR d R d 、-SR d 、-(C=O)R d 、-(C=O)OR d 、-O(C=O)R d 、-O(C=O)OR d or Among them, R d Each occurrence is independently C 1-12 Alkyl, G1 is a branched group with a valence of k+1, j is 0 or 1, and F1 contains a functional group R 01; When j is 0, G1 does not exist; when j is 1, G1 leads to k F1s, and the k F1s are independently the same or different structures, and k is an integer from 2 to 8;

[0016] R4 is a carbocyclic group, a heterocyclic group or R'N(R')-R"-N(R')-; wherein the heterocyclic group is a cyclic group containing 1, 2 or more heteroatoms as ring atoms, wherein the heteroatoms are B, O, N, Si, P or S; wherein each occurrence of R' is independently H or C 1-3 Alkyl, R" is C 2-4 alkylene;

[0017] The alkylene group, aliphatic hydrocarbon group, aliphatic hydrocarbon derivative residue, carbocyclic group, and heterocyclic group are each independently substituted or unsubstituted;

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

[0019] The present invention also provides another embodiment:

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

[0021] The present invention also provides another embodiment:

[0022] A lipid pharmaceutical composition comprises a lipid composition and a drug, wherein the lipid composition comprises a cationic lipid having a structure as shown in formula (1).

[0023] The present invention also provides another embodiment:

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

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The novel cationic lipid compounds of the present invention are cationic lipids containing functional groups on the polar head side chains. This enriches the variety of cationic lipids and provides more options for the construction of lipid nanoparticles. Specifically, they can be applied to the delivery of nucleic acid drugs, anti-tumor drugs, small molecule drugs, peptide drugs, or protein drugs, thereby improving the therapeutic and / or diagnostic effects of these drugs as preventive and / or therapeutic agents. The number, type, and position of the functional groups on the cationic lipid side chains are important entry points for regulating the cellular uptake efficiency, stability under different physiological environments, biomolecule affinity, drug binding capacity, and drug delivery efficiency of the lipid nanoparticles. In particular, the cationic lipids of the present invention can be used in conjunction with conventional PEGylated lipids to achieve lipid nanoparticles of appropriate size, excellent nucleic acid drug complexing capacity and drug encapsulation efficiency, and both serum stability and low toxicity, achieving safe and efficient nucleic acid drug delivery and transfection. In addition, the present invention also provides structurally similar PEGylated lipids derived from the cationic lipids of the present invention, which can be used in conjunction with the cationic lipids of the present invention to further improve the stability and drug delivery efficiency of the lipid nanoparticles.

[0027] Implementation Method

[0028] Terminology

[0029] In the present invention, unless otherwise specified, each term has the following meaning.

[0030] In the present invention, unless otherwise specified, the “selected from” / “preferred” of any two objects are independent of each other. When there are multiple levels of selected from / preferred, the selected from / preferred of any two objects can be of the same level or different levels. For example, “L A , L B Each independently selected from A, B, C", can be L A , L B All are A, can also be L A L for A B is B1 (B1 is a subordinate of B). For example, “L A Preferably A (level 1 is preferred), more preferably A1 to A3 (level 2 is preferred), and most preferably A 11 ~A 13 (Level 3 preferred), L B Preferably B (level 1 is preferred), more preferably B1 to B3 (level 2 is preferred), and most preferably B 11 ~B 13 (3-level preference)", the preference can be A1 to A3 (2-level preference) and B is B 11 ~B 13 (Level 3 preference), or both A and B can be Level 3 preference.

[0031] In the present invention, "each independently is / is selected from / is preferably" not only means that different categories can each independently be / are selected from / are preferably any option in the definition, but also can be added with "each time it appears" to mean that the same category appears at different positions or at different times each time and is independently / is selected from / is preferably any option in the definition, for example, "the divalent linking group is -NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -、-NR c C(=O)S-、-C(R c )=N-NR c -or-NR c -N=C(R c )-; where R c Each occurrence is independently a hydrogen atom or a C 1-12 Alkyl" This description indicates that in "-NR c -N=C(R c )-”, two R c The groups may be the same or different (e.g., one R c is a methyl group, and another R c is a hydrogen atom or an ethyl group), and "-NR c C(=O)NR c -" c Can be used with "-NR c R in "C(=O)O-" c Same or different.

[0032] In the present invention, when at least two items are listed, the "combination" of the listed items refers to a combination of any two or more of the aforementioned listed items; and there is no limitation on the number of items, the number of any item can be one or more than one, and when the number of the same item is greater than 1, it can be the same or different specific forms that satisfy the item. For example, "L is a linking bond or any one or a combination of more than one selected from a hydrocarbylene group, a divalent hydrocarbon group containing heteroatoms, -O-, -S-, -SS-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, and -NH-", L can be any one of the listed items, or a combination of any two or more of them. The combination can be -CH2-O-CH2- (i.e., a combination of -O- and a hydrocarbylene group in the listed items, wherein the specific form of the hydrocarbylene group is 2 methylene groups), or -CH2-NH-CH2CH2- (i.e., a combination of -NH- and a hydrocarbylene group in the listed items, wherein the specific form of the hydrocarbylene group is 1 methylene group and 1 ethylene group). In particular, the combination of a linking bond and any linking group is still the linking group itself, and the "linking group" in the present invention defaults to containing at least one atom.

[0033] In the present invention, unless otherwise specified, the terms "include", "comprising", "containing" and similar expressions should be interpreted in an open and inclusive sense as "including but not limited to" or "including without limitation" in this specification and claims.

[0034] In the present invention, "including but not limited to" a certain range means that the items within the range are optional, but are not limited to the items within the range, and not all structures within the range are applicable. In particular, the items explicitly excluded by the present invention are not among the candidates, and the successful implementation of the present invention is the screening criterion.

[0035] In the present invention, the interpretation of numerical intervals includes numerical intervals marked with a dash (such as 1-6), numerical intervals marked with a wavy line (such as 1 to 6), and numerical intervals marked with "to / to" (such as 1 to 6, 1 to 6). Unless otherwise specified, intervals marked in the form of intervals can represent a group consisting of all integers and non-integers within the interval range, and the range includes two endpoints. For example, the average number of EO units is selected from 22 to 100, and its selection range is not limited to integers within the interval, but can also be any non-integer. For another example, "an integer in 1-3" represents a group consisting of 1, 2, and 3. For another example, -(CH2) 1-4 - represents a group consisting of -CH2-, -(CH2)2-, -(CH2)3-, and -(CH2)4-. For example, express The group composed of.

[0036] The numerical ranges in the present invention include, but are not limited to, numerical ranges expressed as integers, non-integers, percentages, and fractions, and unless otherwise specified, all include both endpoints.

[0037] In the present invention, with respect to polymer molecular weight, "about" or "approximately" generally refers to a numerical range of ±10%, which may be expanded to ±15% in some cases, but not exceeding ±20%. The predetermined value is used as the base number. For example, the deviations between 11 kDa, 12 kDa, and 10 kDa are 10% and 20%, respectively. "About 10 kDa" includes, but is not limited to, 11 kDa and 12 kDa. For another example, when a PEG component in a general formula is specified as having a molecular weight of approximately 5 kDa, the corresponding molecular weight or number average molecular weight is permitted to vary within the range of 5 kDa ± 10%, i.e., 4500 to 5500 Da.

[0038] In the present invention, for percentages, when the numerical value given (excluding the percentage sign) is accurate to the Nth decimal place, "about" or "approximately" generally means ±(0.5*0.1 N )% range. For example, about 1% refers to 1±(0.5*0.10)%, that is, the range of 0.5%-1.5%, and about 2.2% refers to 2.2±(0.5*0.1 1 )% is the range of 2.15%-2.25%, and about 2.33% refers to 2.2±(0.5*0.1 2 )% which is the range of 2.335%-2.325%.

[0039] In the present invention, the degree of polymerization of polyethylene glycol chain is expressed as n i Indicates (i is a natural number selected from 1, 2, 3, ...), n with the same value in the same structure i They can be expressed interchangeably. For example, when n2≈n3, n2 can be expressed as n3, or n3 can be expressed as n2.

[0040] The divalent linking group in the present invention, unless otherwise specified, may select either of the two connecting ends when connecting to other groups. For example, when an amide bond is used as the divalent linking group between Group A and Group B, and no specific connecting end is specified, it may be GroupA-C(=O)NH-GroupB or GroupA-NHC(=O)-GroupB.

[0041] In the present invention, when referring to the structure of a group, To mark the connection key, such as represents a group -G or G-, It represents a group -CH3 or CH3-.

[0042] In the present invention, when a connecting bond or group derived from a cyclic structure is not marked on a specific ring-forming atom but points to the interior of the ring, it means that the connecting bond can be derived from any suitable ring-forming atom, and when the marked ring is part of a parallel ring or condensed ring structure, the connecting bond can be derived from any suitable ring-forming atom in the parallel ring or condensed ring structure. Represents a structure containing any reasonable chemical connection method (including but not limited to etc.)

[0043] In the present invention, the range of carbon atoms in a group is marked in the subscript position of C in the form of a subscript, which indicates the number of carbon atoms in the group. Unless otherwise specified, the carbon number does not include the contribution of substituents. For example, C 1-12 means "having 1 to 12 carbon atoms". For example, C 1-10 Alkylene refers to any alkylene group with the number of carbon atoms in the range indicated by the subscript, i.e. C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 Any of the alkylene groups, including but not limited to linear C 1-10 Alkylene (e.g. -(CH2)6-) and branched C 1-10 Alkylene (e.g. -(CH2)3-CH(CH3)-(CH2)3-). Another example is "substituted C 1-12 "Alkyl" refers to C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 、C 11 or C 12 An alkyl group is obtained by replacing at least one hydrogen atom of the alkyl group with a substituent. The number of carbon atoms and heteroatoms in the substituent is not particularly limited.

[0044] In the present invention, when a structure has isomers, unless otherwise specified, any of the isomers may be present. For example, a structure having cis-trans isomers may be either the cis structure or the trans structure; a structure having E / Z isomers may be either the E structure or the Z structure; and a structure having optical activity may be either levorotatory or dextrorotatory.

[0045] In the present invention, if there is a difference between a structure described herein and the name of the structure, the described structure shall have greater weight.

[0046] In the present invention, the molecular weight of polyethylene glycol and its derivatives refers to the average molecular weight by default, and unless otherwise specified, the "average molecular weight" generally refers to the number-average molecular weight (M n). The number average molecular weight can refer to either the molecular weight of a polydisperse block or substance or the molecular weight of a monodisperse block or substance. Unless otherwise specified, molecular weight is expressed in Daltons (Da). The molecular weight of the polyethylene glycol chain can also be characterized by the "degree of polymerization," specifically the number of repeating units (ethylene oxide units). Accordingly, the "average degree of polymerization" or "number average degree of polymerization" is preferably used to characterize the average or number average of the number of repeating units.

[0047] In the present invention, the term "any suitable" in "any suitable linking group," "any suitable reactive group," etc., refers to a structure that conforms to the basic principles of chemical structure and enables the preparation method of the present invention to be successfully implemented. Chemical structures described in this manner can be considered to have a clear and defined scope.

[0048] In the present invention, the terms "stable existence" and "degradable" are relative concepts.

[0049] In the present invention, "degradable" refers to the breaking of the chemical bond of the invention, and the breaking is at least two residues that are independent of each other. If the structure is changed after chemical change, but the entire linker is still only a complete linker, then the linker still falls into the category of "can exist stably". The conditions for degradation are not particularly limited, and can be physiological conditions in vivo, or simulated physiological environments or other conditions in vitro, preferably physiological conditions in vivo and simulated physiological conditions in vitro. The physiological conditions are not particularly limited, and include but are not limited to serum, heart, liver, spleen, lungs, kidneys, bones, muscles, fat, brain, lymph nodes, small intestine, gonads and other parts. They can refer to cells or extracellular matrix, and can refer to normal physiological tissues or pathological physiological tissues (such as tumors, inflammation, etc.). The simulated in vitro environment is not particularly limited, and includes but is not limited to physiological saline, buffer, culture medium, etc. The speed of degradation is not particularly limited, for example, it can be rapid degradation under the action of enzymes, or it can refer to slow hydrolysis under physiological conditions, etc. The physiological conditions in vivo include physiological conditions during treatment, such as ultraviolet irradiation, heat therapy, etc. Degradation includes, but is not limited to, degradation under conditions such as light, heat, low temperature, enzymes, redox, acidic, alkaline, physiological conditions, and simulated in vitro environments, preferably under light, heat, enzymes, redox, acidic, and alkaline conditions. Degradation refers to degradation occurring under any of the aforementioned conditions. Light conditions include, but are not limited to, visible light, ultraviolet light, infrared light, near-infrared light, and mid-infrared light. Thermal conditions refer to temperatures above normal physiological temperatures, typically above 37°C and typically below 45°C, preferably below 42°C. Low temperature conditions refer to temperatures below human physiological temperature, preferably below 25°C, more preferably ≤10°C, including refrigeration temperatures, freezing temperatures, liquid nitrogen treatment temperatures, 2-10°C, 4-8°C, 4°C, 0°C, and -20±5°C. Enzymatic conditions are not particularly limited and encompass all enzymes that can be produced under physiological conditions, such as peptidases, proteases, and lyases. Redox conditions are not particularly limited, such as redox transitions and hydrogenoreduction transitions between sulfhydryl groups and disulfide bonds. The acidic and alkaline conditions mainly refer to the pH conditions of normal tissues, diseased tissues, organs or tissues in the treatment period, etc., for example, the stomach is acidic, and the tumor site is often acidic. Degradable here means that it can be degraded by metabolic effects in the body (such as physiological effects, such as enzymes, such as redox, etc.), degraded due to microenvironmental stimulation in specific parts of the body (such as acidity, alkalinity), or degraded under clinical treatment stimulation (such as light, heat, low temperature), etc. It should be noted that some extreme conditions in organic chemistry relative to organisms, such as bond breakage under strong acid, strong base, high temperature (such as above 100°C), are not included in the scope of the degradable conditions of the present invention. For example, although the ether bond can be broken under strong acid conditions such as hydrobromic acid, it is always classified as a linking group that can exist stably in the present invention.

[0050] In the present invention, "can stably exist" means that the linking group can remain as a complete linking group (a linking group is stably covalently connected to its adjacent group), which is defined as "can stably exist", wherein chemical changes that can maintain the integrity of the linking group are allowed to occur. The chemical changes are not particularly limited, and include but are not limited to isomerization, oxidation, reduction, ionization, protonation, deprotonation, substitution reactions, etc. There are no special restrictions on the conditions for stable existence, including but not limited to light, heat, low temperature, enzymes, redox, neutral, acidic, alkaline, physiological conditions, in vitro simulated environments, etc., preferably under light, heat, enzymes, redox, acidic, alkaline and other conditions. Stable existence here means that under the conditions of no special stimulation (such as pH conditions of special parts, light, heat, low temperature during treatment, etc.), a stable connection can be maintained in the metabolic cycle in the body, and the molecular weight will not be reduced due to chain scission (as long as the integrity can still be maintained).

[0051] In the present invention, "stable" is not an absolute term for a given linker. For example, an amide bond is much more stable than an ester bond under acidic or alkaline conditions, and thus "stable" linkers in the present invention include amide bonds. However, peptide bonds, for example, formed by the dehydration condensation of the α-carboxyl group of an amino acid and the α-amino group of an amino acid, can be cleaved when exposed to specific enzymes and are therefore also included in "degradable" linkers. Similarly, carbamate and thiocarbamate groups can be both stable and degradable linkers. More generally, carbamate and thiocarbamate groups are more prone to slow degradation, while non-peptide amide bonds can be stable during in vivo circulation. For example, while common ester bonds can degrade under acidic and alkaline conditions, ester bonds contained in specialized structures can also degrade under ultraviolet light. For example, even if certain chemical bonds can be degraded under the action of specific enzymes, if the circulation pathway does not pass through or basically does not pass through the specific enzyme environment during clinical use (such as in the case of targeted drug administration), the corresponding chemical bonds can still be considered to be stable.

[0052] In the present invention, all compounds of the general formula should be understood to include salts of the compounds of the general formula. The term "salt" used is selected from any one, any two, or any combination of two or more of acid addition salts formed with inorganic and / or organic acids and 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". "Salt" can be a pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salt or other salt. Salts of the 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, sulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates, sulfonates, tartrates, thiocyanates, toluenesulfonates, undecanoates, and the like; exemplary base addition salts include ammonium salts, alkali metal salts (such as sodium, lithium, and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts), salts with organic bases (e.g., 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 (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, tetradecyl, and stearyl chlorides, bromides, and iodides), aryl alkyl halides (e.g., benzyl and phenethyl bromides), and others. Both the acid addition salts and the base addition salts are preferably pharmaceutically acceptable salts and, for purposes of this disclosure, are considered equivalent to the free forms of the corresponding compounds of the general formula.

[0053] The heteroatoms in the present invention are not particularly limited, and include but are not limited to O, S, N, P, Si, F, Cl, Br, I, B, etc.

[0054] In the present invention, a group formed by losing some atoms or groups from a compound is also referred to as a residue.

[0055] In the present invention, groups with a valence greater than or equal to 2 are collectively referred to as "linking groups." Linking groups may contain only one atom, such as an ether group (-O-) or a sulfide group (-S-). In particular, when the definition of a group includes "linking bond," it indicates that the group may not contain any atoms and serves solely as a linker.

[0056] In the present invention, with respect to the valence of a group, "multivalent" means a valence of at least 3.

[0057] In the present invention, the word "group" in a divalent linking group can be replaced with "bond" without changing its meaning. For example, a divalent ether group can also be referred to as an ether bond (-O-), a divalent ester group can also be referred to as an ester bond (-OC(=O)- or -C(=O)O-), and a divalent carbamate group can also be referred to as a carbamate bond (-OC(=O)NH- or -NHC(=O)O-).

[0058] In the present invention, when the number of atoms contained in a substituent is 1, it may also be referred to as a "substituent atom".

[0059] In the present invention, when the compound or group is "substituted", it means that the compound or group contains one or more substituents.

[0060] In the present invention, unless otherwise specified, "amino" and "amine" have the same meaning and include monovalent, divalent, trivalent, or tetravalent neutral groups or cationic groups, substituted or unsubstituted. For example, the -NH2 in CH3-NH2 can be referred to as an "amino group," "amine group," or "primary amino group." For another example, the -NH- in CH3-NH-CH3 can be referred to as a "secondary amino group" or "secondary amino group," and the -NH-CH3 therein can also be understood as an amino group substituted with a methyl group.

[0061] In the present invention, "amine group" includes but is not limited to primary amine group, secondary amine group, tertiary amine group and quaternary ammonium ion. For example, -NR t R t and -N + R t R t R t , where each R t Each is independently a hydrogen atom or any hydrocarbon structure.

[0062] In the present invention, when the valence state is not specified, the "hydrocarbon group" can be a monovalent hydrocarbon group, a divalent hydrocarbon group, a trivalent hydrocarbon group, ..., an n-valent hydrocarbon group, wherein n is the highest valence state that the hydrocarbon group can have.

[0063] In the present invention, a "hydrocarbylene group" is a divalent hydrocarbon group.

[0064] The secondary amine bond and hydrazine bond in the present invention refer to "-NH-" or "-NH-NH-" with both ends capped by hydrocarbon groups, such as -CH2-NH-CH2- and -CH2-NH-NH-CH2-; while -C(=O)-NH- is called an amide bond and is not considered to contain a secondary amine bond.

[0065] In the present invention, "functional group" is also referred to as "functional group," preferably a reactive group, a protected reactive group, a precursor of a reactive group, etc. "Polymeric" means the number of functional groups is at least 3, e.g., a polyol refers to a compound containing at least 3 hydroxyl groups, a polythiol refers to a compound containing at least 3 thiol groups, etc. It should be noted that other heterogeneous functional groups are permitted, for example, tris(hydroxymethyl)aminomethane is a trihydric alcohol containing an amino group, and citric acid is a tricarboxylic acid containing a hydroxyl group.

[0066] In the preparation method of the present invention, unless otherwise specified, the reactive groups also include protected forms thereof, and the protected forms can be deprotected in any appropriate step of the actual preparation process to obtain the corresponding active forms.

[0067] In the present invention, ring atoms are atoms that together constitute the ring skeleton.

[0068] In the present invention, "biologically related substances" include, but are not limited to, substances described, listed, and cited in documents CN104877127A, WO / 2016 / 206540A, CN106967213A, CN108530637A, CN108530617A, and the references thereto. In general, biologically related substances include, but are not limited to, the following substances: drugs, proteins, polypeptides, oligopeptides, protein mimetics, fragments and analogs, enzymes, antigens, antibodies and their fragments, receptors, small molecule drugs, nucleosides, nucleotides, oligonucleotides, antisense oligonucleotides, polynucleotides, nucleic acids, aptamers, polysaccharides, proteoglycans, glycoproteins, steroids, steroid compounds, lipid compounds, hormones, vitamins, phospholipids, glycolipids, dyes, fluorescent substances, targeting factors, targeting molecules, cytokines, neurotransmitters, extracellular matrix substances, plant or animal extracts, viruses, vaccines, cells, vesicles, liposomes, micelles, and the like. The bio-related substance can be the bio-related substance itself, or its precursor, activated state, derivative, isomer, mutant, analog, mimetic, polymorph, pharmaceutically acceptable salt, fusion protein, chemically modified substance, genetically recombinant substance, etc., and can also be a corresponding agonist, activator, activator, inhibitor, antagonist, modulator, receptor, ligand or ligand, antibody and its fragment, active enzyme (such as kinase, hydrolase, lyase, redox enzyme, isomerase, transferase, deaminase, deiminase, invertase, synthetase, etc.), enzyme substrate (such as coagulation cascade protease substrate, etc.). The derivative includes but is not limited to glycosides, nucleosides, amino acids, and polypeptide derivatives. Chemically modified products that form new reactive groups, that is, modified products generated by modifying reactive groups to change their type, or by introducing additional functional groups, reactive groups, amino acids or amino acid derivatives, polypeptides, etc., all belong to chemically modified substances of bio-related substances. Before or after being combined with the functionalized polyethylene glycol, the bio-related substance is allowed to have a target molecule, appendage or delivery carrier combined with it to form a modified bio-related substance or a composite bio-related substance. Among them, the pharmaceutically acceptable salt can be an inorganic salt such as hydrochloride, sulfate, phosphate, or an organic salt such as oxalate, malate, citrate, etc. Among them, the "drug" in the present invention includes any agent, compound, composition or mixture that provides a physiological or pharmacological effect in vivo or in vitro, and often provides a beneficial effect. There is no special limitation on its type, including but not limited to drugs, vaccines, antibodies, vitamins, foods, food additives, nutrients, nutritional supplements and other agents that provide beneficial effects. There is no special limitation on the scope of the physiological or pharmacological effect produced by the "drug" in the body, and it can be a systemic effect or only produce an effect locally.The activity of the "drug" is not particularly limited. It primarily refers to active substances that interact with other substances, but can also be inert substances that do not interact. However, inert drugs can be converted into active forms through in vivo effects or certain stimuli. "Small molecule drugs" are biologically relevant substances with a molecular weight of no more than 1000 Da, or small molecule mimics or active fragments of any biologically relevant substance.

[0069] In the present invention, unless otherwise specified, "monosaccharide group" refers to a monosaccharide residue, that is, a monosaccharide backbone, including open-chain monosaccharides and cyclic monosaccharides (such as furanose rings and pyranose rings).

[0070] The monosaccharide group in the present invention can be selected from the residue of any one of the compounds including but not limited to monosaccharides, sugar alcohols, deoxy sugars, amino sugars, amino sugar derivatives (such as amide derivatives), sugar acids, and glycosides, and can be an open-chain structure or a cyclic structure. For example, an amino residue is formed by removing an amino hydrogen atom from an amino sugar, and an acyl group is formed by removing a carboxyl hydroxyl group from a sugar acid. The monosaccharide may include but is not limited to aldoses (polyhydroxy aldehydes) and ketoses (polyhydroxy ketones). For example, alkyl ether derivatives and methyl ether derivatives, such as quebracho. The number of carbon atoms in the monosaccharide group in the present invention is not particularly limited, and includes but is not limited to tetroses, pentoses, hexoses, and heptoses. Pentose and hexose are preferred. Among them, examples of tetroses, pentoses, hexoses, heptoses, sugar alcohols, deoxy sugars, amino sugars, amide derivatives of amino sugars, sugar acids, glycosides, etc. include but are not limited to the structures disclosed in CN106967213A.

[0071] "Minor modification" in the present invention refers to a chemical modification process that can be completed through a simple chemical reaction process. The simple chemical reaction process mainly includes chemical reaction processes such as protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation, or changing the leaving group.

[0072] In the present invention, "minor changes" correspond to "minor modifications" and refer to structures that can form the target reactive group after undergoing simple chemical reactions such as protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation, or leaving group modification. Modification of the leaving group, i.e., the conversion of the leaving group, includes, but is not limited to, the conversion of an ester form to an acyl chloride form.

[0073] In some specific embodiments of the present invention, the reaction process also involves the "protection" and "deprotection" of related groups. To prevent a reactive group from affecting the reaction, the reactive group is typically protected. In some specific embodiments of the present invention, when there are two or more reactive groups, only the target reactive group is selectively reacted, and therefore the other reactive groups are protected. Protecting groups not only remain stable during the target reaction, but can also be removed as needed using conventional techniques in the art.

[0074] In this invention, "protection" of a reactive group refers to a strategy for 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 a protecting group for the hydroxyl group.

[0075] In the present invention, "deprotection" and "deprotection" have the same meaning, and both refer to the process of converting a protected group from a protected form to an unprotected form.

[0076] In the present invention, "hydroxy protecting group" includes all groups that can be used as a protecting group for a common hydroxyl group. The hydroxy protecting group is preferably an alkanoyl group (such as acetyl, tert-butyryl), an aralkanoyl group (such as benzoyl), a benzyl group, a trityl group, a trimethylsilyl group, a tert-butyldimethylsilyl group, an allyl group, an acetal group or a ketal group. The removal of the acetyl group is generally carried out under alkaline conditions, and the most commonly used methods are aminolysis with NH3 / MeOH and methanolysis catalyzed by methanol anion; the benzyl group can be easily removed by palladium-catalyzed hydrogenolysis in a neutral solution at room temperature, and can also be reduced and cleaved by metal sodium in ethanol or liquid ammonia; the trityl group is generally removed by catalytic hydrogenolysis; the trimethylsilyl group is usually removed using a reagent containing fluoride ions (such as tetrabutylammonium fluoride / anhydrous THF, etc.); tert-butyldimethylsilyl ether is relatively stable and can withstand the ester hydrolysis conditions of alcoholic potassium hydroxide and mild reducing conditions (such as Zn / CH3OH, etc.), and can be removed by fluoride ions (such as Bu4N + F - ) is removed in tetrahydrofuran solution or with aqueous acetic acid at room temperature. The diol is preferably protected by forming dioxolane, dioxane, cyclic carbonate or cyclic borate.

[0077] In the present invention, "sulfhydryl protecting group" includes all groups that can be used as a conventional sulfhydryl protecting group. Similar to hydroxyl, sulfhydryl can be protected in the form of thioether and thioester. The sulfhydryl protecting group is preferably tert-butyl, benzyl, substituted benzyl, diphenylmethyl, substituted diphenylmethyl, triphenylmethyl, acetyl, benzoyl, tert-butyloxycarbonyl, benzyloxycarbonyl, thioacetal or thioketal. The deprotection of thioether can be carried out by reducing with Na / NH3 under acid catalysis or by using heavy metal ions such as Ag. + 、Hg + The reaction is then treated with hydrogen sulfide. Some groups, including S-diphenylmethyl, S-triphenylmethyl sulfide, S-2-tetrahydropyranyl, and S-isobutoxymethyl hemimercaptoacetals, can be oxidized to disulfides using (SCN)2, iodine, or thionyl chloride, followed by reduction to thiols. Thioester formation and deprotection methods are similar to those for carboxylates.

[0078] In the present invention, "carboxyl protecting group" refers to a protecting group that can be converted into a carboxyl group by the deprotection reaction of hydrolysis or carboxyl protecting group. Carboxyl protecting group is preferably an alkyl group (such as a methyl group, an ethyl group, a tert-butyl group) or an aralkyl group (such as a benzyl group), more preferably a tert-butyl group (tBu), a methyl group (Me) or an ethyl group (Et). In the present invention, "protected carboxyl" refers to a group formed after a carboxyl group is protected by a suitable carboxyl protecting group, preferably a methoxycarbonyl group, an ethoxycarbonyl group, a tert-butyloxycarbonyl group or a benzyloxycarbonyl group. The carboxyl protecting group can be removed by hydrolysis under the catalysis of an acid or base, and can be eliminated by thermal decomposition reaction occasionally. For example, the tert-butyl group can be removed under mild acidic conditions, and the benzyl group can be removed by hydrogenolysis. The reagent for removing the carboxyl protecting group is selected from TFA, H2O, LiOH, NaOH, KOH, MeOH, EtOH and a combination thereof, preferably a combination of TFA and H2O, a combination of LiOH and MeOH or a combination of LiOH and EtOH. The protected carboxyl group is deprotected to produce the corresponding free acid. The deprotection is carried out in the presence of a base. The base and the free acid formed by the deprotection form a pharmaceutically acceptable salt.

[0079] In the present invention, "amino protecting group" is equivalent to "amine protecting group" and includes all groups that can be used as conventional amino / amine protecting 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, or silyl, etc. The amino protecting group is preferably Boc (tert-butyloxycarbonyl), Moz (p-methoxybenzyloxycarbonyl), and Fmoc (9-fluorenylmethyleneoxycarbonyl). The reagent for removing the amino protecting group is selected from TFA, H2O, LiOH, MeOH, EtOH, and combinations thereof, preferably a combination of TFA and H2O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. The reagent for removing the Boc protection is TFA or HCl / EA; TFA is preferred. The deprotecting agent used for removing the Fmoc protection is a solution of 20% piperidine in N,N-dimethylformamide (DMF).

[0080] In the present invention, "alkynyl protecting group" includes all groups commonly used as alkynyl protecting groups, preferably trimethylsilyl (TMS), triethylsilyl, tert-butyldimethylsilyl (TBS), or biphenyldimethylsilyl. TMS-protected alkynyl groups are easily deprotected under alkaline conditions, such as KCO / MeOH or KOH / MeOH. TBS-protected alkynyl groups can be deprotected in tetra-n-butylammonium fluoride in tetrahydrofuran (TBAF / THF).

[0081] In the present invention, the hydroxyl group protected by the hydroxyl-protecting group is not particularly limited, and may be, for example, an alcoholic hydroxyl group, a phenolic hydroxyl group, or the like. The amino / amine group protected by the amino-protecting group is not particularly limited, and may be, for example, a primary amine, a secondary amine, a hydrazine, an amide, or the like. In the present invention, the amine group is not particularly limited, and includes, but is not limited to, a primary amine group, a secondary amine group, a tertiary amine group, and a quaternary ammonium ion.

[0082] In the present invention, the repeating unit of the polyethylene glycol component is an oxyethylene unit, i.e., -CH2CH2O- or -OCH2CH2-, also referred to as "EO unit", the number of repeating units is also referred to as the number of EO units, and the average number of repeating units is also referred to as the average number of EO units, and preferably the number average number.

[0083] In the present invention, for the case of polydispersity, the molecular weight / polymerization degree of a single molecule of a compound and the number average molecular weight / number average polymerization degree of the compound components in a macroscopic substance are "equal" or "same" or "equal" (including other forms of equivalent expressions), unless otherwise specified, to strictly equal values, but rather to close or approximately equal values. The said close or approximately equal values ​​preferably have a deviation of no more than ±10%, usually based on a preset value.

[0084] In the present invention, for the case of monodispersity, the same or equal number of ethylene oxide units in individual compound molecules and general formulas means that they are strictly equal in value; for example, if the number of EO units of a certain PEG component is set to 11, then a set value of 12 does not fall within the set range; however, for a macroscopic product obtained by a certain preparation method in order to obtain a compound component containing a set number of EO units, due to the limitations of the preparation method and purification method, the macroscopic product may also contain other EO unit components in addition to the target EO unit component. In this case, when the average number of EO units deviates from the preset number of EO units by no more than ±5% (base ≥ 10) or no more than ±0.5 (base < 1 0), it is deemed that a monodisperse macroscopic product containing the target component is obtained; in addition, when the content of the component that meets the range of the number of EO units or the average number of EO units reaches a certain percentage (preferably ≥90%, more preferably >95%, more preferably greater than 96%, more preferably greater than 98%, more preferably 99% to 100%), it is also deemed that a monodisperse macroscopic product containing the target component is obtained; even if the above-mentioned content ratio is not reached, as long as the preparation method of the present invention or a similar method using basically the same preparation concept is adopted, the product with insufficient content obtained for some reason, the component that appears in the form of the main product, co-product or by-product, regardless of whether it is separated and purified, are all within the scope of the present invention.

[0085] In the present invention, when the molecular weight of a compound formula of a polydisperse component is described using Da, kDa, the number of repeating units, or the number of EO units, for a single compound molecule, the molecular weight value is allowed to fall within a certain range of the given value (including the endpoints, preferably within a range of ±10%). When the number of ethylene oxide units is used to describe the predetermined molecular weight of a compound formula of a monodisperse component, there is no range fluctuation and the molecular weight is a discrete point. However, the average number of EO units in the prepared product may fluctuate within a certain range (not exceeding ±10% or ±1, preferably not exceeding ±5% or ±0.5) due to the non-uniform molecular weight. For example, the molecular weight of mPEG (methoxypolyethylene glycol unit) is 5 kDa, which means that the molecular weight of a single molecule in the general formula is between 4500 and 5500 Da, and the average molecular weight of the corresponding components of the corresponding prepared product is 5 kDa. That is, the product with an average molecular weight between 4500 and 5500 Da is the target product, and only the components with a molecular weight falling within this range contribute to the content of the target component. For another example, if mPEG is designed to have 22 ethylene oxide units, the number of EO units of all compound molecules in the general formula should strictly be 22, but the prepared product may be a mixture of compounds with 20, 21, 22, 23, or 24 EO units. In this case, when the average number of EO units falls within the range of 22±2.2 (preferably within the range of 22±1.1), the target component is considered to be obtained, and the components with a molecular weight falling within this numerical range can all be considered as target components for calculating purity.

[0086] In the present invention, unless otherwise specified, "mPEG" refers to a methoxy-terminated polyethylene glycol segment, the structural formula of which is Among them, n i is the degree of polymerization of the polyethylene glycol chain.

[0087] In the present invention, a product with a PDI < 1.005 can be regarded as monodisperse, which can be recorded as PDI = 1.

[0088] In the present invention, the number of repeating units in the "single-chain component" is at least 4.

[0089] In the present invention, " lipid " is also called " lipid " (lipid), includes but not limited to the ester of fatty acid, and is characterized by having poor solubility usually in water, but being soluble in many non-polar organic substances. Although lipid has poor solubility usually in water, some classes of lipid (for example, lipid modified by polar groups such as DMG-PEG2000) have limited water solubility and can be dissolved in water under certain conditions. The known types of lipid include biomolecules, for example fatty acids, waxes, sterols, fat-soluble vitamins (such as vitamin A, D, E and K), monoglycerides, diglycerides, triglycerides and phospholipids.

[0090] In the present invention, lipids include simple esters, complex esters, and derivatized lipids. Simple esters are esters composed of fatty acids and alcohols, which can be further divided into three subcategories: fats, oils, and waxes. Complex esters, also known as "lipidoid compounds," include phospholipids, sphingolipids, glycolipids, steroids and sterols, and lipoproteins. Derivatized lipids, including simple lipid derivatives and complex lipid derivatives, possess the general properties of lipids.

[0091] In the present invention, lipids may be synthetic or derived (isolated or modified) from natural sources or compounds.

[0092] In the present invention, " lipid nanoparticle" or " LNP" (lipid nanoparticle) refers to nanometer scale (such as 1nm to 1000nm) particle comprising one or more types of lipid molecules. In the present invention, LNP can further include at least one non-lipid payload molecule (such as, one or more nucleic acid molecules). In some embodiments, LNP comprises non-lipid payload molecules partially or completely encapsulated in lipid shell inside. Especially, in some embodiments, wherein payload is negatively charged molecule (such as, mRNA encoding viral protein), and the lipid component of LNP comprises at least one cationic lipid and at least one pegylated lipid. It is contemplated that cationic lipid can interact with negatively charged payload molecule, and promotes payload to be incorporated into and / or be encapsulated in LNP during LNP formation. As provided herein, other lipids that can form a part for LNP include but are not limited to neutral lipids and steroid lipids.

[0093] As used herein, "cationic" refers to the ability of a corresponding structure to permanently or non-permanently acquire a positive charge in response to certain conditions (e.g., pH). Thus, cations include both permanent cations and cationizable cations. A permanent cation refers to a compound, group, or atom that is positively charged at any pH or hydrogen ion activity of its environment. Typically, the positive charge is generated by the presence of a quaternary nitrogen atom. When a compound carries multiple such positive charges, it can be referred to as a permanent cation. A cationizable refers to a compound, group, or atom that is positively charged at relatively low pH and uncharged at relatively high pH of its environment. Furthermore, in a non-aqueous environment where pH cannot be determined, a cationizable compound, group, or atom is positively charged at high hydrogen ion concentrations and uncharged at low hydrogen ion concentrations or activity. Whether it is charged or uncharged at a given pH or hydrogen ion concentration depends on the properties of the cationizable or polycationizable compound, particularly the pKa of the corresponding cationizable group or atom. In a dilute aqueous environment, the fraction of cationizable compounds, groups or atoms that are positively charged can be estimated using the so-called Henderson-Hasselbalch equation, which is well known to those skilled in the art. In some embodiments, the whole or part of the cationizable compound is positively charged at physiological pH values ​​(e.g., about 7.0-7.4). In some preferred embodiments, the whole or part of the cationizable compound is neutral at physiological pH values ​​(e.g., about 7.0-7.4), but is positively charged at lower pH values ​​(e.g., about 5.5 to 6.5). In some embodiments, the pKa of the whole or part of the cationizable compound is preferably in the range of about 5 to about 7.

[0094] In the present invention, "cationic lipid" refers to a lipid that is positively charged at any pH value or hydrogen ion activity of its environment, or a lipid that is positively charged in response to the pH value or hydrogen ion activity of its environment (e.g., its intended use environment). Therefore, the term "cationic" encompasses the scope of "permanent cations" and "cationizable". In some embodiments, the positive charge in the cationic lipid is derived from the presence of a quaternary nitrogen atom. In some embodiments, the cationic lipid includes a zwitterionic lipid that is positively charged in the environment in which it is intended to be applied (e.g., at endosomal pH). In some embodiments, if a liposome contains a cationizable lipid, it is preferred that it is at a pH of about 1 to 9, preferably 4 to 9, 5 to 8, or 6 to 8, most preferably at an endosome pH (e.g., about 5.5 to 6.5), wherein about 1% to 100% of the cationizable lipid is cationized. Cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethylamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethylamine ( KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino)butyric acid triheptadecyl-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), any one of the cationic lipids disclosed in CN113402405A and mixtures thereof.

[0095] In the present invention, "PEGylated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion, and can be divided into linear PEGylated lipids and non-linear PEGylated lipids based on their structure. In addition to the PEGylated lipids described in the present invention, PEGylated lipids also include but are not limited to polyethylene glycol-1,2 dimyristin (PEG-DMG), polyethylene glycol-distearyl phosphatidylethanolamine (PEG-DSPE), PEG-cholesterol, polyethylene glycol-diacylglycerol (PEG-DAG), polyethylene glycol-dialkoxypropyl (PEG-DAA), specifically including polyethylene glycol 500-dipalmitoylphosphatidylcholine, polyethylene glycol 2000-dipalmitoylphosphatidylcholine, polyethylene glycol 500-stearoylphosphatidylethanolamine, polyethylene glycol 2000-distearoylphosphatidylethanolamine, polyethylene glycol 500-1,2-oleoylphosphatidylethanolamine, polyethylene glycol 2000-1,2-oleoylphosphatidylethanolamine and polyethylene glycol 2000-2,3-dimyristoylglycerol (PEG-DMG) and the like.

[0096] In the present invention, "neutral lipid" refers to any of a number of lipid substances that exist in the form of no charge or neutral zwitterions at a selected pH, preferably phospholipids. Such lipids include but are not limited to 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (MPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSP ...DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phospho Alkanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0DietherPC), 1-oleoyl-2-cholestyrene-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) Neutral lipids include any of: 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 and lysophosphatidylethanolamine (LPE), and combinations thereof. Neutral lipids may be synthetic or naturally derived.

[0097] In the present invention, the "steroid lipid" is selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and mixtures thereof.

[0098] The liposomes in the present invention can be referred to as "PEGylated liposomes" when they contain PEGylated lipids, and can be referred to as "cationic liposomes" when they contain cationic lipids. When they contain both PEGylated lipids and cationic lipids, they can be referred to as both "PEGylated liposomes" and "cationic liposomes."

[0099] In the present invention, "cationic liposomes" are liposomes containing cationic lipids, and may also contain other types of lipids (including but not limited to pegylated lipids, neutral lipids, steroid lipids, etc.).

[0100] In the present invention, the "N / P ratio" refers to the molar ratio of nitrogen atoms in the cationic lipid to phosphate in the nucleic acid.

[0101] In the present invention, "nucleic acid" refers to DNA or RNA or a modified form thereof, which contains purine or pyrimidine bases (adenine "A", cytosine "C", guanine "G", thymine "T") present in DNA or purine or pyrimidine bases (adenine "A", cytosine "C", guanine "G", uracil "U") present in RNA.

[0102] In the present invention, "RNA" refers to ribonucleic acid that may be naturally occurring or non-naturally occurring. For example, RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides or linkers. RNA may include a cap structure, a chain-terminating nucleoside, a stem-loop, a polyadenylate sequence and / or a polyadenylation signal. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, such as in vivo translation of an mRNA within a mammalian cell, can produce the encoded polypeptide. RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single-stranded guide RNA (sgRNA), cas9 mRNA and mixtures thereof.

[0103] In the present invention, antisense oligonucleotides or small interfering RNA (siRNA) can inhibit the expression of target genes and target proteins in vitro or in vivo.

[0104] In the present invention, FLuc mRNA can express luciferase protein, which emits bioluminescence in the presence of luciferin substrate, so FLuc is often used in mammalian cell culture to measure gene expression and cell activity.

[0105] In the present invention, "inhibiting the expression of a target gene" refers to the ability of nucleic acid to silence, reduce or inhibit the expression of a target gene. To test the degree of gene silencing, a test sample (e.g., a cell sample in a culture medium expressing a target gene) is contacted with a nucleic acid that inhibits the expression of a target gene. The expression of the target gene in a test sample or test animal is compared with the expression of the target gene in a control sample (e.g., a cell sample in a culture medium expressing a target gene) that is not contacted with or not administered with nucleic acid. The expression of the target gene in the control sample can be assigned a value of 100%. In a specific embodiment, when the target gene expression level in the test sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% relative to the target gene expression level in the control sample or control mammal, the expression of the target gene is inhibited.

[0106] In the present invention, methods for determining the expression level of the target gene include, but are not limited to, dot blot, northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme action, and phenotypic assay.

[0107] In the present invention, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0108] In the present invention, "antigen" typically refers to a substance that can be recognized by the immune system, preferably recognized by the adaptive immune system, and can trigger an antigen-specific immune response, for example, by forming antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen can be or can comprise a peptide or protein that can be presented to a T cell by MHC. In the sense of the present invention, an antigen can be a translation product of a provided nucleic acid molecule (preferably an mRNA as defined herein). In this context, fragments, variants, and derivatives of peptides and proteins comprising at least one epitope are also understood to be antigens.

[0109] In the present invention, "delivery" refers to providing an entity to a target, for example, delivering a drug and / or therapeutic agent and / or preventive agent to a subject, which is a tissue and / or cell of a human and / or other animal.

[0110] As used herein, "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 the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical compositions are 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 compositions may also contain small amounts of wetting agents, emulsifiers, or pH buffers, as needed. Oral formulations may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, 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 or coatings, 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.

[0111] The pharmaceutical composition of the present invention can act systemically and / or locally. For this purpose, they can be administered by suitable routes, for example, by injection (such as intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermal administration; or by oral, buccal, nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation administration. For these routes of administration, the pharmaceutical composition of the present invention can be administered in suitable dosage forms. The dosage forms include but are not limited to tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

[0112] In the present invention, a vaccine is a preventive 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.

[0113] In the present invention, 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.

[0114] 1. Cationic lipids

[0115] One embodiment of the present invention:

[0116] A cationic lipid, characterized in that the structure is as shown in the general formula (1):

[0117]

[0118] Wherein, N is a nitrogen branching center;

[0119] L1 and L2 are each independently a connecting bond or a divalent connecting group;

[0120] L3 is a trivalent linking group, and together with R3 forms a divalent linking group -L3(R3)- with the side chain being R3;

[0121] B1 and B2 are each independently a connecting bond or C 1-30 alkylene;

[0122] R1 and R2 are each independently C 2-30 Aliphatic hydrocarbon or C containing 1-2 O 2-30 Aliphatic hydrocarbon derivative residues;

[0123] R3 is -OR d 、-NR d R d 、-SR d 、-(C=O)R d、-(C=O)OR d 、-O(C=O)R d 、-O(C=O)OR d or Among them, R d Each occurrence is independently C 1-12 Alkyl, G1 is a branched group with a valence of k+1, j is 0 or 1, and F1 contains a functional group R 01 ; When j is 0, G1 does not exist; when j is 1, G1 leads to k F1s, and the k F1s are independently the same or different structures, and k is an integer from 2 to 8;

[0124] R4 is a carbocyclic group, a heterocyclic group or R'N(R')-R"-N(R')-; wherein the heterocyclic group is a cyclic group containing 1, 2 or more heteroatoms as ring atoms, wherein the heteroatoms are B, O, N, Si, P or S; wherein each occurrence of R' is independently H or C 1-3 Alkyl, R" is C 2-4 alkylene;

[0125] The alkylene group, aliphatic hydrocarbon group, aliphatic hydrocarbon derivative residue, carbocyclic group, and heterocyclic group are each independently substituted or unsubstituted;

[0126] or a salt, tautomer, stereoisomer or solvate thereof.

[0127] 1.1. Stable and degradable divalent linking groups

[0128] In the present invention, unless otherwise specified, any divalent linking group itself or a divalent linking group composed of any divalent linking group and an adjacent heteroatom group can be either a stable linking group (STAG) or a degradable linking group (DEGG).

[0129] (1) Stable divalent linking group (STAG)

[0130] There is no particular restriction on the conditions under which STAG can exist stably. It can exist stably under any condition including but not limited to light, heat, low temperature, enzymes, redox, acidic, alkaline conditions, physiological conditions, in vitro simulated environments, etc., preferably under any condition including light, heat, enzymes, redox, acidic, alkaline, etc.

[0131] The type of STAG is not particularly limited, and includes but is not limited to alkylene, divalent heteroalkyl, double bond, triple bond, divalent diene, divalent cycloalkyl, divalent cycloalkenyl, divalent cycloalkene, divalent cycloalkenyl, aromatic ring, aliphatic heterocycle, heterophenyl ring, aromatic heterocycle, heterocondensed heterocycle, substituted alkylene, substituted heteroalkyl, substituted divalent heteroalkyl, substituted double bond, substituted triple bond, substituted diene, substituted divalent cycloalkyl, substituted divalent cycloalkenyl, substituted divalent cycloalkene, substituted divalent cycloalkenyl, substituted divalent cycloalkenyl, substituted divalent cycloalkyne, substituted aromatic ring, substituted Aliphatic heterocycle, substituted heterobenzene ring, substituted aromatic heterocycle, substituted condensed heterocycle, ether bond, thioether bond, urea bond, thiourea bond, carbamate group, thiocarbamate group, -P(=O)-, divalent silicon group without active hydrogen, divalent linking group containing boron atom, secondary amino group, tertiary amino group, carbonyl group, thiocarbonyl group, amide group, thioamide group, sulfonamide group, enamine group, triazole, 4,5-dihydroisoxazole, any divalent linking group in the backbone of amino acid and derivatives thereof, or a stable divalent linking group composed of any two or more groups.

[0132] Specifically, STAG includes but is not limited to the structures described and listed in CN104530413A, CN104530415A, and CN104530417A. Taking CN104530417A as an example, the corresponding sections are

[0627] to

[0704] . There is no particular limitation on the manner in which two or more stably existing divalent linking groups can be combined to form a STAG, including but not limited to section

[704] of CN104530417A.

[0133] (2) Degradable divalent linking group (DEGG) in the present invention

[0134] There is no particular limitation on the conditions under which DEGG can be degraded. DEGG can be degraded under any conditions including but not limited to light, heat, low temperature, enzymes, redox, acidity, alkalinity, physiological conditions, and in vitro simulated environments. DEGG can be degraded under any conditions including but not limited to light, heat, enzymes, redox, acidity, alkalinity, and the like.

[0135] The divalent linking group formed by combining any DEGG and any STAG is still a degradable linking group. For the degradable divalent linking group containing an aromatic ring, it can also be formed by combining an aromatic ring and a degradable divalent linking group.

[0136] The type of DEGG is not particularly limited, and includes but is not limited to disulfide bonds, vinyl ether bonds, ester groups, thioester groups, thioester groups, dithioester groups, carbonate groups, thiocarbonate groups, dithiocarbonate groups, trithiocarbonate groups, carbamate groups, thiocarbamate groups, dithiocarbamate groups, acetals, cyclic acetals, thioacetals, azaacetals, azaheterocyclic acetals, azathioacetals, dithioacetals, hemiacetals, thiohemiacetals, azahemiacetals, ketals, thioketals, azaketals, azaheterocyclic ketals, azathioketals, imine bonds, hydrazone bonds, acylhydrazone bonds, oxime bonds, thiooxime ether groups, semicarbazone bonds, thiosemicarbazone bonds, hydrazine groups, hydrazide groups, thiocarbonylhydrazide groups, azocarbonylhydrazide groups, thioazocarbonylhydrazide groups, carbamate groups, hydrazide groups, The invention further comprises a divalent linking group selected from the group consisting of an aminothiocarbamate group, an isourea group, an isothiourea group, an allophanate group, a thioalloy group, a guanidine group, an amidine group, an aminoguanidine group, an aminoamidino group, an imidic acid group, an imidic acid thioester group, a sulfonate group, a sulfinate group, a sulfonylhydrazine group, a sulfonylurea group, a maleimide group, an orthoester group, a phosphate group, a phosphite group, a hypophosphite group, a phosphonate group, a phosphosilane group, a silane group, a carbonamide, a thioamide, a sulfonamide group, a polyamide, a phosphoramide, a phosphoramidite, a pyrophosphamide, a cyclophosphamide, an ifosfamide, a thiophosphamide, an aconityl group, a polypeptide fragment, a nucleotide and its derivative backbone, a deoxynucleotide and its derivative backbone, or a combination of any two or more divalent linking groups.

[0137] The carbamate group, thiocarbamate group, carbonamide, phosphoramide, etc. herein can be used as either a stable linking group or a degradable linking group, depending on the environment in which they are used.

[0138] Specifically, DEGG includes but is not limited to the structures described and listed in documents CN104530413A, CN104530415A, and CN104530417A. Taking CN104530417A as an example, the corresponding sections are

[705] to

[0725] .

[0139] 1.2.L1, L2

[0140] In a specific embodiment of the present invention, L1 and L2 are any of the following:

[0141] Case (1): One of L1 and L2 is a connecting bond, and the other is a divalent connecting group;

[0142] Case (2): L1 and L2 are both connecting bonds;

[0143] Case (3): L1 and L2 are both divalent linking groups, and L1 and L2 have the same or different structures;

[0144] In any of the above cases, the divalent linking group is -O-, -S-, -SS-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -NR c -、-NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -、-NR c C(=O)S-、-C(R c )=N-NR c -、-NR c -N=C(R c )-any one, or any two and C 1-10 A combination of alkylene groups; wherein R c Each occurrence is independently H or methyl, preferably H;

[0145] Preferably, L1 and L2 are each independently -O-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -(C=O)O(CH2) y OC(=O)-、-O(C=O)(CH2) y OC(=O)-、-(C=O)O(CH2) y C(=O)O-, -O(C=O)(CH2) y C(=O)O-, wherein y is an integer of 2-8.

[0146] In a specific embodiment of the present invention, L1 and L2 are each independently selected from any one of -O(C=O)-, -(C=O)O- and -O(C=O)O-.

[0147] In a specific embodiment of the present invention, one of L1 and L2 is -(C=O)-, and the other is -O(C=O)-, -(C=O)O-, -O(C=O)O- or -O-.

[0148] In a specific embodiment of the present invention, one of L1 and L2 is -O(C=O)O-, and the other is -O(C=O)- or -(C=O)O-.

[0149] In one embodiment of the present invention, L1 and L2 are both -O(C=O)- or both -(C=O)O-.

[0150] In one embodiment of the present invention, L1 and L2 are both -O(C=O)O-.

[0151] In one embodiment of the present invention, L1 and L2 are both -O-.

[0152] In a specific embodiment of the present invention, L1 and L2 are each independently selected from any one of -NH(C=O)-, -(C=O)NH-, -O(C=O)-, and -(C=O)O-.

[0153] In one embodiment of the present invention, L1 and L2 are both -(C=O)O(CH2) y OC(=O)-、-O(C=O)(CH2) y OC(=O)-、-(C=O)O(CH2) y C(=O)O-, -O(C=O)(CH2) y C(=O)O-, wherein y is an integer of 2-8.

[0154] 1.3.L3, R3

[0155] In a specific embodiment of the present invention, the divalent linking group -L3(R3)- is selected from any one of -L4-, -Z-L4-, -L5-Z-L4-, -L4-Z-L5-, -Z-L4-Z-L5-, -L5-Z-L4-Z-L5-, -L4-Z-L5-Z-L5-, -Z-L5-Z-L4- and -L5-Z-L5-Z-L4-, and the right end is connected to the nitrogen branching center; wherein L4 is -(CH2) x -CR3H-(CH2) x -, L5 is -(CH2) x -, x is independently an integer from 0 to 3 at each occurrence, and Z is independently -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, -NR c -、-NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-; wherein R cEach occurrence is independently H or methyl, preferably H;

[0156] Preferably -L3(R3)- is -(CH2) x -CR3H-(CH2) x -.

[0157] In a specific embodiment of the present invention, the structure of the aforementioned -L3(R3)- is wherein a1 is a connecting bond connected to R4, a2 is a connecting bond connected to the nitrogen branching center in the general formula (1); Preferably, any one of the following structures:

[0158] In a specific embodiment of the present invention, the aforementioned for Preferably

[0159] In a specific embodiment of the present invention, the divalent linking group -L3(R3)- is composed of 1 L4, 0-4 Z and 0-4 L5 in a linear manner, and the arrangement of L4, Z and L5 is not particularly limited; wherein L4 is -(CH2) x -CR3H-(CH2) x -, L5 is -(CH2) x -, x is independently an integer from 0 to 3 at each occurrence, and Z is independently -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, -NR c -、-NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-; wherein R c Each occurrence is independently H or methyl; preferably -L3(R3)- is any one of the following structures:

[0160]

[0161] 1.4.B1, B2

[0162] In a specific embodiment of the present invention, B1 and B2 are each independently a connecting bond or C 1-20 Alkylene; preferably B1, B2 is any one of the following:

[0163] Case (1): One of B1 and B2 is a connecting bond, and the other is C 1-20 alkylene;

[0164] Case (2): B1 and B2 are both connecting bonds;

[0165] Case (3): B1 and B2 are each independently C 1-20 alkylene;

[0166] The C 1-20 The alkylene group is substituted or unsubstituted and is selected from methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene, eicosylene, 1-CR q A divalent linking group composed of H- and 0-19 -CH2- or 2-CR q A divalent linking group composed of H- and 0-18 -CH2-; the R q Each occurrence is independently selected from -OH, -(CH2) tq OH, -(CH2) tq CH3, -(CH2) tq OCH3, -SH, -(CH2) tq SH, -(CH2) tq SCH3, -NH2, -N((CH2) tq CH3)2, -(CH2) tq NH2 and -(CH2) tq Any one of N(CH3)2, wherein tq is selected from an integer of 0-4; R q Preferred is -OH.

[0167] In a specific embodiment of the present invention, the aforementioned B1 and B2 are each independently selected from any one of the following: a linker, an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, a heptylene group, an octylene group, a nonylene group, a decylene group, a -OH substituted butylene group, a -OH substituted pentylene group, a -OH substituted hexylene group, a -OH substituted heptylene group, and a -OH substituted octylene group;

[0168] Preferably, B1 and B2 are both ethylene, or B1 and B2 are both butylene, or B1 and B2 are both hexylene, or B1 and B2 are both heptylene, or B1 is butylene and B2 is heptylene, or B1 is pentylene and B2 is hexylene, or B1 is pentylene and B2 is heptylene, or B1 is a connecting bond and B2 is hexylene, or B1 is pentylene and B2 is hexylene substituted with -OH, or B1 and B2 are both hexylene substituted with -OH, or B1 is hexylene and B2 is hexylene substituted with -OH.

[0169] In a specific embodiment of the present invention, B1 and B2 are each independently any one of butylene, pentylene, hexylene and heptylene.

[0170] In a specific embodiment of the present invention, one of B1 and B2 is a connecting bond, and the other is selected from any one of pentylene, hexylene and heptylene.

[0171] In a specific embodiment of the present invention, B1 and B2 are selected from any one of pentylene, hexylene and heptylene, and the other one is

[0172] In a specific embodiment of the present invention, B1 and B2 are both ethylene, or both are propylene, or both are butylene, or both are pentylene, or both are hexylene, or both are heptylene, or both are

[0173] In one specific embodiment of the present invention, B1 is pentylene and B2 is heptylene.

[0174] 1.5.R1, R2

[0175] In a specific embodiment of the present invention, R1 and R2 are each independently selected from R L 、R B , Rr; the R L 、R B , Rr each independently contain 0-4 R m Substituent; said R m Each occurrence is independently selected from the linear C 1-12 Hydrocarbon or branched C 1-3 an alkyl group, preferably a methyl group;

[0176] The R L For straight chain C 2-30 An aliphatic hydrocarbon group containing 0-4 carbon-carbon double bonds; preferably any one of the following structures or any cis-trans isomers thereof:

[0177]

[0178] Among them, tL An integer selected from 0-20, t L1 , t L2 are each independently selected from an integer of 2-10;

[0179] The R B The structure is Where t is 0, 1, or 2; t 01 , t 02 , t 03 , t 04 Each independently is 0 or 1; R e 、R f Each independently is C 1-12 Alkyl, C 3-12 Alkenyl and C 3-12 Any of the alkynyl groups; preferably R B Any of the following structures:

[0180]

[0181] The Rr is a C containing a ring structure 3-30 Aliphatic hydrocarbon group; the ring structure is C 3-20 Carbon ring, preferably benzene ring or C 3-20 The residue of a cycloalkane; preferably Rr is any one of the following structures:

[0182] or Among them, t a Each occurrence is independently selected from an integer from 0 to 12, t b An integer selected from 1 to 12, t L An integer selected from 0-20, R g Each occurrence is independently a substituent derived from any position on the ring, the substituent being selected from H, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Any of alkynyl groups;

[0183] Rr is further preferably any one of the following structures:

[0184]

[0185]

[0186] In a specific embodiment of the present invention, R1 and R2 each independently contain 0-8 carbon-carbon double bonds and / or 0-8 carbon-carbon triple bonds. Preferably, R1 and R2 each independently represent any one of a straight-chain alkyl group, a branched-chain alkyl group, a straight-chain alkenyl group, a branched-chain alkenyl group, a straight-chain alkynyl group and a branched-chain alkynyl group. The alkenyl group preferably contains 1-4 carbon-carbon double bonds, and the alkynyl group preferably contains 1-4 carbon-carbon triple bonds.

[0187] 1.6.R3, G1, F1, R 01

[0188] In a specific embodiment of the present invention, R3 is selected from -O(CH2) r CH3, -S(CH2) r CH3, -C(=O)(CH2) r CH3, -C(=O)O(CH2) r CH3, -OC(=O)(CH2) r CH3, -OC(=O)O(CH2) r CH3 and -(CH2) r Any one of N(CH3)2, wherein r is an integer from 0 to 3, preferably 0 or 1.

[0189] In a specific embodiment of the present invention, R3 is The structure of F1 is -(CH2) h -R 01 or -(CH2) f -Z3-(CH2) g -R 01 ; wherein h is an integer from 0 to 6, f is 0, 1 or 2, and g is 2, 3 or 4; wherein Z3 is selected from -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c -、-NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-; wherein R c Each occurrence is independently H or methyl, preferably H;

[0190] Preferably F1 is -R 01 、-CH2-R 01 、-(CH2)2-R 01 、-O-(CH2)2-R 01 、-(C=O)O-(CH2)2-R 01 、-O(C=O)-(CH2)2-R 01 、-O(C=O)O-(CH2)2-R 01Any one of them.

[0191] In a specific embodiment of the present invention, j is 1, R3 is The structure of G1 is -(CH2) f -(Z0) q -(CH2) f -G 01 ; where q is 0 or 1; f is independently 0, 1 or 2 each time it occurs; G 01 is a trivalent branched core or a tetravalent branched core selected from -OCH<, -N< or And the left end and -(CH2) f - connected; Z0 is Z1, Z2 or -(Z2) q -(CH2) g -(Z1) q -, wherein Z1 and Z2 are each independently -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c -、-NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-; R c Each occurrence is independently H or methyl, preferably H; g is 2, 3 or 4;

[0192] Preferably G1 is -(CH2) f O(CH2) f CH<, more preferably -OCH2CH<.

[0193] In a specific embodiment of the present invention, R 01 Any one selected from alkoxy, alcoholic hydroxyl, protected alcoholic hydroxyl, sulfhydryl, protected sulfhydryl, carboxyl, protected carboxyl, amino, protected amino, aldehyde, protected aldehyde, ester, carbonate, carbamate, succinimide, maleimide, protected maleimide, dimethylamino, alkenyl, alkenoate, azido, alkynyl, folic acid, rhodamine and biotinyl.

[0194] In a specific embodiment of the present invention, R 01Selected from: any one of a reactive group, a modified form of a reactive group, a functional group with therapeutic targeting, and a fluorescent functional group; wherein the modified form is selected from any one of a precursor of a reactive group, an active form with a reactive group as a precursor, an active form in which a reactive group is substituted, and an inactive form in which a reactive group is protected; wherein the precursor of the reactive group refers to a structure that can be converted into the reactive group through at least one process of oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, and deprotonation.

[0195] In a specific embodiment of the present invention, the aforementioned R 01 Any functional group selected from the following Class A to Class I or a variation thereof:

[0196] Category A: active ester group, active ester group-like structure; wherein the active ester group is selected from any one of succinimide active ester group, p-nitrobenzene active ester group, o-nitrobenzene active ester group, 1,3,5-trifluorobenzene active ester group, 1,3,5-trichlorobenzene active ester group, 1,3,5-tribromobenzene active ester group, 1,3,5-triiodobenzene active ester group, pentafluorobenzene active ester group, imidazole active ester group, benzotriazole active ester group, thiazolidine-2-thione active ester group, tetrahydropyrrole-2-thione active ester group, 2-mercaptobenzothiazole active ester group, and 1-oxo-3-thioxoisoindoline active ester group; wherein the active ester group-like structure is an active carboxylate ester group or an active acyl group;

[0197] Class B: carboxyl, protected carboxyl, sulfonic acid, sulfonate, sulfinic acid, sulfinic acid, sulfenic acid, ester, thioester, dithioester, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate, xanthate, tetrathiodiester, sulfone, sulfoxide, methacryloyl, hydroxamic acid, thiohydroxamic acid, sulfonyl halide, thiocarboxyl;

[0198] Class C: aldehyde group, hydrated aldehyde group, thialdehyde group, acyl halide group, ketone group, hydrated ketone group, thioketone group, thioketone hydrate group, glyoxal group, acetal group, monothioacetal group, bisthioacetal group, ketal group, monothioketal group, bisthioketal group, hemiacetal group, thiohemiacetal group, hemiketal group, ortho acid group, protected ortho acid group, ortho ester group, cyanate group, thiocyanate group, isocyanate group, isothiocyanate group, oxazoline group, isoxazoline group;

[0199] Class D: primary amino group, secondary amino group, protected amino group, hydroxylamine group, sulfhydryl group, disulfide compound group, halogen atom, haloacetamide group, ammonium salt, hydrazine group, tetramethylpiperidinyloxy group, dioxapiperidinyloxy group, O-carbonylhydroxylamine group, amide group, imide group, hydrazide group, sulfonylhydrazide group, hydrazone group, imine group, enamine group, alkynamine group, carbamate group, monothiocarbamate group, dithiocarbamate group;

[0200] Class E: urea group, thiourea group, guanidine group and its protonated form, amidine group and its protonated form, anhydride group, squaryl group, squarate group, semisquaryl group, semisquarate group, imidazole-1-carboxamide group, imidate group, nitrone group, aldoxime group, ketoxime group;

[0201] Class F: maleimide, furan-protected maleimide, acrylate, N-acrylamide, N-methylacrylamide, methacrylate, maleamic acid, 1,2,4-triazolidine-3,5-dione, linear azo compound, cyclic azo compound;

[0202] Class G: alkenyl, alkenylalkyl, cycloalkene, alkynyl, alkynylalkyl, protected alkynyl, cycloalkynyl, linear conjugated diene, cyclic conjugated diene, cyclic conjugated diene containing heteroatoms, epoxy, 1,2,4,5-tetrazinyl, azido, nitrile oxide, cyano, isocyano, diazo, diazonium ion, azoxy, nitrile imine, N-oxide aldimine, tetrazolyl, 4-acetyl-2-methoxy-5-nitrophenoxy and its diazotized form, imidazolyl, indolyl; wherein the cycloalkene is selected from any one of cyclooctenyl, norbornenyl, norbornadienyl, oxa-norbornenyl, and oxa-norbornadienyl;

[0203] Class H: hydroxyl, protected hydroxyl, protected dihydroxyl, silyl, trihydroxysilyl, protected trihydroxysilyl; wherein the hydroxyl group is selected from any one of alcoholic hydroxyl, phenolic hydroxyl, enol hydroxyl, and hemiacetal hydroxyl;

[0204] Class I: Monosaccharide selected from allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, fucitol, fucosamine, fucose, fucose, galactosamine, galactosamine alcohol, N-acetyl-galactosamine, galactose, glucosamine, N-acetyl-glucosamine, glucosamine alcohol, glucose, glucose-6-phosphate, gulose glyceraldehyde, L-glycero-D-mannoheptose, glycerol, glyceraldehyde, dihydroxyacetone, gulose, arachid ... The residue of any one of dulose, lyxose, mannosamine, mannose, mannose-6-phosphate, mannoheptulose, psicose, quinose, quinosamine, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, deoxyribose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, threose, xylose, and xylulose, or a functional derivative thereof; the monosaccharide group is in D- or L-configuration, is cyclic or chain-like, and is substituted or unsubstituted;

[0205] Among them, the protected hydroxyl group is preferably any one of ether, silyl ether, ester, carbonate, and sulfonate; the protected amino group is preferably any one of carbamate, amide, imide, N-alkylamine, N-arylamine, imine, enamine, imidazole, pyrrole, and indole; the protected thiol group is preferably any one of sulfide, disulfide, silyl sulfide, and thioester; the protected carboxyl group is preferably a form in which the carboxyl group is protected by any one of methyl, ethyl, tert-butyl, and benzyl; the protected alkynyl group is preferably a form in which the alkynyl group is protected by a silyl group; the protected dihydroxyl group is preferably an acetal structure in which its protecting group and two oxygen atoms form a five-membered ring or a six-membered ring; the protecting group of the dihydroxyl group is preferably a methylene or substituted methylene, more preferably any one of methylene, 1-methylmethylene, 1,1-dimethylmethylene, 1,1-cyclopentylene, 1,1-cyclohexylene, 1-phenylmethylene, and 3,4-dimethylphenylmethylene.

[0206] In a specific embodiment of the present invention, R 01 Any functional group selected from the following Class A to Class I or a variation thereof:

[0207] Class A:

[0208]

[0209] Class B:

[0210]

[0211] Class C:

[0212]

[0213] Class D:

[0214]

[0215] Class E:

[0216]

[0217] Class F:

[0218]

[0219]

[0220] Class G:

[0221]

[0222] Class H:

[0223]

[0224] Class I:

[0225]

[0226] Wherein, X is a halogen atom selected from any one of a fluorine atom, a chlorine atom, a bromine atom and an iodine atom;

[0227] Wherein, Y1 is selected from C 1-5 Any of alkyl, vinyl, phenyl, benzyl, p-methylphenyl, 4-(trifluoromethoxy)phenyl, trifluoromethyl and 2,2,2-trifluoroethyl;

[0228] Among them, R d2 is an organic group, preferably each occurrence of which is independently selected from C 1-5 Alkyl, C 2-5 Alkenyl, C 2-5 Any of alkynyl and phenyl, wherein the alkyl, alkenyl, alkynyl and phenyl are each independently substituted or unsubstituted;

[0229] Wherein, W is a leaving group selected from any one of -F, -Cl, -Br, -I and -SPh;

[0230] Wherein, M5 is a ring-forming atom selected from any one of carbon atoms, nitrogen atoms, phosphorus atoms and silicon atoms; the ring structure in which M5 is located is a 3-30-membered ring, preferably a 3-20-membered ring, more preferably a 3-16-membered ring, and more preferably a 5-16-membered ring; the ring structure is preferably any one of the following groups, any substituted form, or any hybrid form: cyclohexane, furanose ring, pyranose ring, benzene, tetrahydrofuran, pyrrolidine, thiazolidine, cyclohexene, tetrahydropyran, piperidine, 1,4-diol ... -dioxane, pyridine, pyridazine, pyrimidine, pyrazine, 1,3,5-triazine, 1,4,7-triazacyclononane, cyclotripeptide, indene, indane, indole, isoindole, purine, naphthalene, dihydroanthracene, xanthene, thioxanthene, dihydrophenanthrene, 10,11-dihydro-5H-dibenzo[a,d]cycloheptane, dibenzocycloheptene, 5-dibenzocycloheptenone, quinoline, isoquinoline, fluorene, carbazole, iminodibenzyl, naphthyl ring, dibenzocyclooctyne, azadibenzocyclooctyne;

[0231] in, The ring structure is a ring structure containing an acetal group, a disulfide bond, an amine group, an imide group, an acid anhydride group, an azo group, a carbon-carbon double bond, a carbon-carbon triple bond, or a conjugated diene on the ring skeleton, wherein the ring structure is selected from a carbocycle, a heterocycle, a benzoheterocycle, a substituted carbocycle, a substituted heterocycle, or a substituted benzoheterocycle;

[0232] Wherein, Q is an atom or substituent that contributes to the induction and conjugation effect of unsaturated bond electrons; when Q is on a ring, the number is one or more; when the number is multiple, they are the same structure or a combination of two or more different structures; when it is a substituent, Q has a straight chain structure, a branched structure containing a side group, or a cyclic structure;

[0233] The modified form is selected from any one of a precursor of a reactive group, an active form with a reactive group as a precursor, an active form in which a reactive group is substituted, and an inactive form in which a reactive group is protected; wherein the precursor of the reactive group refers to a structure that can be converted into the reactive group through at least one process of oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, and deprotonation.

[0234] In a specific embodiment of the present invention, R 01 It is selected from any one of hydroxyl, carboxyl, aldehyde, amino, thiol or their protected forms, or any one of halogen atoms, ester, sulfonate, active ester groups; preferably any one of -OH, -COOH, -C(=O)OCH3.

[0235] 1.7.R4

[0236] In a specific embodiment of the present invention, R4 is R'N(R')-R"-N(R')-, selected from Any one, preferably

[0237] In a specific embodiment of the present invention, R4 is a carbocyclic group or a heterocyclic group, and contains 0-5 substituents, each of which is independently -F, -Cl, -Br, -I, -R d '、-OR d ',-NR d 'R d '、-SR d ', -(C=O)R d ', -(C=O)OR d ', -O(C=O)R d 'or-O(C=O)OR d ', where R d ' for C 1-3 alkyl;

[0238] Preferably, R4 is any one of the following structures:

[0239]

[0240] More preferably, it is any one of the following structures:

[0241]

[0242] In a specific embodiment of the present invention, R4 is selected from phenyl, benzyl, naphthyl, phenanthrenyl, anthracenyl, biphenyl, oxacycloalkyl, epoxyalkyl, furanyl, dihydrofuranyl, tetrahydrofuranyl, pyranyl, dihydropyranyl, tetrahydropyranyl, thienyl, dihydrothienyl, tetrahydrothienyl, thiopyranyl, dihydrothiopyranyl, tetrahydrothiopyranyl, aziridinyl, 2-aziridinyl, azetidinyl, pyrrolidinyl, pyrrolidonyl, piperidine any one or any benzo derivative of 1, 2 or 3 alkyl, 2, 3 or 4 alkyl; R4 is unsubstituted or contains R n Substituent, R n For one or more; R n When the number is multiple, it is the same structure, or a combination of two or more different structures; each R n Independently selected from C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10Alkynyl, phenyl, =O, acetal, -NH2, -OR j 、-NR j R j 、-SR j 、-(C=O)R j 、-(C=O)OR j 、-O(C=O)R j 、-O(C=O)OR j , piperidinyl, piperazinyl and morpholinyl, wherein R j C 1-10 Alkyl, the alkyl, alkenyl, alkynyl is a linear structure or a cyclic structure; preferably, each R n Independently C 3-8 Cycloalkyl, C 3-8 Cycloalkenyl, cyclooctynyl.

[0243] 1.8. Specific structural examples

[0244] In a specific embodiment of the present invention, the structure of the cationic lipid is selected from any one of the following:

[0245]

[0246]

[0247]

[0248] 2. PEGylated lipids

[0249] One embodiment of the present invention:

[0250] A PEGylated lipid derived from the aforementioned cationic lipid, the structure of which is shown in general formula (2):

[0251]

[0252] Wherein, N is a nitrogen branching center;

[0253] L1 and L2 are each independently a connecting bond or a divalent connecting group;

[0254] L3 is a trivalent linking group, and A (A1) n1 OR3 constitutes a side chain of -L A (A1) n1 The divalent linking group of OR3 -L3[L A (A1) n1 OR3]-;

[0255] B1 and B2 are each independently a connecting bond or C 1-30 alkylene;

[0256] R1 and R2 are each independently C 2-30 Aliphatic hydrocarbon or C containing 1-2 O 2-30 Aliphatic hydrocarbon derivative residues;

[0257] R3 is H, -R d 、-(CH2) h NR d R d 、-(CH2) h SR d 、-(C=O)R d 、-(C=O)OR d or Among them, R d Each occurrence is independently C 1-12 Alkyl; h is an integer from 0 to 6; G1 is a branched group with a valence of k+1, j is 0 or 1, and F1 contains a functional group R 01 ; When j is 0, G1 does not exist; when j is 1, G1 leads to k F1s, and the k F1s are independently the same or different structures, and k is an integer from 2 to 8;

[0258] R5 is C 3-14 Alkyl, carbocyclic, heterocyclic or R'N(R')-R"-N(R')-; wherein the heterocyclic group is a cyclic group containing 1, 2 or more heteroatoms, wherein the heteroatoms are B, O, N, Si, P or S; wherein each occurrence of R' is independently H or C 1-3 Alkyl, R" is C 2-4 alkylene;

[0259] L A -(CH2) h -or-(CH2) f -Z3-(CH2) g -; wherein h is an integer from 0 to 6, f is 0, 1 or 2, and g is 2, 3 or 4; wherein Z3 is selected from -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c -、-NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-; wherein Rc Each occurrence is independently H or methyl, preferably H;

[0260] A1 is -OCH2CH2- and its left end is connected to L A connected;

[0261] n1 is the number of times A1 appears repeatedly, selected from integers between 20 and 250;

[0262] The alkyl group, alkylene group, aliphatic hydrocarbon group, aliphatic hydrocarbon derivative residue, carbocyclic group, and heterocyclic group are each independently substituted or unsubstituted.

[0263] In a specific embodiment of the present invention, the structure of the aforementioned PEGylated lipid is any one of the following:

[0264]

[0265]

[0266] 3. Preparation of lipids

[0267] In the present invention, the raw materials used in each preparation method can be purchased or synthesized by oneself.

[0268] In the present invention, two identical or different reactive groups can react to form a divalent linking group. The reaction conditions are related to the type of divalent linking group generated by the reaction, and existing public technologies can be used. For example: amino groups react with active esters, formic acid active esters, sulfonic acid esters, aldehydes, α, β-unsaturated bonds, carboxylic acid groups, epoxides, isocyanates, and isothiocyanates to obtain divalent linking groups such as amide groups, urethane groups, amino groups, imine groups (which can be further reduced to secondary amino groups), amino groups, amide groups, amino alcohols, urea bonds, and thiourea bonds; sulfhydryl groups react with active esters, formic acid active esters, sulfonic acid esters, sulfhydryl groups, maleimides, aldehydes, α, β-unsaturated bonds, carboxylic acid groups, iodoacetamide, and acid anhydrides to obtain divalent linking groups such as thioester groups, thiocarbonates, thioethers, disulfides, thioethers, thiohemiacetals, thioethers, thioesters, thioethers, and imides; unsaturated bonds react with sulfhydryl groups to obtain thioether groups; Carboxyl groups or acyl halides react with sulfhydryl groups and amino groups to produce thioester groups, amide groups and other groups; hydroxyl groups react with carboxyl groups, isocyanates, epoxides, and chloroformyloxy groups to produce divalent linking groups such as ester groups, carbamate groups, ether bonds, and carbonate groups; carbonyl groups or aldehyde groups react with amino groups, hydrazines, and hydrazides to produce divalent linking groups such as imine bonds, hydrazones, and acylhydrazones; click chemistry reactions of reactive groups such as azide, alkynyl, alkenyl, sulfhydryl, azide, diene, maleimide, 1,2,4-triazolin-3,5-dione, dithioesters, hydroxylamine, hydrazides, acrylates, allyloxy groups, isocyanates, and tetrazole can generate various divalent linking groups containing structures including but not limited to triazole, isoxazole, and thioether bonds.

[0269] In the present invention, the type of coupling reaction is not particularly limited, as long as two identical or different reactive groups can form a covalent linking group through the reaction; the same preparation process may contain a single-step or step-by-step coupling reaction, and preferably each coupling reaction step is independently any one of an alkylation reaction, a condensation reaction, an amidation reaction, an esterification reaction, a thioesterification reaction, a ring-opening reaction, a ring-closing condensation reaction, an addition reaction, a cycloaddition reaction, an α,β-unsaturated bond addition reaction, an alkynyl addition reaction, a Schiff base reaction combined with a reduction reaction, a click reaction, an azide-alkyne addition reaction, a 1,3-dipolar cycloaddition reaction, a Diels-Alder addition reaction, a thiol-yne reaction, a thiol-ene reaction, a thiol-vinyl reaction, and a condensation reaction; the reaction conditions of the coupling reaction are related to the type of covalent linking group generated by the reaction, and existing public technologies can be used; the valence state of the covalent linking group generated by the coupling reaction can be divalent or trivalent, preferably divalent; the coupling reaction can generate a stable group or a degradable group.

[0270] In the present invention, the PEGylated lipid is obtained through a preparation process involving a coupling reaction and / or a polymerization reaction. The polymerization reaction involves at least two steps: deprotonation of a small molecule initiator and polymerization of ethylene oxide. The small molecule initiator can be a directly obtained raw material or an intermediate in the preparation process. During the preparation process, when the coupling reaction and the polymerization reaction occur simultaneously, the order of the coupling reaction and the polymerization reaction is not particularly limited.

[0271] In the present invention, in the preparation method of monodisperse PEGylated lipids, the monodisperse raw material containing the polyethylene glycol component can be replaced with a polydisperse raw material of the same component to obtain a corresponding polydisperse product; similarly, in the preparation method of polydisperse PEGylated lipids, the polydisperse raw material containing the polyethylene glycol component can be replaced with a monodisperse raw material of the same component to obtain a corresponding monodisperse product.

[0272] In the present invention, when only the number average degree of polymerization and / or PDI of the polyethylene glycol or its derivative reagent raw material is changed without changing its structural formula, the reaction conditions can be smoothly carried out without any obvious changes, and the changes do not include changes in the amount of solvent used.

[0273] 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, or supercritical extraction. The structure and molecular weight of the final product can be characterized by methods including, but not limited to, nuclear magnetic resonance, electrophoresis, UV-visible spectrophotometry, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism, and the like.

[0274] In the present invention, the step-by-step / multi-step reaction can be completed in multiple actual operations of technicians, or can be completed in one actual operation.

[0275] Preparation of cationic lipids

[0276] In the present invention, any of the aforementioned cationic lipids can be prepared using the following general reaction scheme, which does not represent an actual complete preparation route. The actual preparation process may also include any necessary minor modifications, protection / deprotection, intermediate preparation, post-processing or purification processes familiar to those skilled in the art.

[0277] General Reaction Scheme I:

[0278]

[0279] Wherein, the structures of R3' and R3 may be the same or different; when the specific structures of R3' and R3 are different, R3' may be converted into R3 after slight modification before or after any reaction step, wherein the slight modification is selected from any one, any combination of two, or any combination of two or more of the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group; preferably, R3 is the deprotected form of R3', for example, -OTBS is converted into -OH after deprotection reaction;

[0280] Among them, F N is a reactive group capable of reacting with an amino group or a secondary amino group, preferably -OMs, -OTs, -CHO, -F, -Cl, -Br, -COOH, -COCl or an activated carboxyl group, wherein the activated carboxyl group refers to a carboxyl group activated by a carboxyl activating agent;

[0281] Wherein, the definitions of L1, L2, L3, B1, B2, R3, R4, R1 and R2 are consistent with those described in general formula (1) and are not repeated here;

[0282] Among them, A-2 and A-3 can be prepared by the following method:

[0283]

[0284] Where x is 1 or 2; when x is 1, B-3 is equivalent to A-2; when x is 2, B-3 is equivalent to A-3;

[0285] Among them, F G1 、F G2 is a reactive group, F G1 and F G2 The reaction generates a divalent linking group L1 or L2; preferably F G1 、F G2Each independently represents any one of -OH, -COOH, -F, -Cl, Br, -OMs, -OTs, -CHO, -COCl, -NH2, and active ester groups; for example, B-1 is B-2 B-1 and B-2 undergo esterification to give B-3 Among them F N It is -Br.

[0286] General Reaction Scheme II:

[0287]

[0288] Among them, the transformation relationship between R3' and R3 and F N The definitions refer to General Reaction Scheme I; B1 obtained from B1' contains a hydroxyl substituent, and B2 obtained from B2' contains a hydroxyl substituent;

[0289] For example, A-1 is C-2 is The structure of IM-1 obtained by the reaction is: Wherein B1' is butylene, B1 is

[0290] Wherein, the definitions of L1, L2, L3, B1, B2, R3, R4, R1 and R2 are consistent with those described in general formula (1).

[0291] General Reaction Scheme III:

[0292]

[0293] Wherein, B1 and B2 are both ethylene;

[0294] Among them, the transformation relationship between R3' and R3 and F N The definition of refers to General Reaction Scheme I;

[0295] Wherein, L1 and L2 are both -(C=O)O(CH2) y OC(=O)-, y is an integer from 2 to 8;

[0296] For example, the structure of D-1 is

[0297] Wherein, the definitions of L1, L2, L3, B1, B2, R3, R4, R1 and R2 are consistent with those described in general formula (1).

[0298] 3.2. Description of relevant raw materials and / or steps in the preparation process

[0299] 3.2.1. Carboxyl activators, condensing agents, oxidizing agents, and reducing agents

[0300] In the present invention, "carboxyl activation" refers to the activation treatment of the carboxyl group with a carboxyl activator. After the carboxyl activation, it can promote the condensation reaction to proceed better, such as: inhibiting the generation of racemic impurities in the condensation reaction, catalytically accelerating the reaction rate, etc. "Carboxyl activating group" is the residue of the carboxyl activator. The carboxyl activator is a combination of one or more of N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb) and N,N-dicyclohexylcarbodiimide (DCC), preferably a combination of NHS / EDCI, NHS / DCC or HONb / DCC.

[0301] In the present invention, the condensing agent used in the reaction is not limited, but preferably includes N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU). DCC is most preferred. The amount of the condensing agent used is generally 1 to 20 times the molar equivalent of the carboxylic acid, preferably 5 to 10 times. A suitable catalyst (such as 4-dimethylaminopyridine (DMAP)) may be added to this reaction.

[0302] In the present invention, the oxidant used in the reaction is not particularly limited, as long as it is a compound or a combination of multiple compounds that can increase the valence of the substrate, preferably phenyliodonium di(trifluoroacetate), 1,4-benzoquinone, benzyltrimethylammonium tribromide, pyridinium dichromate, potassium dichromate, ozone, oxygen, hypofluoric acid, sodium hypochlorite, cobalt acetate, cobalt acetate, manganese acetate, palladium acetate, copper acetate, monoperoxyphthalic acid, iodine, N-iodosuccinimide, iodobenzoylbenzene, 2-iodobenzoic acid, dimethyldioxycyclopropane, dimethyl sulfoxide-oxalyl chloride, dimethyl sulfoxide-acetic anhydride, DDQ, dichlorotris(triphenylphosphine)ruthenium, manganese dioxide, diacetoxyiodobenzene, periodic acid, sodium periodate, sodium periodate-osmium tetroxide, potassium permanganate, sodium perborate, peroxybenzoic acid, dibenzoyl peroxide, Nickel peroxide, hydrogen peroxide, cumene hydroperoxide, tert-butyl peroxide, peracetic acid, m-chloroperbenzoic acid, N-chlorosuccinimide, pyridinium chlorochromate, palladium chloride-copper chloride, urea hydrogen peroxide complex, triphenylmethyl tetrafluoroborate, tributyltin oxide, cobalt trifluoride, vanadium trifluoride, chromium trioxide, manganese triacetate, TEMPO, ceric ammonium nitrate, bromine, N-pyridine oxide, silver oxide, O-ethyl peroxycarbonic acid, manganese acetylacetonate, vanadium acetylacetonate, aluminum isopropoxide, potassium persulfate, dichloroiodobenzene, etc., or a combination thereof, more preferably oxygen, sodium hypochlorite, hydrogen peroxide, dichloroiodobenzene, potassium persulfate, etc., or a combination thereof, the amount of the oxidant being 1 to 50 times, preferably 1 to 20 times, and more preferably 5 to 10 times, the molar equivalent of the hydroxyl group in the intermediate compound.

[0303] In the present invention, the reducing agent used in the reaction is not particularly limited, as long as it can reduce the Schiff base generated by ammonia and aldehyde or ketone to an amino group; preferably, one or a combination of sodium borohydride, sodium cyanoborohydride, lithium aluminum hydride, borane, diborane, diisobutylaluminum hydride, diisopinocamphor borane, lithium borohydride, zinc borohydride, borane-pyridine, borane-methyl sulfide, borane-tetrahydrofuran, etc.; more preferably, sodium cyanoborohydride. The equivalent of the reducing agent is 1 to 50 times the molar equivalent of the amino group to be modified, preferably 1 to 20 times, and more preferably 5 to 10 times.

[0304] In the present invention, the reaction temperature is 0 to 200°C, preferably 0 to 100°C, more preferably 0 to 25°C, and the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours. The obtained product can be purified by purification methods such as extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, or supercritical extraction.

[0305] In the present invention, the reaction solvent can be a solvent-free solvent or an aprotic solvent. The aprotic solvent includes toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, methyl tert-butyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, preferably tetrahydrofuran, dichloromethane, dimethyl sulfoxide, and dimethylformamide.

[0306] In the present invention, the base used in the reaction can be an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine), preferably triethylamine or pyridine. The amount of the base is 1 to 50 times the molar equivalent of the carboxylic acid, preferably 1 to 10 times, more preferably 2 to 3 times. It can also be an inorganic base, such as potassium carbonate (K2CO3).

[0307] 3.2.2. Protection and deprotection of related groups during the reaction

[0308] The present invention also involves the "protection" and "deprotection" processes of related groups during the reaction. To prevent the functional groups from affecting the reaction, they are typically protected. Furthermore, when there are two or more functional groups, the remaining functional groups are protected in order to selectively allow only the target functional group to react. Protecting groups not only stably protect the target functional group but also need to be easily removable as needed. Therefore, in organic synthesis, it is important to deprotect only the protecting groups bonded to the intended functional group under appropriate conditions.

[0309] In the present invention, "carboxyl protecting group" refers to a protecting group that can be converted into a carboxyl group by the deprotection reaction of hydrolysis or carboxyl protecting group. Carboxyl protecting group is preferably an alkyl group (such as a methyl group, an ethyl group, a tert-butyl group) or an aralkyl group (such as a benzyl group), more preferably a tert-butyl group (tBu), a methyl group (Me) or an ethyl group (Et). In the present invention, "protected carboxyl" refers to a group formed after a carboxyl group is protected by a suitable carboxyl protecting group, preferably a methoxycarbonyl group, an ethoxycarbonyl group, a tert-butyloxycarbonyl group or a benzyloxycarbonyl group. The carboxyl protecting group can be removed by hydrolysis under the catalysis of an acid or base, and can be eliminated by thermal decomposition reaction occasionally. For example, the tert-butyl group can be removed under mild acidic conditions, and the benzyl group can be removed by hydrogenolysis. The reagent for removing the carboxyl protecting group is selected from TFA, H2O, LiOH, NaOH, KOH, MeOH, EtOH and a combination thereof, preferably a combination of TFA and H2O, a combination of LiOH and MeOH or a combination of LiOH and EtOH. The protected carboxyl group is deprotected to produce the corresponding free acid. The deprotection is carried out in the presence of a base. The base and the free acid formed by the deprotection form a pharmaceutically acceptable salt.

[0310] In the present invention, the hydroxyl group protected by the hydroxyl-protecting group is not particularly limited, and may be, for example, an alcoholic hydroxyl group, a phenolic hydroxyl group, or the like. The amino / amine group protected by the amino-protecting group is not particularly limited, and may be, for example, a primary amine, a secondary amine, a hydrazine, an amide, or the like. In the present invention, the amine group is not particularly limited, and includes, but is not limited to, a primary amine group, a secondary amine group, a tertiary amine group, and a quaternary ammonium ion.

[0311] In the present invention, the deprotection of the protected hydroxyl group is related to the type of hydroxyl protecting group. The type of the hydroxyl protecting group is not particularly limited. Taking benzyl, silyl ether, acetal, and tert-butyl as examples for protecting the terminal hydroxyl group, the corresponding deprotection methods are:

[0312] A: Deprotection of benzyl group

[0313] Benzyl deprotection can be achieved by utilizing the hydrogenation reaction of a hydrogenation reducing agent and a hydrogen donor. The water content in this reaction system should be less than 1% for the reaction to proceed smoothly.

[0314] The hydrogenation reduction catalyst is not limited, and is preferably palladium and nickel, but the carrier is not limited, but preferably aluminum oxide or carbon, more preferably carbon. The amount of palladium is 1 to 100 wt% of the protected hydroxy compound, preferably 1 to 20% wt% of the protected hydroxy compound.

[0315] The reaction solvent is not particularly limited, as long as both the raw materials and the product can be dissolved. However, methanol, ethanol, ethyl acetate, tetrahydrofuran, and acetic acid are preferred, with methanol being more preferred. The hydrogen donor is not particularly limited, but hydrogen, cyclohexene, 2-propanol, ammonium formate, and the like are preferred. The reaction temperature is preferably 25 to 40°C. The reaction time is not particularly limited and is inversely correlated with the amount of catalyst used, but is preferably 1 to 5 hours.

[0316] B: Deprotection of acetals and ketals

[0317] Preferred acetal or ketal compounds for this type of hydroxyl protection include ethyl vinyl ether, tetrahydropyran, acetone, 2,2-dimethoxypropane, and benzaldehyde. Deprotection of these acetals and ketals is achieved under acidic conditions, preferably with a solution pH of 0 to 4. The acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and nitric acid are preferred, with hydrochloric acid being more preferred. The reaction solvent is not particularly limited, as long as it can dissolve the reactants and products, and water is preferred. The reaction temperature is preferably 0 to 30°C.

[0318] C: Deprotection of silyl ethers

[0319] Compounds used for this type of hydroxyl protection include trimethylsilyl ether, triethylsilyl ether, dimethyl tert-butylsilyl ether, tert-butyldiphenylsilyl ether, etc. Deprotection of this type of silyl ether is performed using a fluorine-containing compound, preferably tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid, potassium fluoride, and more preferably tetrabutylammonium fluoride and potassium fluoride. The amount of the fluorine-containing reagent used is 5 to 20 times the molar equivalent of the protected hydroxyl group, preferably 8 to 15 times the molar equivalent of the initiator. If the amount of the fluorine-containing reagent is less than 5 times the molar equivalent of the protected hydroxyl group, incomplete deprotection will result. When the amount of the deprotection reagent is greater than 20 times the molar equivalent of the protected hydroxyl group, the excess reagent or compound will cause problems in purification and may be mixed into subsequent steps, causing side reactions. The reaction solvent is not particularly limited, as long as it can dissolve the reactants and products. Aprotic solvents are preferred, and tetrahydrofuran and dichloromethane are more preferred. The reaction temperature is preferably 0 to 30°C. When the temperature is below 0°C, the reaction rate is slow and the protecting group cannot be completely removed.

[0320] D: Deprotection of tert-butyl group

[0321] Deprotection of the tert-butyl group is carried out under acidic conditions, preferably at a solution pH of 0 to 4. The acid is not particularly limited, but is preferably acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, or nitric acid, with hydrochloric acid being more preferred. The reaction solvent is not particularly limited, as long as it can dissolve the reactants and products, and is preferably water. The reaction temperature is preferably 0 to 30°C.

[0322] In the terminal functionalization method, preferably q=0, q1=1, and Z1 is 1,2-methylene. When q is not 0, A and R 01 When there are linkers such as amino acids and succinyl groups, existing technologies in the art for generating Z2 or Z1 (including but not limited to alkylation, condensation, click reaction, etc.) can be used, and the preparation can be carried out with reference to the following linear functionalization steps.

[0323] 3.2.3. Alkylation reaction

[0324] The alkylation reaction of the present invention is preferably based on the alkylation of hydroxyl, sulfhydryl or amino groups, corresponding to the formation of ether bonds, thioether bonds, secondary amino groups or tertiary amino groups, respectively. Examples are as follows:

[0325] (1) Alkylation of substrate alcohol with sulfonate and halide

[0326] In the presence of a base, an amine intermediate is obtained by nucleophilic substitution of substrate alcohol with sulfonic ester derivatives and halide. Wherein, the molar equivalent of sulfonic ester and halide is 1 to 50 times, preferably 1 to 5 times, of substrate alcohol. When the molar equivalent of the molar equivalent of sulfonic ester and halide is less than 1 times of molar equivalent of substrate alcohol, the reaction substitution is incomplete and is difficult to purify. And when the molar equivalent of sulfonic ester and halide is greater than 50 times of substrate alcohol, excessive reagent brings trouble to purification and may be mixed into subsequent steps, thereby causing next step side reaction to increase, increasing purification difficulty.

[0327] The resulting product is a mixture of an ether intermediate and excess sulfonate and halide, which can be purified by methods such as anion exchange resin, osmosis, and ultrafiltration. The anion exchange resin is not particularly limited, as long as the target product can undergo ion exchange and adsorption on the resin. Ion exchange resins containing tertiary amines or quaternary ammonium salts, such as dextran, agarose, polypropionate, polystyrene, and polystyrene, as backbones are preferred. The solvent for osmosis and ultrafiltration is not limited, and can generally be water or an organic solvent. The organic solvent is not particularly limited, as long as the product can be dissolved therein. Dichloromethane, chloroform, and the like are preferred.

[0328] The reaction solvent is not limited, but is preferably an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, more preferably dimethylformamide, dichloromethane, dimethyl sulfoxide or tetrahydrofuran.

[0329] The base includes an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine) or an inorganic base (such as sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium carbonate or potassium hydroxide), preferably an organic base, more preferably triethylamine or pyridine. The molar amount of the base is 1 to 50 times the molar equivalent of the sulfonic acid ester or halide, preferably 1 to 10 times, more preferably 3 to 5 times.

[0330] (2) Alkylation reaction between substrate amine and aldehyde derivatives

[0331] After the substrate amine reacts with the aldehyde derivative to obtain the imine intermediate, the intermediate is obtained under the action of a reducing agent. The molar equivalent of the aldehyde derivative is 1 to 20 times that of the substrate amine, preferably 1 to 2 times, and more preferably 1 to 1.5 times. When the molar equivalent of the aldehyde derivative is greater than 20 times that of the substrate amine, the excess reagent brings trouble to the purification and may be mixed into the subsequent steps, increasing the difficulty of purification. When the molar equivalent of the aldehyde derivative is less than 1 times that of the substrate amine, the reaction is incomplete, increasing the difficulty of purification. The product after the reaction can be purified by means of cation exchange resin, osmosis, ultrafiltration, etc. to obtain the intermediate. There are no special restrictions on the cation exchange resin, as long as it can exchange with the quaternary ammonium cation to achieve a separation effect. There are no restrictions on the solvent for osmosis and ultrafiltration, and it can generally be water or an organic solvent. The organic solvent is not particularly limited as long as the product can be dissolved in it, preferably dichloromethane, chloroform, etc.

[0332] The reaction solvent is not limited, and is preferably an organic solvent such as methanol, ethanol, water, toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide; more preferably water and methanol.

[0333] The reducing agent is not particularly limited, as long as it can reduce the imine to an amine. Preferred are sodium borohydride, lithium aluminum hydride, sodium cyanoborohydride, Zn / AcOH, and the like, with sodium cyanoborohydride being more preferred. The amount of reducing agent used is generally 0.5 to 50 times, more preferably 1 to 10 times, the amount of the aldehyde derivative.

[0334] 3.2.4. Linear functionalization of the R3 side chain terminal

[0335] The method of linear functionalization of the R3 side chain terminal is not particularly limited and is related to the type of the final functional group or its protected form. R3 that has undergone linear functionalization of the terminal still falls within the scope of R3 of the present invention.

[0336] (1) Functionalization of terminal hydroxyl groups

[0337] Starting from the terminal hydroxyl group of F1, functional groups of type A to type I or their protected forms are obtained by functionalization, for example, from -(CH2) h -R 01 To -(CH2) f -Z3-(CH2) g -R 01 wherein h is an integer from 0 to 6, f is 0, 1 or 2, and g is 2, 3 or 4; wherein Z3 is selected from -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c -、-NR cC(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-; wherein R c Each occurrence is independently H or methyl, preferably H; the specific preparation method includes but is not limited to that described in paragraphs

[0960] to

[1205] of document CN104530417A.

[0338] (2) Based on the transformation of reactive groups to target functional groups or their protected forms

[0339] Method 1: Direct modification: Direct modification of reactive groups to obtain the target functional group or its protected form. Examples include the conversion of carboxyl groups to acyl halides, hydrazides, esters, thioesters, and dithioesters, as well as the conversion of hydroxyl groups, sulfhydryl groups, alkynyl groups, amino groups, and carboxyl groups to their corresponding protected structures. Another example is the modification of hydroxyl groups and amino groups with acid anhydrides.

[0340] Method 2: A coupling reaction between two reactive groups. Using a heterofunctionalizing reagent containing one reactive group and the target functional group or its protected form as a starting material, one of the reactive groups reacts with the reactive group at the end of F1 to introduce the target functional group or its protected form. The reaction between the two reactive groups is not particularly limited in terms of method and reaction conditions. The reaction conditions are related to the type of divalent linker formed by the reaction, and existing publicly available techniques can be employed. Examples include alkylation, alkenyl addition, alkynyl addition, Schiff base reaction combined with reduction, and condensation reactions. Alkylation reactions are preferably based on the alkylation of sulfhydryl or amino groups, corresponding to the formation of thioether bonds, secondary amino groups, or tertiary amino groups, respectively. Condensation reactions include, but are not limited to, those that form ester groups, thioester groups, amide groups, imine bonds, hydrazone bonds, and carbamate groups. Another example is the click reaction, where heterofunctionalized reagents containing groups such as azide, alkynyl, alkenyl, trithioester, sulfhydryl, dienyl, furyl, 1,2,4,5-tetrazine, and cyanate are used as raw materials to introduce the target functional group or its protected form. The reaction between the two reactive groups is accompanied by the formation of a new bond. Typical examples of the newly formed divalent linking groups include amide bonds, urethane bonds, ester groups, secondary amine bonds, thioether bonds, and triazole groups.

[0341] Method 3: Obtain the target functional group or its protected form by combining direct modification and coupling reaction. 4. Lipid composition, lipid pharmaceutical composition and its preparation

[0342] 4.1. Lipid composition

[0343] In one embodiment of the present invention, the lipid composition forms liposomes, preferably lipid nanoparticles (LNPs).

[0344] The lipid compositions of the present invention can be used to deliver biologically active ingredients to one or more of the following in a patient: liver or liver cells (e.g., hepatocytes), kidney or kidney cells, tumor or tumor cells, CNS or CNS cells (central nervous system, such as brain and / or spinal cord), PNS or PNS cells (peripheral nervous system), lung or lung cells, blood vessels or blood vessel cells, skin or skin cells (e.g., dermal cells and / or follicular cells), eye or eye cells (e.g., macula, fovea, cornea, retina), ear or ear cells (e.g., inner ear, middle ear, and / or outer ear cells).

[0345] One embodiment of the present invention:

[0346] A lipid composition comprising any of the aforementioned cationic lipids.

[0347] In a specific embodiment of the present invention, the lipid composition further contains one or more of phospholipids, steroid lipids and PEGylated lipids, selected from any one of the following situations:

[0348] Case (1): also contains phospholipids;

[0349] Case (2): also contains steroid lipids;

[0350] Case (3): also contains PEGylated lipids;

[0351] Case (4): also contains phospholipids and steroid lipids;

[0352] Case (5): also contains phospholipids and PEGylated lipids;

[0353] Case (6): also contains steroid lipids and PEGylated lipids;

[0354] Case (7): also contains phospholipids, steroid lipids and PEGylated lipids;

[0355] It is preferred that the composition also contains three lipids: phospholipids, steroid lipids and PEGylated lipids.

[0356] In a specific embodiment of the present invention, the phospholipids in the aforementioned lipid composition are selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, Phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 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, 1, 2-Diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dioleoyl Any one of acylphosphatidylserine, 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 and lysophosphatidylethanolamine, and combinations thereof.

[0357] In a specific embodiment of the present invention, the steroid lipid in the aforementioned lipid composition is selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and mixtures thereof.

[0358] In a specific embodiment of the present invention, the PEGylated lipid in the aforementioned lipid composition is selected from polyethylene glycol-1,2 dimyristin, polyethylene glycol-distearoyl phosphatidylethanolamine, PEG-cholesterol, polyethylene glycol-diacylglycerol, polyethylene glycol-dialkoxypropyl, specifically including any one of polyethylene glycol 500-dipalmitoylphosphatidylcholine, polyethylene glycol 2000-dipalmitoylphosphatidylcholine, polyethylene glycol 500-stearoylphosphatidylethanolamine, polyethylene glycol 2000-distearoylphosphatidylethanolamine, polyethylene glycol 500-1,2-oleoylphosphatidylethanolamine, polyethylene glycol 2000-1,2-oleoylphosphatidylethanolamine and polyethylene glycol 2000-2,3-dimyristoylglycerol.

[0359] In a specific embodiment of the present invention, the PEGylated lipid in the aforementioned lipid composition is selected from any PEGylated lipid represented by general formula (2).

[0360] In a specific embodiment of the present invention, the structure of the PEGylated lipid in the aforementioned lipid composition is selected from any one of the following:

[0361]

[0362]

[0363]

[0364] wherein n1 is an integer selected from 20-250.

[0365] In a specific embodiment of the present invention, the molar percentage of PEGylated lipids, cationic lipids, neutral lipids, and steroid lipids in the lipid composition is not particularly limited, and is preferably:

[0366] 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%, 1.9%;

[0367] The molar percentage of cationic lipids to total lipids is 30-65%, preferably 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 55%;

[0368] The molar percentage of phospholipids to total lipids is 7.5-13%, preferably 8%, 9%, 10%, 11%, or 12%;

[0369] The molar percentage of steroid lipids in the total lipids is 35-50%, preferably 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%.

[0370] In a specific embodiment of the present invention, the aforementioned lipid composition comprises 20-80% of the cationic lipid represented by formula (1), 5-15% of the neutral lipid, 25-55% of the steroid lipid and 0.5-10% of the PEGylated lipid, wherein the percentages are the molar percentages of each lipid in the total lipids.

[0371] 4.2. Lipid Pharmaceutical Compositions and Their Preparations

[0372] One embodiment of the present invention:

[0373] 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, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs.

[0374] In a specific embodiment of the present invention, the drug in the aforementioned 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, and the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA and siRNA; preferably any one of DNA, mRNA, miRNA and siRNA.

[0375] 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, an antibiotic, an antiviral agent, an antifungal agent, and a vaccine.

[0376] In a specific embodiment of the present invention, the lipid composition in the aforementioned lipid pharmaceutical composition forms LNP; the lipid pharmaceutical composition is an LNP-pharmaceutical composition, preferably an LNP-nucleic acid pharmaceutical composition, and more preferably an LNP-mRNA composition.

[0377] In a specific embodiment of the present invention, the drugs in the aforementioned 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 drugs, vitamins, sedatives and 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.

[0378] In a specific embodiment of the present invention, the drug in the aforementioned 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.

[0379] One embodiment of the present invention:

[0380] A lipid pharmaceutical composition preparation contains any of the aforementioned lipid pharmaceutical compositions and a pharmaceutically acceptable diluent or excipient, wherein the diluent or excipient is 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.

[0381] In a specific embodiment of the present invention, the ratio of the lipid composition to the working solution contained in the aforementioned lipid pharmaceutical composition preparation is not particularly limited. Preferably, the lipid composition: working solution = 0.05-20 g: 100 mL, more preferably the lipid composition: working solution = 0.1-10 g: 100 mL, and most preferably the lipid composition: working solution = 0.2-5 g: 100 mL.

[0382] Preparation method

[0383] In a specific embodiment of the present invention, the preparation of the lipid pharmaceutical composition preparation comprises the following steps:

[0384] (1) equilibrating the lipid component in a diluent or excipient;

[0385] (2) adding the drug to the mixture of the equilibrated liposomes and the diluent or excipient for compounding;

[0386] 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.

[0387] In a specific embodiment of the present invention, the preparation of the LNP-nucleic acid pharmaceutical composition comprises the following steps:

[0388] (1) dissolving the lipid component in an organic solvent to obtain an organic phase solution;

[0389] (2) adding the nucleic acid drug to a buffer solution to obtain an aqueous solution;

[0390] (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;

[0391] 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.

[0392] 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. DETAILED DESCRIPTION

[0393] The preparation of cationic lipids, lipid compositions, lipid pharmaceutical compositions, and the biological activity testing of LNP-nucleic acid pharmaceutical compositions are further described below in conjunction with some specific examples. The specific examples are provided to further illustrate the present invention, but are not intended to limit the scope of the present invention.

[0394] 5.1. Preparation of lipid compounds

[0395] Example 1: Cationic lipid (E1-1)

[0396]

[0397] Corresponding to the general formula (1), in E1-1, R1 and R2 are B1 and B2 are both ethylene, L1 and L2 are both -(C=O)O-(CH2)4-OC(=O)-, -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 938 Da.

[0398] The preparation process is as follows:

[0399] S1-1 (2.53 g, 6.6 mmol, prepared by condensation reaction of 2-hexyldecanoic acid and 4-hydroxybutyl acrylate) was dissolved in 40 mL of methanol, and 1-amino-3-(4-methyl-1-piperazinyl)-2-propanol (S1-2, 0.86 g, 3.0 mmol) containing a TBS-protected hydroxyl group was added. The mixture was stirred at 35°C overnight. After the reaction, the mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 20 mL of tetrahydrofuran was added, followed by 20 mL of a 1 M solution of tetra-n-butylammonium fluoride (TBAF) in tetrahydrofuran (THF). The reaction was allowed to react overnight to remove the TBS protection. After the reaction was completed, the mixture was concentrated, extracted, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to yield E1-1 (2.59 g). The main data of the H NMR spectrum of E1-1 are as follows: 1 H NMR (400MHz, CDCl3) δ: 4.10-4.06 (m, 8H), 3.88-3.81 (m, 1H), 2.79-2.24 (m, 25H) 1.71-1.19 (m, 56H), 0.85 (t, 12H). MS(ESI):m / z=938.82([M+H] + ).

[0400]

[0401] Example 2: Cationic lipid (E2-1)

[0402]

[0403] Corresponding to the general formula (1), in E2-1, R1 is undecyl, R2 is B1 is pentylene, B2 is heptylene, L1 and L2 are both ester groups (-C(=O)O-), -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 838 Da.

[0404] The preparation process is as follows:

[0405] Step a: Dissolve 1-amino-3-chloro-2-propanol (S2-1, 3.23 g, 10.0 mmol) containing a TBS-protected hydroxyl group and a Boc-protected amino group in dry THF (50 mL). Slowly add NaH (60%, 3.98 g, 100.0 mmol) under ice-cooling. Incubate for 1 hour. After completion of the reaction, add trimethyl-1,3-propanediamine (S2-2, 1.39 g, 12.0 mmol). Stir under ice-cooling for 1 hour, then slowly return the reaction mixture to room temperature and allow to react overnight. After the reaction, the reaction was placed in an ice bath, and 3 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (50 mL) was added and stirred, and then extracted twice with dichloromethane (25 mL*2). The organic phases were collected and combined, backwashed once with saturated sodium chloride (50 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and dissolved with dichloromethane. TFA was added to 0.1 M, and after reacting for 4 hours, the pH was adjusted to neutral, the reaction solution was concentrated, purified water was added, and extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and recrystallized to obtain the amino-exposed intermediate S2-3 (2.29 g).

[0406] Step b: Dissolve S2-4 (3.31 g, 7.2 mmol, obtained by condensation of 8-bromooctanoic acid and 9-heptadecanol) in 50 mL of DMF, add S2-3 (1.82 g, 6.0 mmol) and K2CO3 (1.19 g, 8.6 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2). The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to yield S2-5 (3.24 g).

[0407] Step c: Dissolve compound S2-5 (2.74 g, 4.0 mmol) in dry THF (50 mL). Slowly add NaH (60%, 1.59 g, 40.0 mmol) under ice-cooling. Incubate for 1 hour. After completion of the reaction, add S2-6 (1.67 g, 4.8 mmol, obtained by condensation of 6-bromohexanoic acid and 1-undecanol). Stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After completion of the reaction, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (50 mL) and stir to mix. Extract the mixture twice with dichloromethane (25 mL x 2). The organic phases are collected, combined, and backwashed once with saturated sodium chloride (50 mL). The organic phase is concentrated under reduced pressure, and 20 mL of tetrahydrofuran is added. Then, 20 mL of 1 M TBAF solution in tetrahydrofuran is added. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction is complete, the organic phases are concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain the crude product of compound E2-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump affords cationic lipid E2-1 (2.41 g). The main H NMR spectrum data for E2-1 are as follows: 1 H NMR (400MHz, CDCl3) δ: 4.83-4.77(m,1H),4.06(t,2H),3.83-3.77(m,1H),2.66-2.29(m,16H),2.26-2.20(m,9H),1.69-1.21(m,64H),0.86(t,9H). MS(ESI):m / z=838.83([M+H] + ).

[0408]

[0409] Example 3: Cationic lipids (E3-1, E3-2)

[0410] Example 3.1: Preparation of cationic lipid E3-1

[0411]

[0412] Corresponding to the general formula (1), in E3-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 and L2 are both ester groups (-OC(=O)-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 863 Da.

[0413] The preparation process is as follows:

[0414] 6-Bromohexyl 2-heptyldecanoate (S3-1, 2.85 g, 6.6 mmol, obtained by condensation of 6-bromohexanol and 2-heptyldecanoic acid) was dissolved in 50 mL of DMF. 1-Amino-3-(1-piperidinyl)-2-propanol (S3-2, 0.82 g, 3.0 mmol) and K2CO3 (1.09 g, 7.9 mmol) with TBS-protected hydroxyl groups were added. The mixture was stirred overnight at room temperature. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. The mixture was washed sequentially with 10% citric acid (25 mL x 2) and brine (25 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 25 mL of tetrahydrofuran was added, followed by 25 mL of a 1 M solution of TBAF in tetrahydrofuran. The reaction mixture was allowed to react overnight to remove the TBS protection. After completion of the reaction, the mixture was concentrated, extracted, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to give E3-1 (2.10 g). The main data of the H NMR spectrum of E3-1 are as follows: 1H NMR (400 MHz, CDCl3) δ: 4.08-4.01 (m, 4H), 3.84-3.75 (m, 1H), 2.62-2.19 (m, 12H), 1.73-1.51 (m, 12H), 1.46-1.20 (m, 64H), 0.86 (t, 12H). MS (ESI): m / z = 863.88 ([M+H] + ).

[0415]

[0416] Example 3.2: Preparation of cationic lipid E3-2

[0417]

[0418] Corresponding to the general formula (1), in E3-2, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 and L2 are both ester groups (-OC(=O)-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 835 Da.

[0419] Referring to the preparation of E3-1, S3-1 was replaced by S3-3 (obtained by condensation reaction of 6-bromohexanol and 2-hexyldecanoic acid), and the remaining steps were the same to obtain E3-2. The main data of its H NMR spectrum are as follows: 1H NMR (400 MHz, CDCl3) δ: 4.07-4.01 (m, 4H), 3.84-3.75 (m, 1H), 2.62-2.20 (m, 12H), 1.72-1.50 (m, 12H), 1.45-1.20 (m, 60H), 0.86 (t, 12H). MS (ESI): m / z = 835.80 ([M+H] + ).

[0420]

[0421] Example 4: Cationic lipid (E4-1)

[0422]

[0423] Corresponding to the general formula (1), in E4-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 and L2 are both ester groups (-C(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 820 Da.

[0424] The preparation process is as follows:

[0425] Step a: Under argon, DCC (3.09 g, 15.0 mmol) was added to a round-bottom flask containing 7-bromoheptanoic acid (S4-1, 2.08 g, 10.0 mmol), 7-pentadecanol (S4-2, 2.51 g, 11.0 mmol), and DMAP (0.31 g, 2.5 mmol) in dichloromethane (50 mL) in an argon atmosphere. The mixture was allowed to react at room temperature for 16 h. After the reaction, the precipitate was removed by filtration. The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography to afford 1-hexylnonyl-7-bromoheptanoate (S4-3, 3.49 g).

[0426] Step b: Dissolve S4-3 (2.76 g, 6.6 mmol) in 50 mL of DMF, add 2-amino-1-cyclohexylethanol (S4-4, 0.77 g, 3.0 mmol) containing a TBS-protected hydroxyl group and KCO (1.09 g, 7.9 mmol), and stir at room temperature overnight. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2). The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 25 mL of tetrahydrofuran is added, followed by 25 mL of a 1 M solution of TBAF in tetrahydrofuran. The reaction is allowed to react overnight to remove the TBS protection. After completion of the reaction, concentrate, extract, and combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate. The crude product is purified by silica gel column chromatography to yield E4-1 (1.99 g). The main data of H NMR spectrum of E4-1 are as follows: 1H NMR (400 MHz, CDCl3) δ: 4.89-4.82 (m, 2H), 3.35-3.28 (m, 1H), 2.60-2.20 (m, 10H), 1.90 (d, 1H), 1.77-1.39 (m, 20H), 1.32-0.96 (m, 54H), 0.86 (t, 12H). MS (ESI): m / z = 820.58 ([M+H] + ).

[0427]

[0428] Example 5: Cationic lipid (E5-1)

[0429]

[0430] Corresponding to the general formula (1), in E5-1, R1 and R2 are B1 and B2 are both heptylene, L1 and L2 are both ester groups (-C(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 912 Da.

[0431] The preparation process is as follows:

[0432] Step a: Under argon atmosphere, glycerol containing one TBS-protected hydroxyl group (S5-1, 4.12 g, 20.0 mmol), K2CO3 (8.28 g, 60.0 mmol), and bromohexane (S5-2, 3.61 g, 22.0 mmol) were dissolved in 100 mL of DMF. The mixture was stirred at 110°C for 16 hours. After completion of the reaction by thin-layer chromatography, the reaction solution was poured into water (100 mL) for precipitation and filtered. The resulting solid was dissolved in 50 mL of tetrahydrofuran, and 50 mL of TBAF tetrahydrofuran solution (1 M) was added. The reaction was allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting phase was purified by silica gel column chromatography to afford S5-3 (3.02 g).

[0433] Step b: Under argon atmosphere, DCC (2.78 g, 13.5 mmol) was added to a round-bottom flask containing 8-bromooctanoic acid (S5-4, 2.00 g, 9.0 mmol), S5-3 (2.57 g, 9.9 mmol), and DMAP (0.27 g, 2.3 mmol) in dichloromethane (50 mL) and the mixture was allowed to react at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration. The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography to afford S5-5 (3.49 g).

[0434] Step c: Dissolve S5-5 (3.06 g, 6.6 mmol) in 50 mL of DMF, add 2-amino-1-cyclohexylethanol (S4-4, 0.77 g, 3.0 mmol) containing a TBS-protected hydroxyl group and K2CO3 (1.09 g, 7.9 mmol), and stir at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 25 mL of tetrahydrofuran was added, followed by 25 mL of 1 M TBAF solution in tetrahydrofuran. The reaction was allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to yield E5-1 (2.19 g). The key H NMR spectrum data of E5-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:4.97-4.92(m,2H),3.59-3.27(m,17H),2.60-2.19(m ,10H),1.90(d,1H),1.79-1.40(m,20H),1.33-0.95(m,42H),0.86(t,12H). MS(ESI):m / z=912.84([M+H] + ).

[0435]

[0436] Example 6: Cationic lipid (E6-1)

[0437]

[0438] Corresponding to the general formula (1), in E6-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 and L2 are both carbonate groups (-OC(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 882 Da.

[0439] The preparation process is as follows:

[0440] Step a: Under nitrogen, 6-bromohexyl-4-nitrophenyl carbonate (S6-1, 10.35 g, 30.0 mmol, obtained by reacting p-nitrophenyl chloroformate and 6-bromohexanol) was dissolved in DCM (350 mL). S4-2 (27.36 g, 120.0 mmol) was added dropwise with stirring at room temperature. Pyridine (3.02 mL, 37.5 mmol) was then slowly added dropwise over 10 min, followed by the addition of DMAP (0.73 g, 6.0 mmol) all at once. The reaction was stirred at room temperature for 16 h. After completion of the reaction, the mixture was extracted twice with DCM and water. The organic phases were combined, washed with brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified on a silica gel column. The target eluate was collected and concentrated to afford S6-2 (3.39 g).

[0441] Step b: S6-2 (2.86 g, 6.6 mmol) was dissolved in 50 mL of DMF, S1-2 (0.86 g, 3.0 mmol) and K2CO3 (1.09 g, 7.9 mmol) were added, and stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 25 mL of tetrahydrofuran was added, followed by 25 mL of TBAF tetrahydrofuran solution (1 M), and the reaction was allowed to proceed overnight to remove the TBS protection. After the reaction was completed, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to obtain E6-1 (2.20 g). The main data of the H NMR spectrum of E6-1 are as follows: 1H NMR (400MHz, CDCl3) δ: 4.15-4.04(m,6H), 3.38-2.92(m,12H), 2.88-2.37(m,8H), 1.75-1.20(m,64H), 0.88(t,12H). MS(ESI):m / z=882.86([M+H] + ).

[0442]

[0443] Example 7: Cationic lipid (E7-1)

[0444]

[0445] Corresponding to the general formula (1), in E7-1, R1 is undecyl, R2 is B1 is pentylene, B2 is heptylene, L1 and L2 are both ester groups (-C(=O)O-), -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 787 Da.

[0446] The preparation process is as follows:

[0447] Step a: Dissolve S2-4 (4.42 g, 9.6 mmol) in 60 mL of DMF, add 2-amino-1-(4-pyridyl)ethanol (S7-3, 2.02 g, 8.0 mmol) containing a TBS-protected hydroxyl group and K2CO3 (1.59 g, 11.5 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 60 mL of dichloromethane. Wash with 10% citric acid (30 mL x 2) and brine (30 mL x 2), sequentially. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S7-4 (4.15 g).

[0448] Step b: Dissolve compound S7-4 (3.80 g, 6.0 mmol) in dry THF (60 mL). Slowly add NaH (60%, 2.40 g, 60.0 mmol) under ice-cooling and react for 1 hour. After the reaction, add S2-6 (2.51 g, 7.2 mmol) and stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (60 mL) and stir to mix. Extract the mixture twice with dichloromethane (30 mL x 2). The organic phases are combined and backwashed once with saturated sodium chloride (60 mL). The organic phase is concentrated under reduced pressure, and 30 mL of tetrahydrofuran is added. Then, 30 mL of 1 M TBAF solution in tetrahydrofuran is added. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction is complete, the organic phases are concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain the crude product of compound E7-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump affords cationic lipid E7-1 (3.64 g). The main H NMR spectrum data for E7-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:8.44(d,2H),7.24(d,2H),4.89-4.83(m,1H),4.72-4. 68(m,1H),4.07(t,2H),2.56-2.24(m,10H),1.78-1.22(m,62H),0.88(t,9H). MS(ESI):m / z=787.70([M+H] + ).

[0449]

[0450] Example 8: Cationic lipid (E8-1)

[0451]

[0452] Corresponding to the general formula (1), in E8-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 is an ester group (-OC(=O)-), L2 is a carbonate group (-OC(=O)O-), and -L3(R3- is Wherein R3 is -O(CH2)2OH, R4 is The total molecular weight is approximately 910 Da.

[0453] The preparation process is as follows:

[0454] Step a: Dissolve S6-2 (3.12 g, 7.2 mmol) in 50 mL of DMF, add S8-1 (1.73 g, 6.0 mmol, prepared in Reference Example 42) and KCO (1.19 g, 8.6 mmol), and stir overnight at room temperature. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. The mixture was washed sequentially with 10% citric acid (25 mL x 2) and brine (25 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to yield S8-2 (3.09 g).

[0455] Step b: Dissolve the above-mentioned compound S8-2 (2.58 g, 4.0 mmol) in dry THF (50 mL). Slowly add NaH (60%, 1.60 g, 40.0 mmol) under ice-cooling. Incubate for 1 hour. After completion of the reaction, add compound S3-3 (2.01 g, 4.8 mmol, obtained by condensation of 6-bromohexanol and 2-hexyldecanoic acid). Stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction, the reaction was placed in an ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (50 mL) was added and stirred, and then extracted twice with dichloromethane (25 mL * 2). The organic phases were collected and combined, backwashed once with saturated sodium chloride (50 mL), and the organic phase was concentrated under reduced pressure, dissolved with methanol, and 1 M hydrochloric acid was added to pH = 3.5. After reacting for 4 hours, it was concentrated, precipitated, filtered, recrystallized, and dried to obtain the crude product of E8-1 with exposed hydroxyl groups. The cationic lipid E8-1 (3.02 g) was purified by column chromatography, concentrated, and pumped dry with an oil pump. The main data of the nuclear magnetic hydrogen spectrum of E8-1 are as follows: 1 HNMR(400MHz, CDCl3)δ:4.24-4.03(m,5H),3.91-3.82(m,1H),3.70-3.52(m,4H),2.68-2.20(m,20H),1.75-1.22(m,64H),0.89(t,12H). MS(ESI):m / z=910.83([M+H] + ).

[0456]

[0457] Example 9: Cationic lipid (E9-1)

[0458]

[0459] Corresponding to the general formula (1), in E9-1, R1 is undecyl, R2 is B1 is a pentylene group, B2 is a heptylene group, L1 is an ester group (-C(=O)O-), L2 is a carbonate group (-OC(=O)O-), and -L3(R3- is Wherein R3 is -(C=O)O(CH2)2OH, R4 is The total molecular weight is approximately 875 Da.

[0460] The preparation process is as follows:

[0461] Step a: Under argon, DCC (3.09 g, 15.0 mmol) was added to a round-bottom flask containing 3-(3-pyridyl)-L-alanine (S9-1, 2.66 g, 10.0 mmol) containing a Boc-protected amino group, ethylene glycol (S9-2, 1.47 g, 11.0 mmol) containing an EE-protected hydroxyl group (where EE is an α-ethoxyethyl ether group formed by the reaction of ethyl vinyl ether with a hydroxyl group, -CH(CH3)OEt), and DMAP (0.31 g, 2.5 mmol). The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration. The filtrate was concentrated and dissolved with dichloromethane. TFA was added to 0.1 M. After reacting for 4 hours, the pH was adjusted to neutral. The reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. The solution was recrystallized to obtain the amino-exposed intermediate S9-3 (1.79 g).

[0462] Step b: Dissolve S9-4 (3.43 g, 7.2 mmol, obtained by reacting 7-bromoheptyl-4-nitrophenyl carbonate with 9-heptadecanol) in 50 mL of DMF, add S9-3 (1.69 g, 6.0 mmol) and KCO (1.19 g, 8.6 mmol), and stir overnight at room temperature. After the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2). The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to yield S9-5 (3.59 g).

[0463] Step c: Dissolve the above compound S9-5 (2.72 g, 4.0 mmol) in dry THF (50 mL). Slowly add NaH (60%, 1.60 g, 40.0 mmol) under ice-cooling and react for 1 hour. After the reaction, add compound S2-6 (1.67 g, 4.8 mmol) and stir under ice-cooling for 1 hour. The reaction solution is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (50 mL) and stir to mix. Extract the mixture twice with dichloromethane (25 mL x 2). The organic phases are collected, combined, and backwashed once with saturated sodium chloride (50 mL). The organic phases are concentrated under reduced pressure, dissolved in methanol, and 1 M hydrochloric acid is added to a pH of 3.5. After reacting for 4 hours, the mixture is concentrated, the precipitate is filtered, recrystallized, and dried to obtain the crude product E9-1 with exposed hydroxyl groups. The cationic lipid E9-1 (2.70 g) was purified by column chromatography, concentrated, and pumped dry. The main data of E9-1's H NMR spectrum are as follows: 1H NMR (400 MHz, CDCl3) δ: 8.48-7.14 (m, 4H), 4.21-4.05 (m, 7H), 3.86-3.80 (m, 2H), 3.68-3.63 (m, 1H), 3.08-3.01 (m, 2H), 2.69-2.24 (m, 6H), 1.72-1.23 (m, 62H), 0.89 (t, 9H). MS (ESI): m / z = 875.74 ([M+H] + ).

[0464]

[0465] Example 10: Cationic lipids (E10-1, E10-2)

[0466] Example 10.1: Preparation of cationic lipid E10-1

[0467]

[0468] Corresponding to the general formula (1), in E10-1, R1 and R2 are both 8,11-heptadecadienyl, B1 and B2 are both butylene, L1 and L2 are both ester groups (-OC(=O)-), and -L3(R3)- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 829 Da.

[0469] The preparation process is as follows:

[0470] Step a: Under argon atmosphere, DCC (3.09 g, 15.0 mmol) was added to a round-bottom flask containing linoleic acid (S10-1, 2.80 g, 10.0 mmol), 4-bromobutanol (S10-2, 1.67 g, 11.0 mmol), and DMAP (0.31 g, 2.5 mmol) in dichloromethane (50 mL). The mixture was allowed to react at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration. The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography to afford S10-3 (3.45 g).

[0471] Step b: Dissolve S10-3 (2.73 g, 6.6 mmol) in 50 mL of DMF, add 1-amino-3-(4-morpholinyl)-2-propanol (S10-4, 0.82 g, 3.0 mmol) containing a TBS-protected hydroxyl group and K2CO3 (1.09 g, 7.9 mmol), and stir at room temperature overnight. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2). The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 25 mL of tetrahydrofuran is added, followed by 25 mL of a 1 M TBAF solution in tetrahydrofuran. The reaction mixture is allowed to react overnight to remove the TBS protection. After completion of the reaction, concentrate, extract, and combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate to obtain the crude product of compound E10-1. The crude product was purified by silica gel column chromatography to obtain E10-1 (1.96 g). The main data of the H NMR spectrum of E10-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:5.37-5.33(m,8H),4.03(t,4H),3.92-3.86(m,1H),3.72-3.67(m,4H),2.76(t ,4H),2.72-2.42(m,10H),2.39-2.23(m,4H,2H),2.06-2.00(m,8H),1.69-1.21(m,40H),0.87(t,6H). MS(ESI):m / z=829.76([M+H] + ).

[0472]

[0473] Example 10.2: Preparation of cationic lipid E10-2

[0474]

[0475] Corresponding to the general formula (1), in E10-2, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 and L2 are both ester groups (-OC(=O)-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 837 Da.

[0476] Referring to the preparation of E10-1, S10-3 was replaced by S3-3, and the remaining steps were the same to obtain E10-2. The main data of its H NMR spectrum are as follows: 1 H NMR(400MHz, CDCl3)δ:4.03(t,4H),3.94-3.87(m,1H),3.71-3.66(m,4H),2.72-2.42(m,10H),2 .37-2.24(m,2H,2H),1.62-1.50(m,10H),1.43-1.32(m,10H),1.26-1.19(m,44H),0.85(t,12H). MS(ESI):m / z=837.80([M+H] + ).

[0477]

[0478] Example 11: Cationic lipid (E11-1)

[0479]

[0480] Corresponding to the general formula (1), in E11-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 is an ester group (-C(=O)O-), L2 is a carbonate group (-OC(=O)O-), and -L3(R3- is Where R3 is R4 is The total molecular weight is approximately 905 Da.

[0481] The preparation process is as follows:

[0482] Step a: To a sealed, anhydrous, and oxygen-free reactor, add 200 mL of tetrahydrofuran and an excess of potassium diphenylmethyl (14.42 g, 70.0 mmol). Then, add 2-amino-1-(4-pyridyl)ethanol (S11-1, 4.00 g, 16.8 mmol) containing a Boc-protected amino group and compound S11-2 (6.55 g, 16.8 mmol). S11-2 is glycerol with one hydroxyl group substituted with a p-toluenesulfonate (-OTs) group and the other two hydroxyl groups protected with EE. After reacting at 30°C for 12 hours, the reactor was opened, concentrated, washed, and dissolved in dichloromethane. TFA was added to 0.1 M. After reacting for 4 hours, the pH was adjusted to neutral. The reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was concentrated, precipitated, filtered, and dried to obtain intermediate S11-3 (3.51 g) with exposed amino groups.

[0483] Step b: Dissolve S6-2 (4.69 g, 10.8 mmol) in 80 mL of DMF, add S11-3 (3.20 g, 9.0 mmol, prepared in Reference Example 43) and KCO (1.79 g, 13.0 mmol), and stir overnight at room temperature. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 80 mL of dichloromethane. After washing with 10% citric acid (40 mL x 2) and brine (40 mL x 2), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S11-4 (5.31 g).

[0484] Step c: Dissolve the above compound S11-4 (4.27 g, 6.0 mmol) in dry THF (80 mL). Slowly add NaH (60%, 2.40 g, 60.0 mmol) under ice-cooling and react for 1 hour. After the reaction, add compound S4-3 (3.01 g, 7.2 mmol) and stir under ice-cooling for 1 hour. The reaction solution is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (80 mL) and stir to mix. Extract the mixture twice with dichloromethane (40 mL x 2). The organic phases are collected, combined, and backwashed once with saturated sodium chloride (80 mL). The organic phases are concentrated under reduced pressure, dissolved in methanol, and 1 M hydrochloric acid is added to a pH of 3.5. After reacting for 4 hours, the mixture is concentrated, the precipitate is filtered, recrystallized, and dried to obtain the crude product E11-1 with exposed hydroxyl groups. The cationic lipid E11-1 (4.40 g) was obtained by column chromatography, concentration, and pumping to dryness. The main data of the H NMR spectrum of E11-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:8.55(d,2H),7.20(d,2H),4.80-4.75(m,1H),4.30-4.25(m,1H),4.21-4.06(m,3H),3.79-3.73( m,1H),3.64-3.56(m,2H),3.41-3.34(m,2H),3.02-2.93(m,2H),2.60-2.25(m,6H),1.72-1.20(m,64H),0.88(t,12H). MS(ESI):m / z=905.78([M+H] + ).

[0485]

[0486] Example 12: Cationic lipid (E12-1)

[0487]

[0488] Corresponding to the general formula (1), in E12-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 and L2 are both ester groups (-OC(=O)-), and -L3(R3- is Wherein R3 is -(C=O)O(CH2)2OH, R4 is The total molecular weight is approximately 1111 Da.

[0489] The preparation process is as follows:

[0490] Step a: Under argon, DCC (3.09 g, 15.0 mmol) was added to a round-bottom flask containing 3-(3-pyridyl)-L-alanine (S12-1, 2.66 g, 10.0 mmol) containing a Boc-protected amino group, ethylene glycol (S9-2, 1.47 g, 11.0 mmol) containing an EE-protected hydroxyl group, and DMAP (0.31 g, 2.5 mmol) in dichloromethane (50 mL). The mixture was allowed to react at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration. The filtrate was concentrated, dissolved in dichloromethane, and TFA was added to 0.1 M. After reacting for 4 h, the pH was adjusted to neutral. The reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and recrystallized to obtain the amino-exposed intermediate S12-2 (1.79 g).

[0491] Step b: Dissolve S12-3 (3.50 g, 6.6 mmol, obtained by condensation reaction of 6-bromohexanol and 2-decyltetradecanoic acid) in 50 mL of DMF, add S12-2 (0.85 g, 3.0 mmol) and K2CO3 (1.09 g, 7.9 mmol), and stir at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. The mixture was washed sequentially with 10% citric acid (25 mL x 2) and brine (25 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and dissolved in methanol. 1 M hydrochloric acid was added to pH 3.5. After reacting for 4 hours, the mixture was concentrated, precipitated, filtered, recrystallized, and dried to obtain the crude product E12-1 with exposed hydroxyl groups. The crude product was purified by silica gel column chromatography to yield E12-1 (2.77 g). The key H NMR spectrum data of E12-1 are as follows: 1HNMR(400MHz, CDCl3)δ:8.48-7.14(m,4H),4.23-4.19(m,2H),4.10-4.04(m,4H),3.87-3.82(m,2 H),3.69-3.63(m,1H),3.10-3.00(m,2H),2.34-2.25(m,2H),1.74-1.20(m,100H),0.88(t,12H). MS(ESI):m / z=1111.99([M+H] + ).

[0492]

[0493] Example 13: Cationic lipid (E13-1)

[0494]

[0495] Corresponding to the general formula (1), in E13-1, R1 is undecyl, R2 is B1 is pentylene, B2 is heptylene, L1 and L2 are both ester groups (-C(=O)O-), -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 792 Da.

[0496] The preparation process is as follows:

[0497] Step a: Dissolve S2-4 (4.42 g, 9.6 mmol) in 60 mL of DMF, add S4-4 (2.06 g, 8.0 mmol) and K2CO3 (1.59 g, 11.5 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 60 mL of dichloromethane. Wash with 10% citric acid (30 mL x 2) and brine (30 mL x 2), sequentially. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S13-1 (4.44 g).

[0498] Step b: Dissolve compound S13-1 (3.83 g, 6.0 mmol) in dry THF (60 mL). Slowly add NaH (60%, 2.40 g, 60.0 mmol) under ice-cooling and react for 1 hour. After the reaction, add S2-6 (2.51 g, 7.2 mmol) and stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (60 mL) and stir to mix. Extract the mixture twice with dichloromethane (30 mL x 2). The organic phases are collected, combined, and backwashed once with saturated sodium chloride (60 mL). The organic phase is concentrated under reduced pressure, and 30 mL of tetrahydrofuran is added, followed by 30 mL of 1 M TBAF solution in tetrahydrofuran. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction was complete, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to yield the crude product of compound E13-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump afforded cationic lipid E13-1 (3.37 g). The main H NMR spectrum data for E13-1 are as follows: 1 H NMR (400MHz, CDCl3) δ: 4.87-4.81(m,1H),4.09(t,2H),3.35-3.27(m,1H),2.63-2.24(m,10H),1.90(d,1H),1.78-0.97(m,72H),0.88(t,9H). MS(ESI):m / z=792.79([M+H] + ).

[0499]

[0500] Example 14: Cationic lipid (E14-1)

[0501]

[0502] Corresponding to the general formula (1), in E14-1, R1 is undecyl, R2 is B1 is pentylene, B2 is heptylene, L1 and L2 are both ester groups (-C(=O)O-), -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 796 Da.

[0503] The preparation process is as follows:

[0504] Step a: Dissolve S5-5 (4.45 g, 9.6 mmol) in 60 mL of DMF, add S4-4 (2.06 g, 8.0 mmol) and K2CO3 (1.59 g, 11.5 mmol), and stir at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 60 mL of dichloromethane. After washing with 10% citric acid (25 mL x 2) and brine (25 mL x 2), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S14-1 (4.37 g).

[0505] Step b: Dissolve compound S14-1 (3.85 g, 6.0 mmol) in dry THF (60 mL). Slowly add NaH (60%, 2.40 g, 60.0 mmol) under ice-cooling and allow to react for 1 hour. After completion of the reaction, add S2-6 (2.51 g, 7.2 mmol) and stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After completion of the reaction, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (60 mL) and stir to mix. Extract the mixture twice with dichloromethane (30 mL x 2). The organic phases are collected, combined, and backwashed once with saturated sodium chloride (60 mL). The organic phase is concentrated under reduced pressure, and 30 mL of tetrahydrofuran is added, followed by 30 mL of 1 M TBAF solution in tetrahydrofuran. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction was complete, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to yield the crude product of compound E14-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump afforded cationic lipid E14-1 (3.63 g). The main H NMR spectrum data for E14-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:4.94-4.89(m,1H),4.11(t,2H),3.58-3.29(m,9H),2.65-2.23(m,10H),1.92(d,1H),1.79-0.99(m,50H),0.87(t,9H). MS(ESI):m / z=796.77([M+H] + ).

[0506]

[0507] Example 15: Cationic lipid (E15-1)

[0508]

[0509] Corresponding to the general formula (1), in E15-1, R1 is undecyl, R2 is B1 is pentylene, B2 is heptylene, L1 and L2 are both ester groups (-C(=O)O-), -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 822 Da.

[0510] The preparation process is as follows:

[0511] Step a: Dissolve S2-4 (4.42 g, 9.6 mmol) in 60 mL of DMF, add S1-2 (2.30 g, 8.0 mmol) and K2CO3 (1.59 g, 11.5 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 60 mL of dichloromethane. Wash with 10% citric acid (30 mL x 2) and brine (30 mL x 2), sequentially. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S15-1 (4.36 g).

[0512] Step b: Dissolve compound S15-1 (4.01 g, 6.0 mmol) in dry THF (60 mL). Slowly add NaH (60%, 2.40 g, 60.0 mmol) under ice-cooling and react for 1 hour. After the reaction, add S2-6 (2.51 g, 7.2 mmol) and stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (60 mL) and stir to mix. Extract the mixture twice with dichloromethane (30 mL x 2). The organic phases are collected, combined, and backwashed once with saturated sodium chloride (60 mL). The organic phase is concentrated under reduced pressure, and 30 mL of tetrahydrofuran is added, followed by 30 mL of 1 M TBAF solution in tetrahydrofuran. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction was complete, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to yield the crude product of compound E15-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump afforded cationic lipid E15-1 (3.92 g). The main H NMR spectrum data for E15-1 are as follows: 1 H NMR (400MHz, CDCl3) δ: 4.88-4.80 (m, 1H), 4.08 (t, 2H), 3.84-3.73 (m, 1H), 2.77-2.19 (m, 23H), 1.72-1.18 (m, 62H), 0.87 (t, 9H). MS(ESI):m / z=822.81([M+H] + ).

[0513]

[0514] Example 16: Cationic lipid (E16-1)

[0515]

[0516] Corresponding to the general formula (1), in E16-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 and L2 are both ester groups (-OC(=O)-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 850 Da.

[0517] The preparation process is as follows:

[0518] S3-3 (2.76 g, 6.6 mmol) was dissolved in 50 mL of DMF, and S1-2 (0.86 g, 3.0 mmol) and K2CO3 (1.09 g, 7.9 mmol) were added and stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 25 mL of tetrahydrofuran was added, followed by 25 mL of TBAF tetrahydrofuran solution (1 M). The reaction was allowed to react overnight to remove the TBS protection. After the reaction was completed, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to obtain E16-1 (2.07 g). The main data of the H NMR spectrum of E16-1 are as follows: 1 H NMR (400MHz, CDCl3) δ: 4.04 (t, 4H), 3.85-3.77 (m, 1H), 2.80-2.20 (m, 21H), 1.66-1.39 (m, 16H), 1.35-1.20 (m, 48H), 0.86 (t, 12H). MS(ESI):m / z=850.83([M+H] + ).

[0519]

[0520] Example 17: Cationic lipid (E17-1)

[0521]

[0522] Corresponding to the general formula (1), in E17-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 and L2 are both carbonate groups (-OC(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 1022 Da.

[0523] The preparation process is as follows:

[0524] Step a: Under nitrogen, 6-bromohexyl-4-nitrophenyl carbonate (S6-1, 10.35 g, 30.0 mmol) was dissolved in DCM (400 mL). 2-Octyldodecanol (S17-1, 35.76 g, 120.0 mmol) was added dropwise with stirring at room temperature. Pyridine (3.02 mL, 37.5 mmol) was then slowly added dropwise over 10 min, followed by the addition of DMAP (0.73 g, 6.0 mmol) all at once. The reaction was stirred at room temperature for 16 h. After completion of the reaction, the mixture was extracted twice with DCM and water. The organic phases were combined, washed with brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography. The target eluate was collected and concentrated to afford S17-2 (3.63 g).

[0525] Step b: S17-2 (3.33 g, 6.6 mmol) was dissolved in 50 mL of DMF, S1-2 (0.86 g, 3.0 mmol) and K2CO3 (1.09 g, 7.9 mmol) were added, and stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 25 mL of tetrahydrofuran was added, followed by 25 mL of TBAF tetrahydrofuran solution (1 M), and the reaction was allowed to proceed overnight to remove the TBS protection. After the reaction was completed, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to obtain E17-1 (2.42 g). The main data of the H NMR spectrum of E17-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:4.13-3.99(m,8H),3.33-2.94(m,12H),2.92-2.57(m,6 H),2.55-2.41(m,2H),1.76-1.38(m,16H),1.34-1.22(m,66H),0.88(t,12H). MS(ESI):m / z=1022.95([M+H] + ).

[0526]

[0527] Example 18: Cationic lipid (E18-1)

[0528]

[0529] Corresponding to the general formula (1), in E18-1, R1 is an undecyl group, R2 is a cyclohexadecyl group, B1 is a pentyl group, B2 is a hexyl group, L1 is an ester group (-C(=O)O-), L2 is a carbonate group (-OC(=O)O-), and -L3(R3)- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 824 Da.

[0530] The preparation process is as follows:

[0531] Step a: Under nitrogen, S6-1 (10.35 g, 30.0 mmol) was dissolved in DCM (400 mL). Cyclohexadecyl-1-ol (S18-1, 28.80 g, 120.0 mmol) was added dropwise with stirring at room temperature. Pyridine (3.02 mL, 37.5 mmol) was then slowly added dropwise over 10 min, followed by the addition of DMAP (0.73 g, 6.0 mmol) all at once. The reaction was stirred at room temperature for 16 hours. After completion of the reaction, the mixture was extracted twice with DCM and water. The organic phases were combined, washed with brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography. The target eluate was collected and concentrated to afford S18-2 (3.75 g).

[0532] Step b: Dissolve S18-2 (3.21 g, 7.2 mmol) in 50 mL of DMF, add S2-3 (1.82 g, 6.0 mmol) containing a TBS-protected hydroxyl group and K2CO3 (1.19 g, 8.6 mmol), and stir overnight at room temperature. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL x 2) and brine (25 mL x 2), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S18-3 (3.34 g).

[0533] Step c: Dissolve the above compound S18-3 (2.68 g, 4.0 mmol) in dry THF (40 mL). Slowly add NaH (60%, 1.59 g, 40.0 mmol) under ice-cooling and react for 1 hour. After the reaction, add S2-6 (1.67 g, 4.8 mmol) and stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (40 mL) and stir to mix. Extract the mixture twice with dichloromethane (20 mL x 2). The organic phases are collected, combined, and backwashed once with saturated sodium chloride (40 mL). The organic phases are concentrated under reduced pressure, and 20 mL of tetrahydrofuran is added. Then, 20 mL of 1 M TBAF solution in tetrahydrofuran is added. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction was complete, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to yield the crude product of compound E18-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump afforded cationic lipid E18-1 (2.49 g). The main H NMR spectrum data for E18-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:4.20-4.07(m,2H),4.05(t,2H),3.82-3.77(m,1H),2.65-2.31(m,14H),2.26-2.20(m,9H),1.74-1.25(m,64H),0.89(t,3H). MS(ESI):m / z=824.78([M+H] + ).

[0534]

[0535] Example 19: Cationic lipid (E19-1)

[0536]

[0537] Corresponding to the general formula (1), in E19-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 and L2 are both ester groups (-C(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 814 Da.

[0538] The preparation process is as follows:

[0539] Step a: Under argon atmosphere, DCC (3.09 g, 15.0 mmol) was added to a round-bottom flask containing 7-bromoheptanoic acid (S4-1, 2.08 g, 10.0 mmol), 8-pentadecanol (S19-1, 2.51 g, 11.0 mmol), and DMAP (0.31 g, 2.5 mmol) in dichloromethane (50 mL). The mixture was allowed to react at room temperature for 16 h. After the reaction, the precipitate was removed by filtration. The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography to afford S19-2 (3.68 g).

[0540] Step b: Dissolve S19-2 (2.76 g, 6.6 mmol) in 50 mL of DMF, add 2-amino-1-phenylethanol (S19-3, 0.75 g, 3.0 mmol) containing a TBS-protected hydroxyl group and K2CO3 (1.09 g, 7.9 mmol), and stir at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 25 mL of tetrahydrofuran was added, followed by 25 mL of 1 M TBAF solution in tetrahydrofuran. The reaction was allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to yield E19-1 (1.95 g). The main data of the H NMR spectrum of E19-1 are as follows: 1 H NMR (400MHz, CDCl3) δ: 7.40-7.22(m,5H), 4.86-4.77(m,2H), 4.72-4.65(m,1H), 2.53-2.24(m,10H), 1.69-1.21(m,64H), 0.88(t,12H). MS(ESI):m / z=814.77([M+H] + ).

[0541]

[0542] Example 20: Cationic lipid (E20-1)

[0543]

[0544] Corresponding to the general formula (1), in E20-1, R1 is undecyl, R2 is B1 is a pentylene group, B2 is a hexylene group substituted with -OH, L1 and L2 are both ester groups (-C(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 840 Da.

[0545] The preparation process is as follows:

[0546] Step a: Under nitrogen, 6-heptenoic acid (S20-2, 2.56 g, 20.0 mmol) was dissolved in DCM (100 mL), and N,N-diisopropylethylamine (DIEA, 7.74 g, 60.0 mmol), S20-1 (7.68 g, 30.0 mmol), EDCI (7.68 g, 40.0 mmol), and DMAP (0.73 g, 6.0 mmol) were added. The reaction mixture was stirred at 50°C for 10 hours. After completion of the reaction, the mixture was extracted twice with DCM and water. The organic phases were combined, washed with brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography. The target eluate was collected and concentrated to afford S20-3 (6.15 g).

[0547] Step b: Dissolve the above compound S20-3 (5.12 g, 14.0 mmol) in 70 mL of DCM, add m-chloroperbenzoic acid (m-CPBA, 4.84 g, 28.0 mmol), and stir at room temperature for 10 hours. After the reaction, pour the mixture into 70 mL of saturated sodium bicarbonate solution and mix thoroughly. The aqueous phase is separated and extracted with DCM (35 mL x 2). The organic phases are combined and washed with saturated brine, then dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography. The target eluate is collected and concentrated to obtain S20-4 (4.44 g).

[0548] Step c: Dissolve the above compound S20-4 (3.67 g, 9.6 mmol) in 50 mL of ethanol, add S2-3 (2.42 g, 8.0 mmol) containing a TBS-protected hydroxyl group, and stir at 50°C for 4 hours. After completion of the reaction, dilute with 50 mL of dichloromethane, backwash with 100 mL of saturated sodium chloride solution, separate the organic phase, and dry it over anhydrous sodium sulfate. Filter, concentrate, and purify by silica gel column chromatography to obtain S20-5 (3.62 g).

[0549] Step d: The above compound S20-5 (3.43 g, 5.0 mmol) and imidazole (0.54 g, 8.0 mmol) were dissolved in 50 mL of dichloromethane, and tert-butyldimethylsilyl chloride (1.05 g, 7.0 mmol) was added under stirring in an ice bath. After reacting for 2 hours, the mixture was filtered and rinsed with 50 mL of dichloromethane. The filtrates were combined, dried over anhydrous magnesium sulfate, and concentrated to obtain the intermediate S20-6 (3.92 g) containing two TBS-protected hydroxyl groups.

[0550] Step e: Dissolve S20-6 (3.20 g, 4.0 mmol) in dry THF (40 mL). Slowly add NaH (60%, 1.59 g, 40.0 mmol) under ice-cooling and react for 1 hour. After the reaction, add S2-6 (1.67 g, 4.8 mmol) and stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (40 mL) and stir to mix. Extract the mixture twice with dichloromethane (20 mL x 2). The organic phases are collected, combined, and backwashed once with saturated sodium chloride (40 mL). The organic phases are concentrated under reduced pressure, and 20 mL of tetrahydrofuran is added. Then, 20 mL of 1 M TBAF solution in tetrahydrofuran is added. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction was complete, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to yield the crude product of compound E20-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump afforded cationic lipid E20-1 (2.72 g). The main H NMR spectrum data for E20-1 are as follows: 1 HNMR(400MHz, CDCl3)δ:4.85-4.80(m,1H),4.07(t,2H),3.83-3.78(m,1H) ,3.63-3.59(m,1H),2.67-2.21(m,25H),1.73-1.23(m,60H),0.87(t,9H). MS(ESI):m / z=840.82([M+H] + ).

[0551]

[0552] Example 21: Cationic lipid (E21-1)

[0553]

[0554] Corresponding to the general formula (1), in E21-1, R1 is undecyl, R2 is B1 and B2 are both hexamethylene substituted with -OH, L1 is an amide group (-NHC(=O)-), L2 is an ester group (-C(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 841 Da.

[0555] The preparation process is as follows:

[0556] Step a: Under nitrogen, 1-amino-5-hexene (S21-1, 1.98 g, 20.0 mmol) was dissolved in 50 mL of dichloromethane. The solution was placed in an ice bath and triethylamine (2.77 mL, 20.0 mmol) was added. Decanoyl chloride (S21-2, 3.80 g, 20.0 mmol) was added over 1 hour with stirring. The ice bath was removed and the solution was warmed to room temperature. After 1 hour, 50 mL of water was added and stirred. The organic layer was separated and the aqueous layer was extracted with DCM (25 mL x 2). The combined organic phases were washed with 100 mL of saturated sodium chloride solution and dried over anhydrous sodium sulfate. The solvent and residual triethylamine were removed under reduced pressure, and the product was purified by silica gel column chromatography to yield S21-3 (4.45 g).

[0557] Step b: Dissolve the above compound S21-3 (3.54 g, 14.0 mmol) in 60 mL of DCM, add m-chloroperbenzoic acid (m-CPBA, 4.84 g, 28.0 mmol), and stir at room temperature for 10 hours. After the reaction, pour the mixture into 60 mL of saturated sodium bicarbonate solution and mix thoroughly. The aqueous phase is separated and extracted with DCM (20 mL x 3). The organic phases are combined and washed with saturated brine, then dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain S21-4 (3.13 g).

[0558] Step c: Dissolve the above compound S21-4 (2.58 g, 9.6 mmol) in 50 mL of ethanol, add S2-3 (2.42 g, 8.0 mmol) containing a TBS-protected hydroxyl group, and stir at 50°C for 4 hours. After completion of the reaction, dilute with 50 mL of dichloromethane, backwash with 100 mL of saturated sodium chloride solution, separate the organic phase, and dry it over anhydrous sodium sulfate. Filter, concentrate, and purify by silica gel column chromatography to obtain S21-5 (3.12 g).

[0559] Step d: The above compound S21-5 (2.87 g, 5.0 mmol) and imidazole (0.54 g, 8.0 mmol) were dissolved in 50 mL of dichloromethane, and tert-butyldimethylsilyl chloride (1.05 g, 7.0 mmol) was added under stirring in an ice bath. After reacting for 2 hours, the mixture was filtered and rinsed with 50 mL of dichloromethane. The filtrate was combined, dried over anhydrous magnesium sulfate, and concentrated to obtain the intermediate S21-6 (3.37 g) containing two TBS-protected hydroxyl groups.

[0560] Step e: The above compound S21-6 (2.75 g, 4.0 mmol) was dissolved in 50 mL of ethanol, and S20-4 (1.67 g, 4.8 mmol) was added and stirred at 50 ° C for 8 hours. After dilution with 50 mL of dichloromethane, the mixture was backwashed with 100 mL of saturated sodium chloride solution, and the organic phase was separated and concentrated under reduced pressure. 30 mL of tetrahydrofuran was added, and then 30 mL of TBAF tetrahydrofuran solution (1 M) was added. The reaction was allowed to react overnight to remove the TBS protection. After the reaction was completed, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by column chromatography, concentrated, and pumped dry to obtain cationic lipid E21-1 (2.39 g). The main data of the H NMR spectrum of E21-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:4.86-4.81(m,1H),3.84-3.78(m,1H),3.66-3.60(m,2H),3.20- 3.15(m,2H),2.67-2.19(m,23H),2.13-2.08(m,2H),1.71-1.22(m,56H),0.86(t,9H). MS(ESI):m / z=841.81([M+H] + ).

[0561]

[0562] Example 22: Cationic lipid (E22-1)

[0563]

[0564] Corresponding to the general formula (1), in E22-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 is an ester group (-OC(=O)-), L2 is a carbonate group (-OC(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 866 Da.

[0565] The preparation process is as follows:

[0566] Step a: Dissolve S6-2 (3.12 g, 7.2 mmol) in 50 mL of DMF, add S1-2 (1.72 g, 6.0 mmol) and K2CO3 (1.19 g, 8.6 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2). The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S22-1 (3.02 g).

[0567] Step b: Dissolve compound S22-1 (2.57 g, 4.0 mmol) in dry THF (40 mL). Slowly add NaH (60%, 1.60 g, 40.0 mmol) under ice-cooling and allow to react for 1 hour. After the reaction, add compound S3-3 (2.01 g, 4.8 mmol) and stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (40 mL) and stir to mix. Extract the mixture twice with dichloromethane (20 mL x 2). The organic phases are collected, combined, and backwashed once with saturated sodium chloride (40 mL). The organic phase is concentrated under reduced pressure, and 20 mL of tetrahydrofuran is added, followed by 20 mL of 1 M TBAF solution in tetrahydrofuran. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction is complete, the organic phases are concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain the crude product of compound E22-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump affords cationic lipid E22-1 (2.60 g). The main H NMR spectrum data for E22-1 are as follows: 1 H NMR (400MHz, CDCl3) δ: 4.18-4.06 (m, 5H), 3.87-3.78 (m, 1H), 2.76-2.20 (m, 20H), 1.68-1.21 (m, 64H), 0.88 (t, 12H). MS(ESI):m / z=866.84([M+H] + ).

[0568]

[0569] Example 23: Cationic lipid (E23-1)

[0570]

[0571] Corresponding to the general formula (1), in E23-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 is an ester group (-OC(=O)-), L2 is a carbonate group (-OC(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 853 Da.

[0572] The preparation process is as follows:

[0573] Step a: Dissolve S6-2 (3.12 g, 7.2 mmol) in 50 mL of DMF, add 1-amino-3-(4-morpholino)-2-propanol (S10-4, 1.64 g, 6.0 mmol) containing a TBS-protected hydroxyl group and K2CO3 (1.19 g, 8.6 mmol), and stir at room temperature overnight. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2), sequentially. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S23-1 (2.94 g).

[0574] Step b: Dissolve compound S23-1 (2.52 g, 4.0 mmol) in dry THF (40 mL). Slowly add NaH (60%, 1.60 g, 40.0 mmol) under ice-cooling and allow to react for 1 hour. After the reaction, add compound S3-3 (2.01 g, 4.8 mmol) and stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (40 mL) and stir to mix. Extract the mixture twice with dichloromethane (20 mL x 2). The organic phases are collected, combined, and backwashed once with saturated sodium chloride (40 mL). The organic phase is concentrated under reduced pressure, and 20 mL of tetrahydrofuran is added, followed by 20 mL of 1 M TBAF solution in tetrahydrofuran. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction was complete, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to yield the crude product of compound E23-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump afforded cationic lipid E23-1 (2.63 g). The main H NMR spectrum data for E23-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:4.21-4.02(m,5H),3.92-3.84(m,1H),3.72-3.64(m,4 H),2.71-2.39(m,10H),2.37-2.27(m,3H),1.67-1.21(m,64H),0.86(t,12H). MS(ESI):m / z=853.81([M+H] + ).

[0575]

[0576] Example 24: Cationic lipid (E24-1)

[0577]

[0578] Corresponding to the general formula (1), in E24-1, R1 and R2 are B1 and B2 are both hexamethylene, L1 and L2 are both -O-, -L3(R3)- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 865 Da.

[0579] The preparation process is as follows:

[0580] Step a: 2-Heptylundecanol (S24-1, 4.05 g, 15.0 mmol) was dissolved in dry tetrahydrofuran (60 mL). Under nitrogen, NaH (60%, 0.60 g, 15.0 mmol) was slowly added with stirring in an ice bath. The mixture was allowed to react for 1 hour in an ice bath. 1,6-Dibromohexane (S24-2, 4.36 g, 18.0 mmol) was then added and allowed to react at room temperature overnight. 3 mL of water was added and stirred for 10 min. The reaction solution was poured into 60 mL of water and extracted twice with dichloromethane (30 mL x 2). The organic phases were combined and backwashed once with saturated NaCl. The organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to afford the small molecule intermediate S24-3 (3.50 g).

[0581] Step b: Dissolve S24-3 (2.85 g, 6.6 mmol) in 50 mL of DMF, add S10-4 (0.82 g, 3.0 mmol) containing a TBS-protected hydroxyl group and K2CO3 (1.09 g, 7.9 mmol), and stir at room temperature overnight. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2). The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 25 mL of tetrahydrofuran is added, followed by 25 mL of a 1 M TBAF solution in tetrahydrofuran. The reaction is allowed to react overnight to remove the TBS protection. After completion of the reaction, concentrate, extract, and combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate to obtain the crude product of compound E24-1. The crude product is purified by silica gel column chromatography to obtain E24-1 (2.26 g). The main data of the H-NMR spectrum of E24-1 are as follows: 1 H NMR (400MHz, CDCl3) δ: 3.80-3.41(m,13H), 2.72-2.30(m,12H), 1.82-1.22(m,74H), 0.86(t,12H). MS(ESI):m / z=865.91([M+H] + ).

[0582]

[0583] Example 25: Cationic lipid (E25-1)

[0584]

[0585] Corresponding to the general formula (1), in E25-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 and L2 are both ester groups (-OC(=O)-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 818 Da.

[0586] The preparation process is as follows:

[0587] S3-3 (2.76 g, 6.6 mmol) was dissolved in 50 mL of DMF, and 1-amino-3-(1-imidazolyl)-2-propanol (S25-1, 0.77 g, 3.0 mmol) and K2CO3 (1.09 g, 7.9 mmol) were added. The mixture was stirred at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 25 mL of tetrahydrofuran was added, followed by 25 mL of 1 M TBAF solution in tetrahydrofuran. The reaction was allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to yield E25-1 (1.99 g). The main data of the H NMR spectrum of E25-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:7.61-6.88(m,3H),4.20-4.14(m,2H),4.09-4.03(m,5H),2.68- 2.63(m,2H),2.49-2.42(m,4H),2.36-2.25(m,2H),1.70-1.21(m,64H),0.87(t,12H). MS(ESI):m / z=818.74([M+H] + ).

[0588]

[0589] Example 26: Cationic lipid (E26-1)

[0590]

[0591] Corresponding to the general formula (1), in E26-1, R1 and R2 are B1 and B2 are both heptylene, L1 and L2 are both ester groups (-C(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 902 Da.

[0592] The preparation process is as follows:

[0593] Step a: Under argon atmosphere, DCC (3.09 g, 15.0 mmol) was added to a round-bottom flask containing 8-bromooctanoic acid (S5-4, 2.22 g, 10.0 mmol), 8-heptadecanol (S26-1, 2.82 g, 11.0 mmol), and DMAP (0.31 g, 2.5 mmol) dissolved in dichloromethane (50 mL) in an argon atmosphere. The mixture was reacted at room temperature for 16 h. After the reaction, the precipitate was removed by filtration. The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography to afford 8-bromooctanoic acid-1-heptyldecyl ester (S26-2, 3.80 g).

[0594] Step b: Dissolve S26-2 (3.04 g, 6.6 mmol) in 50 mL of DMF, add 2-amino-1-[2-(1-methylimidazolyl)]ethanol (S26-3, 0.77 g, 3.0 mmol) and KCO (1.09 g, 7.9 mmol), and stir at room temperature overnight. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2). The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Then, 25 mL of tetrahydrofuran is added, followed by 25 mL of 1 M TBAF solution in tetrahydrofuran. The reaction is allowed to react overnight to remove the TBS protection. After completion of the reaction, concentrate, extract, and combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate. The crude product is purified by silica gel column chromatography to yield E26-1 (2.19 g). The main data of the H-NMR spectrum of E26-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:6.94-6.91(m,1H),6.88-6.84(m,1H),4.90-4.84(m,2H),4.82-4.78( m,1H),3.77(s,3H),2.90-2.75(m,2H),2.53-2.22(m,8H),1.75-1.22(m,76H),0.87(t,12H). MS(ESI):m / z=902.82([M+H] + ).

[0595]

[0596] Example 27: Cationic lipid (E27-1)

[0597]

[0598] Corresponding to the general formula (1), in E27-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 is an ester group (-C(=O)O-), L2 is a carbonate group (-OC(=O)O-), and L3 (R3) is Wherein R3 is -(C=O)OH, R4 is The total molecular weight is approximately 850 Da.

[0599] The preparation process is as follows:

[0600] Step a: Dissolve S6-2 (4.69 g, 10.8 mmol) in 60 mL of DMF, add 3-(2-tetrazolyl)-L-alanine (S27-1, 1.41 g, 9.0 mmol) and K2CO3 (1.79 g, 13.0 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 60 mL of dichloromethane. Wash with 10% citric acid (30 mL x 2) and brine (30 mL x 2), sequentially. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S27-2 (3.59 g).

[0601] Step b: Compound S27-2 (3.07 g, 6.0 mmol) was dissolved in dry THF (50 mL). NaH (60%, 2.40 g, 60.0 mmol) was slowly added under ice-cooling and allowed to react for 1 hour. After the reaction, compound S4-3 (3.01 g, 7.2 mmol) was added and stirred under ice-cooling for 1 hour. The reaction mixture was then slowly returned to room temperature and allowed to react overnight. After the reaction, the reaction mixture was placed under ice-cooling and quenched by the slow addition of 2 mL of water. After stirring for 30 minutes, water (50 mL) was added and stirred. The mixture was then extracted twice with dichloromethane (25 mL x 2). The organic phases were combined and backwashed once with saturated sodium chloride (50 mL). The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to yield the crude product of compound E27-1. The product was purified by column chromatography, concentrated, and pumped to dryness to obtain cationic lipid E27-1 (4.13 g). The main data of the H-NMR spectrum of E27-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:8.40(s,1H),4.98-4.75(m,3H),4.21-4.09(m,3H),3.50-3. 45(m,1H),2.51-2.42(m,4H),2.38-2.32(t,2H),1.72-1.24(m,64H),0.89(t,12H). MS(ESI):m / z=850.84([M+H] + ).

[0602]

[0603] Example 28: Cationic lipid (E28-1)

[0604]

[0605] Corresponding to the general formula (1), in E28-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 and L2 are both carbonate groups (-OC(=O)O-), and -L3(R3- is Wherein R3 is -(C=O)OH, R4 is The total molecular weight is approximately 864 Da.

[0606] The preparation process is as follows:

[0607] S6-2 (2.86 g, 6.6 mmol) was dissolved in 50 mL of DMF, and 3-imidazolyl-L-alanine (S28-1, 0.47 g, 3.0 mmol) and K2CO3 (1.09 g, 7.9 mmol) were added. The mixture was stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain E28-1 (2.10 g). The main data of the H NMR spectrum of E28-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:7.63-6.89(m,3H),4.61-4.40(m,2H),4.18-4.06(m,6 H),3.49-3.44(m,1H),2.56-2.47(m,4H),1.72-1.22(m,64H),0.88(t,12H). MS(ESI):m / z=864.80([M+H] + ).

[0608]

[0609] Example 29: Cationic lipid (E29-1)

[0610]

[0611] Corresponding to the general formula (1), in E29-1, R1 is undecyl, R2 is B1 is pentylene, B2 is heptylene, L1 and L2 are both ester groups (-C(=O)O-), -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 779 Da.

[0612] The preparation process is as follows:

[0613] Step a: Under argon atmosphere, DCC (3.71 g, 18.0 mmol) was added to a round-bottom flask containing S5-4 (2.66 g, 12.0 mmol), S19-1 (3.01 g, 13.2 mmol), and DMAP (0.37 g, 3.0 mmol) in dichloromethane (50 mL). The mixture was allowed to react at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration. The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography to afford S29-1 (4.28 g).

[0614] Step b: Dissolve S29-1 (4.15 g, 9.6 mmol) in 60 mL of DMF, add 1-(3-amino-2-hydroxypropyl)pyrrolidin-2-one (S29-2, 2.18 g, 8.0 mmol) containing a TBS-protected hydroxyl group and K2CO3 (1.59 g, 11.5 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 60 mL of dichloromethane. Wash with 10% citric acid (30 mL x 2) and brine (30 mL x 2), sequentially. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S29-3 (4.15 g).

[0615] Step c: Dissolve compound S29-3 (3.75 g, 6.0 mmol) in dry THF (50 mL). Slowly add NaH (60%, 2.40 g, 60.0 mmol) under ice-cooling and react for 1 hour. After the reaction, add S2-6 (2.51 g, 7.2 mmol) and stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (50 mL) and stir to mix. Extract the mixture twice with dichloromethane (25 mL x 2). The organic phases are collected, combined, and backwashed once with saturated sodium chloride (50 mL). The organic phase is concentrated under reduced pressure, and 25 mL of tetrahydrofuran is added, followed by 25 mL of 1 M TBAF solution in tetrahydrofuran. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction was complete, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to yield the crude product of compound E29-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump afforded cationic lipid E29-1 (3.65 g). The main H NMR spectrum data for E29-1 are as follows: 1H NMR(400MHz, CDCl3)δ:4.90-4.85(m,1H),4.06(t,2H),3.96-3.87(m,1H),3.67-3.52(m,4H) ,2.64-2.58(m,2H),2.52-2.26(m,10H),2.09-1.94(m,2H),1.74-1.23(m,58H),0.89(t,9H). MS(ESI):m / z=779.73([M+H] + ).

[0616]

[0617] Example 30: Cationic lipid (E30-1)

[0618]

[0619] Corresponding to the general formula (1), in E30-1, R1 is undecyl, R2 is B1 is a pentylene group, B2 is a heptylene group, L1 is an ester group (-C(=O)O-), L2 is a carbonate group (-OC(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 856 Da.

[0620] The preparation process is as follows:

[0621] Step a: Under nitrogen, 7-bromoheptyl-4-nitrophenyl carbonate (S30-1, 10.77 g, 30.0 mmol, obtained by reacting p-nitrophenyl chloroformate and 7-bromo-n-heptanol) was dissolved in DCM (400 mL). S20-1 (30.72 g, 120.0 mmol) was added dropwise with stirring at room temperature. Pyridine (3.02 mL, 37.5 mmol) was then slowly added dropwise over 10 min, followed by the addition of DMAP (0.73 g, 6.0 mmol) all at once. The reaction was stirred at room temperature for 16 h. After completion of the reaction, the mixture was extracted twice with DCM and water. The organic phases were combined, washed with brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by silica gel column chromatography. The target eluate was collected and concentrated to afford S30-2 (3.71 g).

[0622] Step b: Dissolve S30-2 (3.43 g, 7.2 mmol) in 50 mL of DMF, add 1-amino-3-(1-benzimidazolyl)-2-propanol (S30-3, 1.83 g, 6.0 mmol) containing a TBS-protected hydroxyl group and KCO (1.19 g, 8.6 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2), sequentially. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S30-4 (3.30 g).

[0623] Step c: Dissolve compound S30-4 (2.81 g, 4.0 mmol) in dry THF (50 mL). Slowly add NaH (60%, 1.60 g, 40.0 mmol) under ice-cooling and react for 1 hour. After the reaction, add compound S2-6 (1.67 g, 4.8 mmol) and stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (50 mL) and stir to mix. Extract the mixture twice with dichloromethane (25 mL x 2). The organic phases are collected, combined, and backwashed once with saturated sodium chloride (50 mL). The organic phase is concentrated under reduced pressure, and 25 mL of tetrahydrofuran is added, followed by 25 mL of 1 M TBAF solution in tetrahydrofuran. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction was complete, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to yield the crude product of compound E30-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump afforded cationic lipid E30-1 (2.57 g). The main H NMR spectrum data for E30-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:8.20(s,1H),7.65(d,1H),7.60(d,1H),7.23-7.20(m,2H),4.45-4.39(m,1H),4 .36-3.32(m,2H),4.21-4.08(m,3H),4.07(t,2H),2.63-2.23(m,8H),1.70-1.21(m,62H),0.87(t,9H). MS(ESI):m / z=856.83([M+H] + ).

[0624]

[0625] Example 31: Cationic lipid (E31-1)

[0626]

[0627] Corresponding to the general formula (1), in E31-1, R1 is 8,11-heptadecadienyl, and R2 is B1 is a butylene group, B2 is a heptylene group, L1 is an ester group (-OC(=O)-), L2 is an ester group (-C(=O)O-), -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 853 Da.

[0628] The preparation process is as follows:

[0629] Step a: Dissolve S2-4 (4.42 g, 9.6 mmol) in 60 mL of DMF, add S19-3 (2.01 g, 8.0 mmol) containing a TBS-protected hydroxyl group and K2CO3 (1.59 g, 11.5 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 60 mL of dichloromethane. Wash with 10% citric acid (30 mL x 2) and brine (30 mL x 2), sequentially. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S31-1 (4.41 g).

[0630] Step b: Dissolve the above compound S31-1 (3.80 g, 6.0 mmol) in dry THF (60 mL). Slowly add NaH (60%, 2.40 g, 60.0 mmol) under ice-cooling and react for 1 hour. After the reaction, add S10-3 (2.98 g, 7.2 mmol) and stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (60 mL) and stir to mix. Extract the mixture twice with dichloromethane (30 mL x 2). The organic phases are collected, combined, and backwashed once with saturated sodium chloride (60 mL). The organic phase is concentrated under reduced pressure, followed by the addition of 30 mL of tetrahydrofuran and 30 mL of 1 M TBAF solution in tetrahydrofuran. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction was complete, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to yield the crude product of compound E31-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump afforded cationic lipid E31-1 (4.04 g). The main H NMR spectrum data for E31-1 are as follows: 1H NMR(400MHz, CDCl3)δ:8.42(d,2H),7.21(d,2H),5.39-5.34(m,4H),4.86-4.81(m,1H),4.73-4.66(m,1 H),4.05(t,2H),2.73(t,2H),2.57-2.23(m,10H),2.04-1.96(m,4H),1.72-1.23(m,58H),0.87(t,9H). MS(ESI):m / z=853.80([M+H] + ).

[0631]

[0632] Example 32: Cationic lipid (E32-1)

[0633]

[0634] Corresponding to the general formula (1), in E32-1, R1 and R2 are both 7-phenylheptyl, B1 and B2 are both butylene, L1 and L2 are both ester groups (-OC(=O)-), and -L3(R3)- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 709 Da.

[0635] The preparation process is as follows:

[0636] Step a: Under argon, to a round-bottom flask containing 8-phenyloctanoic acid (S32-1, 2.20 g, 10.0 mmol), 4-bromobutanol (S10-2, 1.67 g, 11.0 mmol), and DMAP (0.31 g, 2.5 mmol) in dichloromethane (40 mL) was added DCC (3.09 g, 15.0 mmol). The mixture was allowed to react at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration. The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography to afford S32-2 (3.08 g).

[0637] Step b: Dissolve S32-2 (2.34 g, 6.6 mmol) in 40 mL of DMF, add S10-4 (0.82 g, 3.0 mmol) containing a TBS-protected hydroxyl group and K2CO3 (1.09 g, 7.9 mmol), and stir at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 40 mL of dichloromethane. After washing with 10% citric acid (20 mL*2) and brine (20 mL*2), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 20 mL of tetrahydrofuran was added, followed by 20 mL of TBAF tetrahydrofuran solution (1 M), and the reaction was allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography to yield E32-1 (1.75 g). The main data of the H NMR spectrum of E32-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:7.24-7.19(m,10H),3.90-3.85(m,1H),3.74-3.68(m,4H),4.05(t,4H),2.68-2.23(m,20H),1.69-1.24(m,28H). MS (ESI): m / z=709.66 Da([M+H]+).

[0638]

[0639] Example 33: Cationic lipid (E33-1)

[0640]

[0641] Corresponding to the general formula (1), in E33-1, R1 is undecyl, R2 is B1 is a pentylene group, B2 is a heptylene group, L1 and L2 are both carbonate groups (-OC(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 985 Da.

[0642] The preparation process is as follows:

[0643] Step a: Dissolve S8-3 (2.62 g, 7.2 mmol, obtained by the reaction of 5-bromopentyl-4-nitrophenyl carbonate and 1-undecanol) in 50 mL of DMF. Add 2-(3-amino-2-hydroxypropyl)-2,3-dihydro-1H-isoindole-1,3-dione (S33-1, 2.00 g, 6.0 mmol) containing a TBS-protected hydroxyl group and KCO (1.19 g, 8.6 mmol). Stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2). The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S33-2 (2.89 g).

[0644] Step b: Dissolve compound S33-2 (2.47 g, 4.0 mmol) in dry THF (40 mL). Slowly add NaH (60%, 1.60 g, 40.0 mmol) under ice-cooling. Incubate the mixture for 1 hour. Add compound S33-3 (2.69 g, 4.8 mmol, obtained by reacting 7-bromoheptyl-4-nitrophenyl carbonate and 3-decyltridecanol). Stir and react for 1 hour under ice-cooling. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction is complete, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (40 mL) and stir to mix. Extract the mixture twice with dichloromethane (20 mL x 2). Combine the organic phases and backwash once with saturated sodium chloride (40 mL). The organic phase was concentrated under reduced pressure and 20 mL of tetrahydrofuran was added, followed by 20 mL of a 1 M TBAF tetrahydrofuran solution. The reaction was allowed to proceed overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to yield the crude product of compound E33-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump afforded the cationic lipid E33-1 (2.99 g). The key H NMR spectrum data for E33-1 are as follows: 1 HNMR(400MHz, CDCl3)δ:7.90-7.65(m,4H),4.20-3.98(m,9H),3.66-3.59(m,2H),2.61-2.44(m,6H),1.78-1.24(m,73H),0.90(t,9H). MS(ESI):m / z=985.79([M+H] + ).

[0645]

[0646] Example 34: Cationic lipid (E34-1)

[0647]

[0648] Corresponding to the general formula (1), in E34-1, R1 is tridecyl, R2 is B1 is a pentylene group, B2 is a heptylene group, L1 is an ester group (-C(=O)O-), L2 is a carbonate group (-OC(=O)O-), and -L3(R3- is Wherein R3 is -(C=O)OH, R4 is The total molecular weight is approximately 859 Da.

[0649] The preparation process is as follows:

[0650] Step a: Dissolve S30-2 (3.43 g, 7.2 mmol) in 50 mL of DMF, add 3-(3-pyridyl)-L-alanine (S34-1, 1.00 g, 6.0 mmol) and K2CO3 (1.19 g, 8.6 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and then brine (25 mL x 2). The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S34-2 (2.66 g).

[0651] Step b: Dissolve compound S34-2 (2.25 g, 4.0 mmol) in dry THF (40 mL). Slowly add NaH (60%, 1.60 g, 40.0 mmol) under ice-cooling. Incubate for 1 hour. Add tridecyl 6-bromohexanoate (S34-3, 1.80 g, 7.2 mmol, obtained by condensation of 6-bromohexanoic acid and 1-tridecanol). Stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction is complete, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (400 mL) and stir to mix. Extract the mixture twice with dichloromethane (20 mL x 2). Combine the organic phases, backwash once with saturated sodium chloride (40 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate to yield crude compound E34-1. The cationic lipid E34-1 (2.56 g) was obtained by column chromatography, concentration, and pumping to dryness. The main data of the H NMR spectrum of E34-1 are as follows: 1H NMR(400MHz, CDCl3)δ:8.48-7.14(m,4H),4.22-4.06(m,3H),4.09(t,2H),3.73-3. 69(m,1H),3.08-3.03(m,2H),2.54-2.23(m,6H),1.70-1.20(m,66H),0.87(t,9H). MS(ESI):m / z=859.75([M+H] + ).

[0652]

[0653] Example 35: Cationic lipid (E35-1)

[0654]

[0655] Corresponding to the general formula (1), in E35-1, R1 is heptadecyl, R2 is B1 is a connecting bond, B2 is a hexamethylene group, L1 is a carbonyl group (-C(=O)-), L2 is an ester group (-OC(=O)-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 771 Da.

[0656] The preparation process is as follows:

[0657] Step a: Under argon, to a round-bottom flask containing isostearic acid (S35-1, 5.68 g, 20.0 mmol), 6-bromohexanol (S35-6, 3.96 g, 22.0 mmol), and DMAP (0.61 g, 5.0 mmol) in dichloromethane (70 mL) was added DCC (6.18 g, 30.0 mmol). The mixture was allowed to react at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration. The filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography to afford S35-2 (5.98 g).

[0658] Step b: Dissolve S35-2 (5.35 g, 12.0 mmol) in 60 mL of DMF, add 2-amino-1-(2-pyridyl)ethanol (S35-3, 2.52 g, 10.0 mmol) containing a TBS-protected hydroxyl group and KCO (1.99 g, 14.4 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 60 mL of dichloromethane. Wash with 10% citric acid (30 mL x 2) and then brine (30 mL x 2). The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S35-4 (4.85 g).

[0659] Step c: Compound S35-4 (4.64 g, 7.5 mmol), stearic acid (S35-5, 1.42 g, 5.0 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.44 g, 7.5 mmol), 1-hydroxybenzotriazole (HOBt, 0.88 g, 6.5 mmol), and triethanolamine (TEA, 1.01 g, 10.0 mmol) were dissolved in dichloromethane (60 mL) and stirred overnight at room temperature. After completion of the reaction, the reaction solution was backwashed twice with 0.1 mol / L aqueous HCl (10% NaCl) (60 mL x 2) and once with saturated NaCl (60 mL). The organic phase was concentrated under reduced pressure, and 30 mL of tetrahydrofuran was added, followed by 30 mL of 1 M TBAF solution in tetrahydrofuran. The reaction was allowed to react overnight to remove the TBS protection. After the reaction is complete, the organic phases are concentrated, extracted, and combined, dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated to obtain a crude product. Column chromatography is then used to purify the target compound E35-1 (2.97 g). The main H NMR spectrum data of E35-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:8.56-8.52(m,1H),7.73-7.65(m,1H),7.55-7.51(m,1H),7.20-7.15(m,1H),4.79-7.76(m,1H) ,4.10-4.05(t,2H),3.71-3.59(m,2H),3.16(t,2H),2.35-2.28(m,1H),2.23(t,2H),1.70-1.19(m,66H),0.88(t,9H). MS(ESI):m / z=771.74([M+H] + ).

[0660]

[0661] Example 36: Cationic lipid (E36-1)

[0662]

[0663] Corresponding to the general formula (1), in E36-1, R1 is undecyl, R2 is B1 is a pentylene group, B2 is a heptylene group, L1 is a carbonate group (-OC(=O)O-), L2 is an ester group (-C(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 825 Da.

[0664] The preparation process is as follows:

[0665] Step a: Dissolve S8-3 (2.62 g, 7.2 mmol) in 50 mL of DMF, add S10-4 (1.64 g, 6.0 mmol) containing a TBS-protected hydroxyl group and K2CO3 (1.19 g, 8.6 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2), sequentially. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S36-1 (2.73 g).

[0666] Step b: Dissolve compound S36-1 (2.23 g, 4.0 mmol) in dry THF (30 mL). Slowly add NaH (60%, 1.60 g, 40.0 mmol) under ice-cooling and allow to react for 1 hour. After completion of the reaction, add compound S2-4 (2.21 g, 4.8 mmol) and stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After completion of the reaction, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (30 mL) and stir to mix. Extract the mixture twice with dichloromethane (15 mL x 2). The organic phases are combined and backwashed once with saturated sodium chloride (30 mL). The organic phase is concentrated under reduced pressure, followed by the addition of 15 mL of tetrahydrofuran and 15 mL of 1 M TBAF solution in tetrahydrofuran. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction is complete, the organic phases are concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain the crude product of compound E36-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump affords cationic lipid E36-1 (2.61 g). The main H NMR spectrum data for E36-1 are as follows: 1H NMR (400 MHz, CDCl3) δ: 4.89-4.85 (m, 1H), 4.22-4.03 (m, 4H), 3.93-3.85 (m, 1H), 3.73-3.67 (m, 4H), 2.69-2.22 (m, 14H), 1.76-1.22 (m, 62H), 0.87 (t, 9H). MS (ESI): m / z = 825.78 ([M+H] + ).

[0667]

[0668] Example 37: Cationic lipid (E37-1)

[0669]

[0670] Corresponding to the general formula (1), in E37-1, R1 is heptadecyl, R2 is B1 is a connecting bond, B2 is a hexamethylene group, L1 is a carbonyl group (-C(=O)-), L2 is an ester group (-OC(=O)-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 793 Da.

[0671] The preparation process is as follows:

[0672] Step a: Dissolve S35-2 (5.35 g, 12.0 mmol) in 60 mL of DMF, add S10-4 (2.74 g, 10.0 mmol) containing a TBS-protected hydroxyl group and K2CO3 (1.99 g, 14.4 mmol), and stir at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 60 mL of dichloromethane. After washing with 10% citric acid (30 mL x 2) and brine (30 mL x 2), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S37-1 (5.06 g).

[0673] Step b: Compound S37-1 (4.81 g, 7.5 mmol), S35-5 (1.42 g, 5.0 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.44 g, 7.5 mmol), 1-hydroxybenzotriazole (HOBt, 0.88 g, 6.5 mmol), and TEA (1.01 g, 10.0 mmol) were dissolved in dichloromethane (80 mL) and stirred overnight at room temperature. After completion of the reaction, the reaction solution was backwashed twice with 0.1 mol / L aqueous HCl (10% NaCl) (80 mL x 2) and once with saturated NaCl (80 mL). The organic phase was concentrated under reduced pressure, and 40 mL of tetrahydrofuran was added, followed by 40 mL of 1 M TBAF solution in tetrahydrofuran. The reaction was allowed to react overnight to remove the TBS protection. After the reaction is complete, the organic phases are concentrated, extracted, and combined, dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated to obtain a crude product. The crude product is purified by column chromatography, concentrated, and pumped dry to obtain the target compound E37-1 (4.16 g). The main H NMR spectrum data of E37-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:4.05(t,2H),3.86-3.80(m,1H),3.69-3.65(m,4H),3.63 -3.20(m,2H),3.14(t,2H),2.66-2.19(m,9H),1.68-1.18(m,66H),0.87(t,9H). MS(ESI):m / z=793.75([M+H] + ).

[0674]

[0675] Example 38: Cationic lipid (E38-1)

[0676]

[0677] Corresponding to the general formula (1), in E38-1, R1 is heptadecyl, R2 is B1 is a connecting bond, B2 is a hexamethylene group, L1 is a carbonyl group (-C(=O)-), L2 is -O-, and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 757 Da.

[0678] The preparation process is as follows:

[0679] Step a: Dissolve S24-3 (3.11 g, 7.2 mmol) in 50 mL of DMF, add S7-3 (1.51 g, 6.0 mmol) containing a TBS-protected hydroxyl group and K2CO3 (1.19 g, 8.6 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2), sequentially. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S38-1 (2.90 g).

[0680] Step b: Compound S38-1 (2.72 g, 4.5 mmol), S35-5 (0.85 g, 3.0 mmol), EDCI (0.86 g, 4.5 mmol), HOBt (0.53 g, 3.9 mmol), and TEA (0.61 g, 6.0 mmol) were dissolved in dichloromethane (50 mL) and stirred overnight at room temperature. After completion of the reaction, the reaction solution was backwashed twice with 0.1 mol / L aqueous HCl (10% NaCl) (50 mL x 2) and once with saturated NaCl (50 mL). The organic phase was concentrated under reduced pressure, and 25 mL of tetrahydrofuran was added, followed by 25 mL of 1 M TBAF solution in tetrahydrofuran. The reaction was allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous magnesium sulfate, filtered, and the filtrate concentrated to obtain the crude product. Purify by column chromatography, concentrate, and pump dry to obtain the target compound E38-1 (1.75 g). The main data of the H NMR spectrum of E38-1 are as follows: 1H NMR(400MHz, CDCl3)δ:8.44(d,2H),7.24(d,2H),4.74-4.69(m,1H),3.71-3.41(m,6H),3.18(t,2H),2.24(t,2H),1.55-1.21(m,67H),0.88(t,9H). MS(ESI):m / z=757.77([M+H] + ).

[0681]

[0682] Example 39: Cationic lipid (E39-1)

[0683]

[0684] Corresponding to the general formula (1), in E39-1, R1 is heptadecyl, R2 is B1 is a connecting bond, B2 is a hexamethylene group, L1 is a carbonyl group (-C(=O)-), L2 is -O-, and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 763 Da.

[0685] The preparation process is as follows:

[0686] Step a: Dissolve S24-3 (3.11 g, 7.2 mmol) in 50 mL of DMF, add 1-amino-3-(1-pyrrolidinyl)-2-propanol (S39-1, 1.55 g, 6.0 mmol) containing a TBS-protected hydroxyl group and KCO (1.19 g, 8.6 mmol), and stir overnight at room temperature. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2), sequentially. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S39-2 (3.01 g).

[0687] Step b: Compound S39-2 (2.75 g, 4.5 mmol), S35-5 (0.85 g, 3.0 mmol), EDCI (0.86 g, 4.5 mmol), HOBt (0.53 g, 3.9 mmol), and TEA (0.61 g, 6.0 mmol) were dissolved in dichloromethane (50 mL) and stirred at room temperature overnight. After completion, the reaction solution was backwashed twice with 0.1 mol / L aqueous HCl (10% NaCl) (50 mL x 2) and once with saturated NaCl (50 mL). The organic phase was retained and concentrated under reduced pressure. 25 mL of tetrahydrofuran was added, followed by 25 mL of 1 M TBAF solution in tetrahydrofuran. The reaction was allowed to react overnight to remove the TBS protection. After completion, the reaction was concentrated, extracted, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, and concentrated to yield crude E39-1. Purify by column chromatography, concentrate, and pump dry to obtain the target compound E39-1 (1.79 g). The main data of the H NMR spectrum of E39-1 are as follows: 1 H NMR (400MHz, CDCl3) δ: 3.82-3.21(m,7H), 3.19(t,2H), 2.45-2.38(m,2H), 2.25-2.19(m,6H), 1.63-1.20(m,71H), 0.87(t,9H). MS(ESI):m / z=763.81([M+H] + ).

[0688]

[0689] Example 40: Cationic lipid (E40-1)

[0690]

[0691] Corresponding to the general formula (1), in E40-1, R1 is heptadecyl, R2 is B1 is a connecting bond, B2 is a hexamethylene group, L1 is a carbonyl group (-C(=O)-), L2 is a carbonate group (-OC(=O)O-), and -L3(R3- is Wherein R3 is -OH, R4 is The total molecular weight is approximately 794 Da.

[0692] The preparation process is as follows:

[0693] Compound S22-1 (2.89 g, 4.5 mmol), S35-5 (0.85 g, 3.0 mmol), EDCI (0.86 g, 4.5 mmol), HOBt (0.53 g, 3.9 mmol), and TEA (0.61 g, 6.0 mmol) were sequentially dissolved in dichloromethane (50 mL) and stirred at room temperature overnight. After completion, the reaction solution was backwashed twice with 0.1 mol / L aqueous HCl (10% NaCl) (50 mL x 2) and once with saturated NaCl (50 mL). The organic phase was concentrated under reduced pressure, followed by the addition of 25 mL of tetrahydrofuran and 25 mL of a 1 M solution of TBAF in tetrahydrofuran. The reaction was allowed to proceed overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous magnesium sulfate, filtered, and the filtrate concentrated to yield the crude product. Purify by column chromatography, concentrate, and pump dry to obtain the target compound E40-1 (2.05 g). The main data of the H NMR spectrum of E40-1 are as follows: 1 H NMR (400MHz, CDCl3) δ: 4.24-4.09 (m, 3H), 3.86-3.79 (m, 1H), 3.64-3.22 (m, 2H), 3.13 (t, 2H), 2.68-2.18 (m, 15H), 1.70-1.19 (m, 62H), 0.86 (t, 9H). MS(ESI):m / z=794.77([M+H] + ).

[0694]

[0695] Example 41: Cationic lipid (E41-1)

[0696]

[0697] Corresponding to the general formula (1), in E41-1, R1 is R2 is B1 is a hexamethylene group, B2 is a hexamethylene group substituted with -OH, L1 is an ester group (-OC(=O)-), L2 is an ester group (-C(=O)O-), and -L3(R3- is Where R3 is G1 is -OCH2CH<, k is 2, the two F1 are -OH and -C(=O)OCH3, R4 is The total molecular weight is approximately 1012 Da.

[0698] The preparation process is as follows:

[0699] Step a: Add S2-3 (3.40 g, 18.0 mmol) to a dry 500 mL round-bottom flask, add 60 mL of dichloromethane solution, add di-tert-butyl dicarbonate (4.71 g, 21.6 mmol), react at room temperature overnight, add saturated sodium bicarbonate solution, extract with dichloromethane (20 mL*3), combine the organic phases, wash with saturated brine, dry, filter, concentrate, and recrystallize to obtain tert-butyloxycarbonyl (Boc)-protected amine derivative S41-1 (4.42 g).

[0700] Step b: To a sealed, anhydrous, and oxygen-free reactor, add 150 mL of tetrahydrofuran and an excess of potassium diphenylmethyl (10.30 g, 50.0 mmol), followed by compound S41-1 (3.76 g, 13.0 mmol) and compound S41-2 (5.04 g, 13.0 mmol). S41-2 is methyl 2,3-dihydroxypropionate with one hydroxyl group substituted with a p-toluenesulfonate group and the other hydroxyl group protected with TBS. After reacting at 30°C for 12 hours, the reactor was opened, concentrated, washed, and dissolved in dichloromethane. Trifluoroacetic acid (TFA) was added to 0.1 M. After reacting for 4 hours, the pH was adjusted to neutral. The reaction solution was concentrated, purified water was added, and the extract was extracted with dichloromethane. The extract was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and recrystallized to obtain intermediate S41-3 (3.56 g) with exposed amino groups.

[0701] Step c: Dissolve S20-4 (3.21 g, 8.4 mmol) in 50 mL of ethanol, add S41-3 (2.84 g, 7.0 mmol), and stir at 50°C for 4 hours. After the reaction, dilute with 50 mL of dichloromethane, backwash with 100 mL of saturated sodium chloride solution, separate the organic phase, and dry it over anhydrous sodium sulfate. Filter, concentrate, and purify by silica gel column chromatography to afford S41-4 (3.64 g).

[0702] Step d: The above compound S41-4 (3.15 g, 4.0 mmol) and imidazole (0.45 g, 6.6 mmol) were dissolved in 50 mL of dichloromethane, and tert-butyldimethylsilyl chloride (0.84 g, 5.6 mmol) was added under stirring in an ice bath. After reacting for 2 hours, the mixture was filtered and rinsed with 50 mL of dichloromethane. The filtrates were combined, dried over anhydrous magnesium sulfate, and concentrated to obtain the intermediate S41-5 (3.54 g) containing two TBS-protected hydroxyl groups.

[0703] Step e: Dissolve compound S41-5 (2.71 g, 3.0 mmol) in dry THF (50 mL). Slowly add NaH (60%, 1.20 g, 30.0 mmol) under ice-cooling and react for 1 hour. After the reaction, add S3-3 (1.50 g, 3.6 mmol) and stir under ice-cooling for 1 hour. The reaction mixture is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (50 mL) and stir to mix. Extract the mixture twice with dichloromethane (25 mL x 2). The organic phases are combined and backwashed once with saturated sodium chloride (50 mL). The organic phase is concentrated under reduced pressure, and 25 mL of tetrahydrofuran is added, followed by 25 mL of 1 M TBAF solution in tetrahydrofuran. The reaction mixture is allowed to react overnight to remove the TBS protection. After the reaction was complete, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to yield the crude product of compound E41-1. Purification by column chromatography, concentration, and pumping to dryness with an oil pump afforded cationic lipid E41-1 (2.46 g). The main H NMR spectrum data for E41-1 are as follows: 1 H NMR(400MHz, CDCl3)δ:4.82-4.75(m,1H),4.15-4.10(m,1H),4.08(t,2H),3.86-3.80(m,1 H),3.68(s,3H),3.65-3.47(m,3H),2.70-2.19(m,24H),1.73-1.23(m,68H),0.87(t,12H). MS(ESI):m / z=1012.93([M+H] + ).

[0704]

[0705] Example 42: Cationic lipid (E42-1)

[0706]

[0707] Corresponding to the general formula (1), in E42-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 and L2 are both ester groups (-OC(=O)-), and -L3(R3- is Wherein R3 is -O(CH2)2OH, R4 is The total molecular weight is approximately 958 Da.

[0708] The preparation process is as follows:

[0709] Step a: To a sealed, anhydrous, and oxygen-free reactor, add 150 mL of tetrahydrofuran and an excess of potassium diphenylmethyl (10.30 g, 50.0 mmol). Compound S42-1 (2.73 g, 10.0 mmol) and compound S42-2 (2.88 g, 10.0 mmol) were then added. S42-2 is ethylene glycol with one hydroxyl group substituted with a p-toluenesulfonate (-OTs) group and the other hydroxyl group protected with EE. After reacting at 30°C for 12 hours, the reactor was opened and concentrated, washed, and purified by column chromatography to yield compound S8-1 (3.27 g), which has two protected hydroxyl groups and one protected amino group.

[0710] Step b: S8-1 (2.33 g, 6.0 mmol) was added to a dry and clean container, dissolved in dichloromethane, and TFA was added to 0.1 M. After reacting for 4 hours, the pH was adjusted to neutral, the reaction solution was concentrated, purified water was added, and the solution was extracted with dichloromethane. The extract was concentrated, precipitated, filtered, and dried to obtain the amino-exposed intermediate S42-3 (1.42 g).

[0711] Step c: Dissolve S42-4 (2.97 g, 6.6 mmol, obtained by condensation reaction of S5-3 and S4-1) in 50 mL of DMF, add S42-3 (0.87 g, 3.0 mmol) and K2CO3 (1.09 g, 7.9 mmol), and stir at room temperature overnight. After the reaction, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. The organic phase was washed sequentially with 10% citric acid (25 mL x 2) and brine (25 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and dissolved in methanol. 1 M hydrochloric acid was added to pH 3.5. After reacting for 4 hours, the mixture was concentrated, precipitated, filtered, recrystallized, and dried to obtain the crude product E42-1 with exposed hydroxyl groups. The crude product was purified by silica gel column chromatography to yield E42-1 (2.34 g). The key H NMR spectrum data of E42-1 are as follows: 1 H NMR (400MHz, CDCl3) δ: 5.04-4.96 (m, 2H), 3.89-3.83 (m, 1H), 3.71-3.33 (m, 20H), 2.74-2.24 (m, 23H), 1.77-1.16 (m, 48H), 0.85 (t, 12H). MS(ESI):m / z=958.80([M+H] + ).

[0712]

[0713] Example 43: Cationic lipid (E43-1)

[0714]

[0715] Corresponding to the general formula (1), in E43-1, R1 is undecyl, R2 is B1 is pentylene, B2 is heptylene, L1 and L2 are both ester groups (-C(=O)O-), -L3(R3- is Where R3 is R4 is The total molecular weight is approximately 861 Da.

[0716] The preparation process is as follows:

[0717] Step a: Dissolve S2-4 (3.31 g, 7.2 mmol) in 50 mL of DMF, add S11-3 (2.14 g, 6.0 mmol) and K2CO3 (1.19 g, 8.6 mmol), and stir at room temperature overnight. After completion of the reaction, concentrate the reaction mixture under reduced pressure and pour into 50 mL of dichloromethane. Wash with 10% citric acid (25 mL x 2) and brine (25 mL x 2), sequentially. The organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to afford S43-5 (3.63 g).

[0718] Step b: Dissolve the above compound S43-5 (2.95 g, 4.0 mmol) in dry THF (50 mL). Slowly add NaH (60%, 1.60 g, 40.0 mmol) under ice-cooling and react for 1 hour. After the reaction, add S2-6 (1.67 g, 4.8 mmol) and stir under ice-cooling for 1 hour. The reaction solution is then slowly returned to room temperature and allowed to react overnight. After the reaction is completed, place the reaction mixture under ice-cooling and slowly add 2 mL of water to quench the reaction. After stirring for 30 minutes, add water (50 mL) and stir to mix. Extract the mixture twice with dichloromethane (25 mL x 2). The organic phases are combined and backwashed once with saturated sodium chloride (50 mL). The organic phases are concentrated under reduced pressure, dissolved in methanol, and 1 M hydrochloric acid is added to pH 3.5. After reacting for 4 hours, the mixture is concentrated, the precipitate is filtered, recrystallized, and dried to obtain the crude product E43-1 with exposed hydroxyl groups. Cationic lipid E43-1 (2.65 g) was purified by column chromatography. The main data of E43-1 are as follows: 1H NMR (400 MHz, CDCl3) δ: 8.55 (d, 2H), 7.20 (d, 2H), 4.84-4.77 (m, 1H), 4.29-4.25 (m, 1H), 4.07 (t, 2H), 3.77-3.72 (m, 1H), 3.63-3.56 (m, 2H), 3.42-3.36 (m, 2H), 3.01-2.94 (m, 2H), 2.57-2.24 (m, 8H), 1.75-1.23 (m, 62H), 0.89 (t, 9H). MS (ESI): m / z = 861.76 ([M+H]+ ).

[0719]

[0720] Example 44: PEGylated lipid (E44-1)

[0721]

[0722] Corresponding to the general formula (2), in E44-1, R1 and R2 are both tetradecyl, B1 and B2 are both connecting bonds, L1 and L2 are both connecting bonds, and L3 is L A -(CH2) h - and h is 0, A1 is -OCH2CH2-, n1≈45, R3 is methyl, R5 is hexyl, and the total molecular weight is about 2.5 kDa.

[0723] The preparation process is as follows:

[0724] Step a: Into an anhydrous and oxygen-free sealed reaction vessel, 120 mL of tetrahydrofuran and an excess of diphenylmethyl potassium (10.30 g, 50.0 mmol) were added, followed by the addition of compound S44-2 (2.45 g, 10.0 mmol) and the sulfonate derivative of methoxypolyethylene glycol mPEG-CH2CH2OTs (S44-1, 12.96 g, 6.0 mmol, M n ≈2.2 kDa, n1≈45, PDI=1.03). After reacting at 30°C for 12 hours, the reactor was opened, concentrated, washed, and the Boc protection was removed with a TFA / DCM mixed solution (1:1 v / v). The product was washed with purified water and extracted with dichloromethane. The extract was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the PEGylated intermediate S44-3 (5.91 g).

[0725] Step b: S44-4 (1.21 g, 4.4 mmol) was dissolved in 30 mL of DMF, and S44-3 (4.28 g, 2.0 mmol, M n≈2.1 kDa, n1≈45, PDI=1.03) and K2CO3 (0.73 g, 5.3 mmol) were stirred at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 30 mL of dichloromethane. After washing with 10% citric acid (15 mL*2) and brine (15 mL*2), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to yield E44-1 (2.92 g). The key H NMR data for E44-1 are as follows: 1H NMR (400 MHz, CDCl3) δ: 3.85-3.45 (m, PEG), 3.37 (s, 3H), 3.31-3.26 (m, 1H), 2.67-2.60 (m, 2H), 2.50-2.41 (m, 4H), 1.68-1.19 (m, 58H), 0.88 (t, 9H). GPC analysis revealed a molecular weight of approximately 2.5 kDa, with a PDI of 1.03.

[0726]

[0727] 5.2. Lipid Pharmaceutical Compositions

[0728] Example 45: Preparation of LNP-mRNA Pharmaceutical Composition

[0729] In this example, LNP-mRNA pharmaceutical compositions containing Fluc-mRNA (LNP / Fluc-mRNA) were prepared. The neutral lipids contained therein were all DSPC, the sterol lipids contained therein were all cholesterol, the cationic lipids contained therein were the cationic lipids of the present invention, DLin-MC3-DMA (abbreviated as MC3), or the reference compounds d-1 to d-4, the PEGylated lipids contained therein were the PEGylated lipids of the present invention or PEG2k-DMG (abbreviated as DMG), and the nucleic acid drugs contained therein were all Fluc-mRNA; wherein the structures of d-1 to d-4 are as follows:

[0730]

[0731] The preparation method of the LNP-mRNA pharmaceutical composition is as follows:

[0732] Step (1): Cationic lipid, DSPC, cholesterol and PEGylated lipid (specifically as shown in Table 1 of Example 46) are dissolved in ethanol at a molar ratio of 50:10:38:1.5 to obtain an ethanol phase solution.

[0733] Step (2): Add Fluc-mRNA to 10-50 mM citrate buffer (pH = 4) to obtain an aqueous solution.

[0734] 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.

[0735] The above steps (1-3) were used to prepare LNP / Fluc-mRNA, except that the molar ratio of each lipid was changed to cationic lipid:DSPC:cholesterol:PEGylated lipid = 48:9:42:1.5, where E3-2 was used as the cationic lipid and E44-1 as the PEGylated lipid. Other conditions remained unchanged.

[0736] Example 46: Biological Activity Test of Lipid Pharmaceutical Composition

[0737] (1) Determination of nanoparticle size and nucleic acid complexing ability

[0738] Nucleic acid complexing ability assay: Gel permeation electrophoresis was used to investigate the nucleic acid complexing ability of LNP / Fluc-mRNA. 0.8 g of agarose was weighed and dissolved in 40 mL of TAE solution. The agarose particles were heated in a microwave oven to completely dissolve the solution. After cooling, 5 μL of the nucleic acid dye GelGreen was added to the cooled agarose gel. The gel was placed in a gel tank and allowed to dry naturally. A mixture of LNP / Fluc-mRNA and 2 μL of Loading Buffer was 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 from the LNP / Fluc-mRNA experimental groups (L-1 to L-43, L-3-2-d, PL-0, PL-3-2, PL-4, PL-10-2, PL-16, PL-17) and the LNP / Fluc-mRNA control group D-0, indicating that the LNPs of the present invention have excellent nucleic acid drug complexing ability.

[0739] Encapsulation efficiency determination: LNP / Fluc-mRNA was ultracentrifuged (4°C, 60,000 rpm, 1 hour). The concentration of unencapsulated Fluc-mRNA in the supernatant was measured using a nucleic acid quantifier. The encapsulation efficiency of the liposomes for Fluc-mRNA was calculated. The results are summarized in Table 1. The liposomes of the present invention exhibited high encapsulation efficiencies for nucleic acid drugs, with encapsulation efficiencies exceeding 80% in the experimental groups. Specifically, compared to E16-1, the polar head of d-1 lacks side chain hydroxyl groups, the polar head of d-2 lacks branched structures, and the polar head of d-3 lacks nitrogen heterocycles. Consequently, the encapsulation efficiency of the experimental group, L-16 (95%), was significantly greater than that of the control groups, D-1, D-2, and D-3 (81%, 91%, and 83%). E1-1 and d-4 also contain multiple ester bonds, and accordingly, the encapsulation efficiency of L-1 (87%) was greater than that of D-4 (81%). Compared with L3-2, L3-2-d contains the same types of lipids and has a higher encapsulation efficiency by adjusting the ratio of each lipid to a certain extent.

[0740] 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 particle size of LNP / Fluc-mRNA prepared using the cationic lipid of the present invention is in the range of about 70 nm to about 113 nm, which is a particle size that can achieve better efficacy.

[0741] Table 1: Particle size and encapsulation efficiency of LNP / Fluc-mRNA

[0742]

[0743]

[0744] (2) Serum stability evaluation

[0745] The above-mentioned LNP / Fluc-mRNA (control group D-0, D-1, D-2, D-3, D-4; experimental group L-1, L-3-2, L-4, L-10-2, L-16, L-17, PL-0, PL-3-2, PL-4, PL-10-2, PL-16, PL-17) was added to a culture medium containing 10% fetal bovine serum (FBS) and stirred at 37°C. Samples were taken at regular intervals to measure the particle size change of LNP / Fluc-mRNA. The serum stability of the nucleic acid drug preparation was analyzed by testing its particle size change. The experimental results show (Table 2) that within 7 days, the particle size of PL-3-2, PL-4, PL-16, and PL-10-2, which contain both the cationic lipids of the present invention and the PEGylated lipids of the present invention, changed minimally (0-3%). The particle size of the remaining experimental and control groups changed by 5-16%. Among them, the cationic lipids D-4, L-1, L-17, and PL-17, which contain carbonate groups or multiple ester groups, showed slightly greater changes in particle size. These results indicate that the cationic lipids of the present invention can be used to prepare lipid pharmaceutical compositions, and the resulting lipid nanoparticles can be sufficiently stable under physiological conditions, and the effect is even better when used in combination with the PEGylated lipids of the present invention.

[0746] Table 2: Stability evaluation of LNP / Fluc-mRNA

[0747]

[0748]

[0749] (3) Cytotoxicity evaluation

[0750] The cells used were Hela cells, and the method used was the CCK-8 kit to determine cell viability.

[0751] The commercial transfection reagent Lipofectamine 2000 (L2K) was used to prepare the L2K / Fluc-mRNA complex according to the method of Example 45.

[0752] Hela press 1×10 4Cells were seeded in 96-well plates at a ratio of 100 μL per well and divided into a control group (blank control), an L2K / Fluc-mRNA group (positive control), and an LNP / Fluc-mRNA group (experimental groups listed in Table 1). The cells were incubated at 37°C and 5% CO₂. After 24 hours of incubation, 3.3 μg / mL of L2K / Fluc-mRNA and 3.3 μg / mL of LNP / Fluc-mRNA were added to the positive control and experimental groups, respectively, and incubation continued at 37°C and 5% CO₂. After 24 hours of incubation, the 96-well plates were removed from the plate in the dark, the culture medium was aspirated, and 120 μL of diluted CCK-8 solution was added to each well. The cells were then incubated at 37°C and 5% CO₂ for 1-4 hours.

[0753] The sample was taken out and the absorbance of each well was measured at a wavelength of 450 nm using an enzyme-labeled instrument.

[0754] The results of three repeated tests were averaged. The cell survival rate of the positive control group L2K / Fluc-mRNA was 92%, the cell survival rates of the experimental groups were all greater than 91%, and the cell survival rates of L3-2, L-16, PL-3-2, PL-4 and PL-16 were 96% and above.

[0755] The results showed that compared with L2K / Fluc-mRNA transfected using the commercial transfection reagent Lipofectamine 2000, the LNP / Fluc-mRNA of the present invention also had no obvious toxicity to cells.

[0756] (4) Evaluation of transfection activity at the cell level

[0757] Table 3: Cell transfection test results

[0758] Serial number Relative fluorescence value Serial number Relative fluorescence value Serial number Relative fluorescence value blank 1 L-1 3.8 L-12 2.9 D-0 2.2 L-2 3.3 L-13 2.5 D-1 2.5 L-3-2 3.5 L-14 2.8 D-2 3.0 L-4 3.0 L-15 2.3 D-3 2.7 L-5 3.2 L-16 3.6 D-4 2.6 L-6 3.1 L-17 3.1 L-7 2.7 L-22 3.2 L-8 3.0 L-36 3.1 L-9 2.5 PL-0 2.1 L-10-2 3.7 PL-1 3.9 L-11 2.4 PL-3-2 3.7

[0759] In order to investigate the mRNA transfection efficiency of each group of LNP / Fluc-mRNA compositions prepared in Example 45 of the present invention at the cellular level, Luciferase bioluminescence was used for testing. The LNP / Fluc-mRNA composition preparation was dissolved in culture medium to prepare the required dose. Hela cells were used as a cell model, and 100 μL / well of the cell suspension was inoculated into a 96-well plate with a black-edged transparent bottom at a seeding density of 6000 cells / well. After inoculation, the cells were incubated in a cell culture incubator for 24 hours, and then administered at a dose of 0.2 μg mRNA per well. The blank control group was added with a corresponding dose of free Fluc-mRNA. After 24 hours of transfection, the old culture medium was removed and replaced with a new culture medium containing D-fluorescein sodium (1.5 mg / mL) substrate. After incubation for 5 minutes, bioluminescence was detected using a microplate reader. The stronger the fluorescence, the more Fluc-mRNA was transported into the cytoplasm and translated into the corresponding fluorescent protein. The experimental results are shown in Table 3, where the relative value of fluorescence intensity is the ratio of the fluorescence intensity value of each group to the fluorescence intensity of the blank control group. The results showed that the LNP / Fluc-mRNA compositions prepared by the present invention all had excellent in vitro transfection effects. The LNPs in both the control and experimental groups were effective delivery vehicles, and the transfection effects of most experimental groups were superior to those of the control group. When the PEGylated lipid in the LNP / Fluc-mRNA composition was DMG, the cationic lipids L-1, L-3-2, L-10-2, and L-16, each containing a nitrogen heterocycle in the polar head, showed the best transfection effects. Among them, L-1 had the best transfection effect, likely because its multiple ester bonds facilitated the degradation of the cationic lipid within the cell, promoting the release of the nucleic acid drug Fluc-mRNA into the cytoplasm. When the PEGylated lipid in the LNP / Fluc-mRNA composition was E44-1 of the present invention, the transfection efficiency was not significantly reduced (e.g., the transfection efficiencies of L-0 and PL-0 were comparable), and when combined with the cationic lipids of the present invention, the transfection efficiency was improved to a certain extent (e.g., PL-1 and PL-3-2).

[0760] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

[0761] For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, can be equivalent parameters, concentration and conditions, implement the present invention in a wider range. Although the present invention provides special embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any variation, purposes or improvements to the present invention, including departing from the disclosed scope in the application, and the changes made with conventional techniques known in the art.

Claims

1. A cationic lipid, characterized in that The structure is shown in the general formula (1): Wherein, N is a nitrogen branching center; L1 and L2 are each independently -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)O(CH2) y OC(=O)-、-OC(=O)(CH2) y OC(=O)-、-C(=O)O(CH2) y C(=O)O-、-OC(=O)(CH2) y Any one of C(=O)O-, wherein y is an integer from 2 to 8; L3 is a trivalent linking group, and together with R3 forms a divalent linking group -L3(R3)- whose side chain is R3; B1 and B2 are each independently C 1-30 Alkylene; R1 and R2 are each independently C 2-30 Aliphatic hydrocarbon or C containing 1-2 O 2-30 Aliphatic hydrocarbon derivative residues; R3 is -(CH2) h -R 01 or -(CH2) f -Z3-(CH2) g -R 01 ; wherein h is an integer of 0-6, f is 0, 1 or 2, g is 2, 3 or 4, Z3 is -O-, R 01 is -OH; R4 is any of the following structures: The structure of -L3(R3)- is The alkylene group, aliphatic hydrocarbon group, and aliphatic hydrocarbon derivative residue are each independently unsubstituted; or a salt, tautomer, stereoisomer or solvate thereof.

2. The cationic lipid according to claim 1, characterized in that The structure of -L3(R3)- is 3. The cationic lipid according to claim 1, characterized in that The B1 and B2 are independently C 1-20 Alkylene.

4. The cationic lipid according to claim 3, characterized in that The B1 and B2 are each independently selected from any one of methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene and eicosylene.

5. The cationic lipid according to claim 3, wherein B1 and B2 are each independently selected from the following any one: a linker, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, and decylene.

6. The cationic lipid according to claim 5, characterized in that B1 and B2 are both ethylene, or B1 and B2 are both butylene, or B1 and B2 are both hexylene, or B1 and B2 are both heptylene, or B1 is butylene and B2 is heptylene, or B1 is pentylene and B2 is hexylene, or B1 is pentylene and B2 is heptylene.

7. The cationic lipid according to claim 1, characterized in that The R1 and R2 are each independently selected from R L , R B , Rr; The R L For straight chain C 2-30 Aliphatic hydrocarbon group, containing 0-4 carbon-carbon double bonds; The R B The structure is Where t is 0, 1 or 2; t 01 ,t 02 ,t 03 ,t 04 Each independently is 0 or 1; R e , R f Each independently is C 1-12 Alkyl, C 3-12 Alkenyl and C 3-12 Any of the alkynyl groups; The Rr is a C containing a ring structure 3-30 Aliphatic hydrocarbon group; the ring structure is C 3-20 Carbon ring.

8. The cationic lipid according to claim 7, characterized in that R L Any one of the following structures or any cis-trans isomers thereof: Among them, t L An integer selected from 0-20.

9. The cationic lipid according to claim 7, characterized in that R B Any of the following structures:

10. The cationic lipid according to claim 7, characterized in that The ring structure is a benzene ring or a C 3-20 Cycloalkane residues.

11. The cationic lipid according to claim 7, characterized in that The Rr is further any one of the following structures:

12. The cationic lipid according to claim 1, characterized in that The R4 is any one of the following structures:

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

14. A lipid composition, characterized in that Contains the cationic lipid according to any one of claims 1 to 13.

15. The lipid composition according to claim 14, characterized in that It also contains one or more of phospholipids, steroid lipids and pegylated lipids, selected from any one of the following situations: Case (1): also contains phospholipids; Case (2): also contains steroid lipids; Case (3): also contains PEGylated lipids; Case (4): also contains phospholipids and steroid lipids; Case (5): also contains phospholipids and PEGylated lipids; Case (6): also contains steroid lipids and PEGylated lipids; Case (7): Also contains phospholipids, steroid lipids and PEGylated lipids.

16. The lipid composition according to claim 15, characterized in that It also contains three types of lipids: phospholipids, steroid lipids and PEGylated lipids.

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

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

19. The lipid composition according to claim 15, characterized in that The PEGylated lipid is selected from polyethylene glycol-1,2 dimyristin, polyethylene glycol-distearoyl phosphatidylethanolamine, PEG-cholesterol, polyethylene glycol-diacylglycerol, polyethylene glycol-dialkoxypropyl, specifically including any one of polyethylene glycol 500-dipalmitoyl phosphatidylcholine, polyethylene glycol 2000-dipalmitoyl phosphatidylcholine, polyethylene glycol 500-stearoyl phosphatidylethanolamine, polyethylene glycol 2000-distearoyl phosphatidylethanolamine, polyethylene glycol 500-1,2-dioleoyl phosphatidylethanolamine, polyethylene glycol 2000-1,2-dioleoyl phosphatidylethanolamine and polyethylene glycol 2000-2,3-dimyristoyl glycerol.

20. The lipid composition according to claim 15, characterized in that The PEGylated lipid is selected from any one of the following: Wherein n1 is selected from an integer of 20-250.

21. The lipid composition according to claim 15, characterized in that The structure of the PEGylated lipid is selected from any one of the following: wherein n1 is an integer selected from 20-250.

22. The lipid composition according to claim 15, characterized in that The molar percentage of PEGylated lipids to total lipids is 0.5-5%; The molar percentage of cationic lipids in total lipids is 30-65%; The molar percentage of phospholipids in total lipids is 7.5-13%; The molar percentage of steroid lipids in total lipids is 35-50%.

23. The lipid composition according to claim 22, characterized in that The molar percentage of the PEGylated lipids to the total lipids is 1-3%.

24. The lipid composition according to claim 22, characterized in that The molar percentage of the PEGylated lipids to the total lipids is 1.5%, 1.6%, 1.7%, 1.8% and 1.9%.

25. The lipid composition according to claim 22, characterized in that The molar percentage of the cationic lipid to the total lipids is 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, and 55%.

26. The lipid composition according to claim 22, characterized in that The molar percentage of the phospholipids to the total lipids is 8%, 9%, 10%, 11% and 12%.

27. The lipid composition according to claim 22, characterized in that The molar percentage of the steroid lipids in the total lipids is 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, and 50%.

28. A lipid pharmaceutical composition, characterized in that A lipid composition and a drug according to any one of claims 14 to 27, wherein the drug is selected from any one of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs.

29. The lipid pharmaceutical composition according to claim 28, 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, and the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA and siRNA.

30. The lipid pharmaceutical composition according to claim 29, characterized in that The drug is any one of DNA, mRNA, miRNA and siRNA.

31. The lipid pharmaceutical composition according to claim 28, 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, an antibiotic, an antiviral agent, an antifungal agent, and a vaccine.

32. The lipid pharmaceutical composition according to claim 28, characterized in that The lipid pharmaceutical composition is a LNP-pharmaceutical composition.

33. The lipid pharmaceutical composition according to claim 32, characterized in that The lipid pharmaceutical composition is a LNP-nucleic acid pharmaceutical composition.

34. The lipid pharmaceutical composition according to claim 32, characterized in that The lipid pharmaceutical composition is a LNP-mRNA composition.

35. A lipid pharmaceutical composition preparation, characterized in that: Contains the lipid pharmaceutical composition according to any one of claims 28 to 34 and a pharmaceutically acceptable diluent or excipient.

36. The lipid pharmaceutical composition preparation according to claim 35, characterized in that The diluent or excipient is any one of deionized water, ultrapure water, phosphate buffer and physiological saline.

37. The lipid pharmaceutical composition preparation according to claim 35, characterized in that The diluent or excipient is phosphate buffer or physiological saline.

38. The lipid pharmaceutical composition preparation according to claim 35, characterized in that The diluent or excipient is physiological saline.

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