Asymmetric cationic lipid and use thereof

Abstract

The present application discloses an asymmetric cationic lipid as represented by formula (1), wherein each X is independently O, S, or NH, and other symbols are as defined herein. The asymmetric cationic lipid may be regarded as being formed by linking two lipid monomers of different structures by means of a degradable group. The asymmetric cationic lipid of the present application has higher nucleic acid drug complexation ability, can broaden a drug loading range of nanoparticles, and can also promote endosomal escape of drugs and improve drug delivery efficiency, thereby improving the immunological or therapeutic effect of pharmaceutical formulations.

Description

An asymmetric cationic lipid and its applications Technical Field

[0001] This application belongs to the field of drug delivery, specifically relating to a pharmaceutical carrier cationic lipid, and more particularly to an asymmetric cationic lipid and its applications. Background Technology

[0002] Cationic lipids are an important component of lipid nanoparticle (LNP) drug delivery systems, particularly suitable for delivering negatively charged drugs, such as nucleic acids. To avoid excessive interference with biological membranes and resulting in significant toxicity during systemic circulation, cationic lipids used for drug delivery typically employ structures that are electrically neutral under physiological conditions and positively charged, capable of ionization at low pH levels. Tertiary amine groups, as typical ionizable groups, have been used in various common cationic lipid structures, such as SM-102 and ALC-0315.

[0003] The specific structure of cationic lipids, including ionizable heads, hydrophobic tails, linker portions, and side groups, all affect the drug delivery efficiency of LNPs to varying degrees. LNPs taken up by cells via endocytosis undergo structural remodeling or even destruction in the acidic environment of the endosome. The cationic lipids interact with the negatively charged endosome membrane, causing membrane fusion or endosome membrane destabilization, ultimately releasing the drug into the cytoplasm. If LNPs cannot effectively remodel under endosome pH conditions (typically 1-2 lower than the cytoplasmic pH), their accumulation in the endosome inhibits endosome acidification, hindering drug escape from the endosome.

[0004] Gemini-type cationic lipids can be considered as cationic lipids with two polar heads formed by linking two cationic lipid monomers together through a spacer group. In 1997, Engberts et al. first reported Gemini-type surfactants for efficient and non-toxic in vitro gene delivery (Proc. Natl. Acad. Sci. USA 1997, 94, 1160-1165). In recent years, with the development of LNP delivery technology, Gemini-type cationic lipids have attracted attention due to their excellent nucleic acid drug complexing ability. Utilizing the stability of carbonate or carbamate bonds under neutral conditions and their tendency to undergo acid-catalyzed hydrolysis, they can be used as spacer groups in the structure of Gemini-type cationic lipids. This allows the lipids to initially degrade under endosome pH conditions into two lipid monomers no longer bound together by covalent bonds. This not only accelerates the rearrangement of LNP lipid components but also allows more cationic lipids originally located inside the LNP to migrate to the LNP surface and interact with the endosome membrane, thereby promoting endosome escape.

[0005] Currently disclosed Gemini-type cationic lipids using carbonate or carbamate bonds as spacer groups are generally symmetrical in structure; that is, excluding the spacer group, the structures of the two lipid monomers are essentially identical, as disclosed in WO2023091787A1. However, this type of symmetrical structure has some limitations. On the one hand, this type of cationic lipid has a limited number of head types capable of ionization and electrostatic interaction with nucleic acid drugs, resulting in a narrow pH range suitable for encapsulation. On the other hand, the two lipid products formed after the spacer group breaks in the symmetrical structure are essentially identical, limiting the impact on the rearrangement of LNP lipid components and potentially hindering the promotion of membrane fusion or endosome membrane destabilization.

[0006] To address the aforementioned issues, substantial structural improvements are needed to the Gemini-type cationic lipids containing carbonate or carbamate bonds. Summary of the Invention

[0007] The purpose of this application is to provide novel Gemini-type cationic lipids, as well as compositions and pharmaceutical formulations containing such cationic lipids, for use in the field of drug delivery.

[0008] The above objectives are achieved through the following technical solutions:

[0009] An asymmetric cationic lipid, the structure of which is shown in formula (1):

[0010] Or its salts, tautomers, stereoisomers, isotopic substitutes, or solvates; wherein...

[0011] Each X is independently O, S, or NH;

[0012] M1 and M2 are each selected independently. Any one of the following; Q is CH or N; p is 0, 1 or 2; Indicates a substituted or unsubstituted nitrogen heterocycle; either M1 or M2 is connected to R1 or R2; M1 and M2 have different specific structures;

[0013] Each R M Independently for R S or R L And it is bonded to any cyclic atom of a nitrogen atom or a nitrogen heterocycle; each R S Independently for C 1-5 Alkyl or C 1-5 Hydroxyalkyl; each R L Independently for substituted or unsubstituted C 6-40 hydrocarbon group, and the C 6-40 The skeleton of the hydrocarbon group may optionally be interrupted by one or two Z0s;

[0014] L1, L2, L3, and L4 are each independently -(CH2). t -Z0-(CH2) t -; Each t is an independent integer from 0 to 6;

[0015] R1 and R2 are each independent of each other. Each R0 is independently a substituted or unsubstituted C 1-40 hydrocarbon group, C 1-40 Heteroalkyl groups or fat-soluble vitamin residues; q is an integer from 0 to 3; each B0 is an independent linking bond, C 1-21 Alkylene or -(CH2) k -P0-(CH2) k - and any B0 that is a connecting bond is not simultaneously connected to two Z0; each k is an independent integer from 0 to 10; P0 is ortho-phenylene, meta-phenylene, or para-phenylene; the C 1-21 The alkylene group may be substituted or unsubstituted;

[0016] Each Z0 is independently one of the following: a linker bond, -Q-, -QQ-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, and -QC(=Q)Q-; Q is independently O, S, or NR' each time it appears; R' is a hydrogen atom or C. 1-4 alkyl.

[0017] This application also provides another implementation scheme:

[0018] A lipid composition comprising an asymmetric cationic lipid of formula (1).

[0019] This application also provides another implementation scheme:

[0020] A lipid pharmaceutical composition comprising a lipid composition and a pharmaceutical active ingredient, wherein the lipid composition comprises an asymmetric cationic lipid represented by formula (1).

[0021] This application also provides another implementation scheme:

[0022] A lipid pharmaceutical composition formulation comprising the aforementioned lipid pharmaceutical composition and a pharmaceutically acceptable diluent or excipient.

[0023] Compared with the prior art, this application has the following beneficial effects:

[0024] The asymmetric cationic lipid structure of this application can be considered as two lipid monomers with different ionizable heads linked by a spacer group containing a carbonate bond, a carbamate bond, or a thiourea bond. Compared with symmetrical structures, the asymmetric cationic lipid of this application can bind to nucleic acid drugs through electrostatic interactions over a wider pH range, which not only makes drug-loaded nanoparticles easier to prepare but also improves their systemic circulation stability.

[0025] The asymmetric cationic lipids of this application can also broaden the adjustable range of drug loading. Symmetrical structures usually require the ionization of both identical heads under specific pH conditions to recombine with nucleic acids via electrostatic interactions; in contrast, asymmetric structures can obtain one or two cationized groups (or even more, such as structures with multiple tertiary amine groups) by adjusting the pH conditions of the nanoparticle preparation process, thereby meeting different drug loading requirements.

[0026] When the spacer group breaks within the cell, the asymmetric cationic lipid of this application initially degrades into two separate cationic lipid products with different ionizable groups. Because they are no longer bound by covalent bonds, and the interactions between the different ionizable groups and the nucleic acid drug differ, the independent cationic lipid products tend to redistribute, promoting the rearrangement of the LNP components. Specifically, the rearrangement of lipid components on the LNP surface can, to some extent, promote fusion with the endosomal membrane, and the cationic lipids originally located inside will migrate more to the surface and interact with the endosomal membrane, accelerating its destabilization and ultimately promoting drug release. In other words, the asymmetric cationic lipid of this application enables nucleic acid drugs to escape from the endosomal membrane more effectively, improving drug delivery efficiency and thus enhancing the immunogenic or therapeutic effects of the drug formulation. Attached Figure Description

[0027] Figure 1 shows the cytotoxicity test results of the LNP-mRNA drug composition L-11 prepared in Example 30.

[0028] Figure 2 shows the imaging results of mice after injection of the LNP-mRNA drug composition L-11 prepared in Example 30.

[0029] Invention Details

[0030] 1. Terminology Explanation

[0031] Unless otherwise specified, all technical and scientific terms used herein have their usual meaning within the field to which the claimed subject matter pertains. Where multiple definitions exist for a term, the definition herein shall prevail. Unless otherwise specified, the terms are explained below.

[0032] In this application, the word "including" and similar expressions should be interpreted in an open and inclusive sense as "including but not limited to" or "inclusively including".

[0033] In this application, when at least two items are listed, the "combination" of the listed items refers to any two or more combinations of the aforementioned listed items, or any two or more combinations of the same type. Each item in the combination can be one or more independent specific forms. Combinations formed by linking bases are assumed to be formed by the linking bases being interconnected. Combinations of linking keys with any linking base remain the linking base itself.

[0034] In this application, the "linking bond" contains no atoms and serves only a connecting function. Specifically, when the selection range of a certain group includes the linking bond, it means that the group can be replaced by the linking bond. When one end of the linking bond points into the ring, for example... This linker bond can be derived from any suitable cyclic atom.

[0035] In this application, a "linking group" is a group containing two, three, or more connecting ends, and is referred to as a divalent, trivalent, or higher-valent linking group depending on the number of connecting ends. In some cases, the "group" in a divalent linking group can be replaced with "bond" without changing its meaning. For example, a divalent ether group (-O-) can also be called an ether bond, a divalent ester group (-OC(=O)-) can also be called an ester bond, and a divalent carbamate group (-OC(=O)NH-) can also be called a carbamate bond. Unless otherwise specified, a linking group can be connected to any adjacent group through any of its connecting ends, provided that the chemical structure is reasonable. Unless otherwise specified, a linking group contains at least one atom. In particular, when the selection range of linking groups explicitly includes a linking bond, or when the general formula of the linking group, in a specific form under certain parameters, does not contain any atoms, the linking bond can be considered a special type of linking group.

[0036] In this application, numerical ranges can be represented by hyphens or wavy lines, such as 1-6 or 1~6. The numerical types within the range include, but are not limited to, integers, non-integers, percentages, and fractions. Numerical ranges representing the number of functional groups are by default composed of integers, such as -(CH2). 1-4 - Selected from groups consisting of -CH2-, -(CH2)2-, -(CH2)3-, and -(CH2)4-, such as Selected from The group that makes up the group.

[0037] In this application, the numerical range indicated by the C subscript position represents the number of carbon atoms in the group, and by default does not include the contribution of substituents. For example, C 1-5 The alkyl group is selected from the group consisting of methyl, ethyl, propyl, butyl, and pentyl, including isomers (such as n-propyl, isobutyl, etc.).

[0038] In this application, the unit of measurement for molecular weight is Daltons (Da). The molecular weight of the polymer is the number-average molecular weight (M). n ).

[0039] In this application, all compounds of the general formula should be understood to include their salts. A "salt" of a compound is selected from any one, any two, or any combination of two or more of the following: acid addition salts formed by the compound with inorganic and / or organic acids and base addition salts formed with inorganic and / or organic bases. When a compound contains a basic moiety (e.g., but not limited to pyridine or imidazole) and an acidic moiety (e.g., but not limited to carboxylic acids), an amphoteric ion ("internal salt") may be formed and included in the term "salt" as used. A "salt" may be a pharmaceutically acceptable (i.e., non-toxic and physiologically acceptable) salt or other salts. Salts of compounds may be formed by reacting the compound with a certain amount (such as an equivalent) of an acid or base in a medium such as a salt precipitation medium or in an aqueous medium, followed by lyophilization. Exemplary acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, hydrogen sulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, gluconate, glyceryl phosphate, hemisulfate, heptaate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, propionate, salicylate, succinate, sulfate, sulfonate, tartrate, thiocyanate, toluenesulfonate, undecanoate, etc. 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 containing organic bases (e.g., organic amines), and salts containing amino acids (such as arginine or lysine). The basic nitrogen-containing group 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 dipentyl sulfates), long-chain halides (e.g., decyl, lauryl, tetradecyl, and stearyl chlorides, bromides, and iodides), arylalkyl halides (e.g., benzyl and phenethyl bromides), and others. Both acid and base addition salts are preferably pharmaceutically acceptable salts and, unless otherwise specified, are considered equivalent to the free form of the corresponding compound.

[0040] In this application, "tautomer" refers to an isomer of a compound obtained by the transfer of hydrogen atoms or protons within the molecule, often involving the conversion of single and double bonds. Types of tautomers include, but are not limited to, keto-enol tautomers, amide-imino acid tautomers, lactam-lactamimide tautomers, enamine-imide tautomers, enamine-enamine tautomers, proton transfer tautomers, and valence tautomers.

[0041] In this application, "stereoisomer" refers to isomers having the same atomic connection sequence but different three-dimensional structures, including but not limited to cis / trans isomers, E- / Z- isomers, and levorotatory / dextral isomers. Unless otherwise stated, stereoisomers of any structure herein are considered to be disclosed together.

[0042] In this application, "isotope-substituted product" refers to a derivative form obtained by replacing one or more atoms in a compound molecule with their respective isotopes.

[0043] In this application, "solvent" refers to an aggregate comprising one or more compound molecules and one or more solvent molecules. The solvent may be water, and the corresponding solvate may be called a hydrate, including monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate, etc. The solvent may also be an organic solvent.

[0044] In this application, heteroatoms include, but are not limited to, O, S, N, P, Si, F, Cl, Br, I, B, etc.

[0045] In this application, when a compound structure (including molecules, groups, and fragments) is described as "substituted," it means that the structure contains one or more substituents. A "substituted" structure is also referred to as a "substituted form" in contrast to an "unsubstituted" structure.

[0046] In this application, a "substituent" can be a single-atom or multi-atom group. Unless otherwise specified, substituents do not include hydrogen atoms. For substituted compound structures, unless otherwise specified, substituents include, but are not limited to, substituents commonly found in the art, such as alkyl, alkoxy, hydroxyl, mercapto, carboxyl, amino, amine, aldehyde, ester, carbonate, carbamate, succinimide, maleimide, alkenyl, acrylate, azide, alkynyl, folic acid, rhodamine, biotin, monosaccharide, polysaccharide, etc. Substituents can be in unprotected or protected forms. Substituted groups can also be used as substituents, such as substituted hydrocarbon or heterohydrocarbon groups; more specifically, -CH2COOH can be considered a carboxyl-substituted methyl group and can also be used as a substituent.

[0047] In this application, both "amino" and "amine group" include neutral or cationic forms with monovalent, divalent, trivalent, and tetravalent oxidation states. "Amino" includes primary amino (-NH₂), secondary amino (-NH₃), tertiary amino (-N<), and quaternary amino (>N). + <). Amine compounds refer to hydrocarbon-substituted products of NH3, including primary amines (monoalkyl substitution, such as CH3NH2), secondary amines (dialkyl substitution, such as NH(CH3)2), tertiary amines (trialkyl substitution, such as N(CH3)3), and quaternary amines (tetraalkyl substitution, such as N... + (CH3)3). A group formed by the loss of one or more hydrogen atoms from an amine compound is collectively called an "amine group," where the hydrogen atoms can come from amino, hydrocarbon, or other groups. The cationic form of an amino or amine group is also called a quaternary ammonium group.

[0048] In this application, "hydrocarbon" refers to compounds containing only carbon and hydrogen. Hydrocarbons without a benzene ring or other aromatic rings are collectively referred to as "aliphatic hydrocarbons," while hydrocarbons containing a benzene ring or other aromatic rings are collectively referred to as "aromatic hydrocarbons." Aliphatic hydrocarbons can be divided into open-chain hydrocarbons and alicyclic hydrocarbons, with open-chain hydrocarbons further divided into straight-chain hydrocarbons and branched-chain hydrocarbons. Based on the degree of unsaturation, aliphatic hydrocarbons can be divided into saturated aliphatic hydrocarbons and unsaturated aliphatic hydrocarbons, and further divided into alkanes, alkenes, and alkynes, where alkenes contain at least one carbon-carbon double bond and alkynes contain at least one carbon-carbon triple bond.

[0049] In this application, "hydrocarbon group" refers to a group formed after a hydrocarbon loses at least one hydrogen atom, and can be monovalent, divalent, trivalent, tetravalent or higher valence.

[0050] In this application, "alkylene group" is a divalent hydrocarbon group, "alkylene group" is a divalent alkyl group, "alkenyl group" is a divalent alkenyl group, and "alkynyl group" is a divalent alkynyl group.

[0051] In this application, "hydroxyalkyl" refers to a hydroxyl-substituted alkyl group. "Arylalkyl" refers to an aryl-substituted alkyl group, such as benzyl.

[0052] In this application, "ring-forming atom" refers to an atom that participates in the formation of a ring-shaped framework.

[0053] In this application, the "skeleton" of a compound or group refers to its core or basic framework, constituting the main topological structure of the compound or group. It can be linear or nonlinear, and by default does not include groups attached to the skeleton. For example, the skeleton of a hydrocarbon group consists only of carbon atoms, while the skeleton of a heterohydrocarbon group also contains heteroatoms. An "interrupted" skeleton should be understood as the form of embedding a specific linker in the original skeleton, and the embedding position is not particularly restricted; for example, a linear butylene group whose skeleton is interrupted by a Z0 can be in the form of -CH2Z0(CH2)3- or -(CH2)2Z0(CH2)2-.

[0054] In this application, "heterocycle" refers to a ring structure in which the ring-forming atoms include a carbon atom and at least one heteroatom. Heterocycles containing nitrogen atoms are also called "nitrogen heterocycles". Heterocycles can be aliphatic heterocycles (such as tetrahydrofuran) or aromatic heterocycles (such as pyridine). Heterocycles can be monocyclic or formed by two or more monocyclic rings sharing one, two or more ring-forming atoms (such as spirocyclic, bridged, fused, etc.); the latter only requires that any of the monocyclic rings constituting the heterocycle contains a heteroatom.

[0055] In this application, "phenylene" refers to a divalent group formed by removing two hydrogen atoms from a benzene ring, with the following structure: The two hydrogen atoms can be located at the para, ortho, or meta positions of the benzene ring, corresponding to para-phenylene (1,4-phenylene), ortho-phenylene (1,2-phenylene), and meta-phenylene (1,3-phenylene), respectively.

[0056] In this application, amino acids can be of natural or non-natural origin. Amino acids can be classified into protein amino acids and non-protein amino acids. Protein amino acids can directly participate in protein molecule synthesis. Non-protein amino acids require modification before they can participate in protein synthesis, such as citrulline, ornithine, and hydroxyproline. Based on the different positions of the amino group on the carbon chain, amino acids can be classified into α-, β-, γ-, etc. Unless otherwise specified, "side chain of an amino acid" in this application refers to the side chain of α-amino acids.

[0057] In this application, "lipid nanoparticles" refers to nanoscale (e.g., 1 nm to 1000 nm) particles containing one or more lipids, which can carry one or more payloads (e.g., nucleic acid drugs).

[0058] In this application, "cationic lipid" can be a lipid that carries a positive charge at any pH or under hydrogen ion activity, or a lipid that can carry a positive charge in response to the pH or hydrogen ion activity of its intended use environment, the latter containing one or more cationizable groups. Cationic lipids preferably partially or completely convert to a cationic form at a pH of about 1 to 9, more preferably at a pH of 4 to 9, 5 to 8, or 6 to 8, and most preferably at an endogenous pH (about 5.5 to 6.5). Zwitterionic lipids that exist primarily in a cationic form under specific pH conditions are also considered cationic lipids.

[0059] In this application, "polyglycolic lipid" refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety. Polyglycolic lipids modified with targeting groups are also known as targeted polyglycolic lipids.

[0060] In this application, "neutral lipids" refers to lipids that exist in an overall electrically neutral nonionic or zwitterionic form.

[0061] In this application, "steroid lipid" refers to a fused ring structure composed of three cyclohexanes and one cyclopentane. Lipids are characterized by their main properties.

[0062] In this application, a "targeting group" refers to a group that provides a strong affinity for a selected target (e.g., a cell, tissue, organ, body region or compartment, such as a cell, tissue, or organ compartment). Exemplary targeting groups include, but are not limited to, residues of the following substances: antibodies, antigens, peptides, vitamins, carbohydrates (including but not limited to monosaccharides such as N-acetylgalactosamine (GalNAc)), folic acid, aptamers, receptor ligands, transferrin, biotin, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.

[0063] In this application, "N / P ratio" refers to the molar ratio of cationic groups (such as tertiary amine groups) in cationic lipids to phosphate groups in nucleic acids.

[0064] In this application, "pharmaceutical active ingredient" refers to biologically related substances that have therapeutic or preventive effects in pharmaceutical preparations, including but not limited to nucleic acids, small molecules, oligopeptides, polypeptides and proteins.

[0065] In this application, "nucleic acid" is composed of nucleotide units, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and their derivatives. RNA can be naturally occurring or non-naturally occurring ribonucleic acid, including but not limited to small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), single-stranded guide RNA (sgRNA), and Cas9 mRNA. "antagomir," also known as "anti-miR," is a class of chemically engineered oligonucleotides designed to silence endogenous miRNAs.

[0066] In this application, small molecule compounds refer to compounds whose molecular weight is typically below 1000 Da. Small molecule drugs include, but are not limited to, enzyme inhibitors, receptor agonists or antagonists, ion channel modulators, and small molecule kinase inhibitors.

[0067] In this application, "oligopeptide" refers to a short-chain peptide composed of 2 to 20 amino acids linked by peptide bonds. Oligopeptides with a molecular weight below 1000 Da also belong to small molecule compounds.

[0068] In this application, "polypeptide" is a macromolecule formed by 21 to 50 amino acids linked by peptide bonds.

[0069] In this application, "protein" refers to a macromolecule composed of more than 50 amino acids linked by peptide bonds, which folds into a three-dimensional structure with a specific function.

[0070] In this application, "delivery" means providing an entity to a target. For example, delivering a drug to a subject, said subject being an organ and / or tissue and / or cell of a human or other animal.

[0071] In this application, "pharmaceutically acceptable carrier" refers to a diluent, excipient, vehicle, or medium administered co-administered with a drug, and which, to the extent of reasonable medical judgment, is suitable for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio. Exemplary diluents include, but are not limited to, water, saline, ethanol, propylene glycol, glycerin, and vegetable oils (such as soybean oil and sesame oil). Exemplary excipients include, but are not limited to, antioxidants (such as vitamin E, BHT, and BHA), preservatives (such as sodium benzoate and parabens), buffers (such as phosphates and citrates), and solubilizers (such as poloxamer and Tween 80). Exemplary excipients include, but are not limited to, thickeners (such as hydroxypropyl methylcellulose and carbomer), disintegrants (such as crosvinylpyrrolidone and starch), binders (such as microcrystalline cellulose and hydroxypropyl cellulose), and lubricants (such as magnesium stearate and talc). Exemplary media include, but are not limited to, solvents (such as dimethyl sulfoxide, ethanol), emulsifiers (such as lecithin, Span), suspending agents (such as gelatin, methylcellulose) and oil bases (such as cocoa butter, stearates).

[0072] In this application, "vaccine" refers to a prophylactic or therapeutic material that provides at least one antigen or antigenic function. The antigen or antigenic function can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0073] In this application, "treatment" refers to the handling and care of a patient in order to combat a disease, obstacle, or symptom, intended to include delaying the progression of the disease, obstacle, or symptom, alleviating or mitigating symptoms and complications, and / or curing or eliminating the disease, obstacle, or symptom. The patient to be treated is preferably a mammal, especially a human.

[0074] 2. Asymmetric cationic lipids

[0075] One embodiment of this application:

[0076] An asymmetric cationic lipid, the structure of which is shown in formula (1):

[0077] Or its salts, tautomers, stereoisomers, isotopic substitutes, or solvates; wherein...

[0078] Each X is independently O, S, or NH;

[0079] M1 and M2 are each selected independently. Any one of the following; Q is CH or N; p is 0, 1 or 2; Indicates a substituted or unsubstituted nitrogen heterocycle; either M1 or M2 is connected to R1 or R2; M1 and M2 have different specific structures;

[0080] Each R M Independently for R S or R L And it is bonded to any cyclic atom of a nitrogen atom or a nitrogen heterocycle; each R S Independently for C 1-5 Alkyl or C 1-5 Hydroxyalkyl; each R L Independently for substituted or unsubstituted C 6-40 hydrocarbon group, and the C 6-40 The skeleton of the hydrocarbon group may optionally be interrupted by one or two Z0s;

[0081] L1, L2, L3, and L4 are each independently -(CH2). t -Z0-(CH2) t -; Each t is an independent integer from 0 to 6;

[0082] R1 and R2 are each independent of each other. Each R0 is independently a substituted or unsubstituted C 1-40 hydrocarbon group, C 1-40 Heteroalkyl groups or fat-soluble vitamin residues; q is an integer from 0 to 3; each B0 is an independent linking bond, C 1-21 Alkylene or -(CH2) k -P0-(CH2) k - and any B0 that is a connecting bond is not simultaneously connected to two Z0; each k is an independent integer from 0 to 10; P0 is ortho-phenylene, meta-phenylene, or para-phenylene; the C 1-21 The alkylene group may be substituted or unsubstituted;

[0083] Each Z0 is independently one of the following: a linker bond, -Q-, -QQ-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, and -QC(=Q)Q-; Q is independently O, S, or NR' each time it appears; R' is a hydrogen atom or C. 1-4 alkyl.

[0084] In this application, for the asymmetric cationic lipids represented by formula (1), These are respectively considered as divalent linkers containing acyclic ionizable side chains, acyclic ionizable main chains, cyclic ionizable side chains, and cyclic ionizable main chains.

[0085] 2.1.R S and R L

[0086] In one specific implementation scheme, each R S It can be independently -CH3, -CH2CH3 or -CH2CH2OH, preferably -CH3.

[0087] In one specific implementation, all R in the asymmetric cationic lipid represented by formula (1) M All are R S .

[0088] In one specific implementation scheme, each R L C is independently substituted or unsubstituted with a hydroxyl group. 6-40 Alkyl, C 6-40 alkenyl or C 6-40 Alkyne group, and the C 6-40 The alkyl skeleton is optionally interrupted by a Z0; preferably, each R L Independently selected from any of the following structures:

[0089] Where each tm is an independent integer from 0 to 20, and each R m Independently linear or branched C 1-20 Alkyl, C 3-20 alkenyl or C 3-20 alkynyl group; the C 3-20 The alkenyl group contains one or two carbon-carbon double bonds; the C 3-20 The alkynyl group contains one or two carbon-carbon triple bonds.

[0090] 2.2.B0

[0091] In one specific implementation, each B0 is independently a connection key, R B The side chain is a hydrogen atom, a hydroxyl group, or an amino acid; the side chain of the amino acid is preferably any of the following structures:

[0092] 2.3.

[0093] In one specific implementation plan, It contains at least one nitrogen atom as a cyclic atom, and optionally also contains one oxygen atom, one sulfur atom or another nitrogen atom as a cyclic atom, with the remaining cyclic atoms being carbon atoms; It has a single-ring or double-ring structure, and each ring is independently a four-, five-, six-, or seven-membered ring; preferably, the two rings in the double-ring structure share at least two ring-forming atoms; preferably, Selected from Any of the following, wherein the *-marked N atom is associated with L1, L2, R1, R2, L4, or R M Connected;

[0094] When substitution is used, it is preferable to use a form in which one hydrogen atom on the ring is replaced by a hydroxyl group;

[0095] Preferably, Choose from any of the following structures:

[0096] Preferably, Choose from any of the following structures:

[0097] 2.4. In one specific implementation plan, Choose from any of the following structures:

[0098] 2.5. General Structural Formula

[0099] In one specific implementation, the structure of the asymmetric cationic lipid is selected from any one of formulas (2) to (5):

[0100] Formula (2) contains a non-cyclic ionizable side chain group. and a main-chain acyclic ionizable group Formula (3) contains a side-chain acyclic ionizable group. and a main-chain cyclic ionizable group Formula (4) contains a side-chain cyclic ionizable group. and a main-chain acyclic ionizable group Formula (5) contains a side-chain cyclic ionizable group. and a main-chain cyclic ionizable group

[0101] In one specific implementation, the structure of the asymmetric cationic lipid is selected from either formula (6) or (7):

[0102] Formula (6) contains a main-chain non-cyclic ionizable group. and a main-chain cyclic ionizable group Formula (7) contains a side-chain acyclic ionizable group. and a side-chain cyclic ionizable group

[0103] 2.6. M1 and M2

[0104] In one specific implementation scheme, both M1 and M2 are or all or all or all Among them, M1 contains at least one R M With any R in M2 M They are not the same.

[0105] 2.7.R0

[0106] In one specific implementation, each R0 is independently a linear, branched, or cyclic C 1-40 Alkyl, C 2-40 alkenyl, C 2-40 alkynyl group, C 1-40 Heteroalkyl or fat-soluble vitamin residues; each R0 is independently substituted or unsubstituted; in the substituted form of said R0, each substituent is independently C 1-6 Alkyl, hydroxyl, C 1-6 Hydroxyalkyl, halogen, aryl or aralkyl, preferably methyl, hydroxy, hydroxymethyl, fluorine atom or benzyl;

[0107] Preferably, R0 is selected from any of the following structures or alternative forms thereof:

[0108] Vitamin A residues, vitamin D residues, vitamin E residues, and vitamin K residues; wherein each tp is an independent integer from 0 to 20, and each R p Independently linear C 1-20 Alkyl, C 2-20 alkenyl or C 2-20 alkynyl group; the C 2-20 The alkenyl group contains one or two carbon-carbon double bonds; the C 2-20 The alkynyl group contains one or two carbon-carbon triple bonds;

[0109] More preferably, R0 is selected from any of the following structures:

[0110] 2.8. R1 and R2

[0111] In one specific implementation, q in R1 and R2 is 0, 1, or 2.

[0112] In one specific implementation, R1 and R2 are each independently selected from any of the following structures:

[0113] 2.9. Z0, L1, L2, L3 and L4

[0114] In one specific implementation, t in L1, L2, L3, and L4 is 0, 1, 2, or 3.

[0115] In one specific implementation, Z0 in L1 and L2 is independently selected from any one of the following: linking bond, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)NH-, -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, and -NHC(=S)NH-; preferably, L1 and L2 are each independently -(CH2). t -(CH2) t -More preferably, L1 and L2 are each independently selected from any one of the following: a connecting bond, -CH2-, -(CH2)2-, and -(CH2)3-.

[0116] In one specific implementation, each Z0 in R1 and R2 is independently selected from any one of the following: linker, -SS-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -OC(=O)O-, -NHC(=O)O-, and -OC(=O)NH-.

[0117] In one specific implementation, Z0 in L3 and L4 is independently selected from any one of the following: linker bond, -C(=O)-, -N(CH3)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, and -NHC(=S)NH-.

[0118] 2.10. Combination of Variables

[0119] In one embodiment, the definitions of the variables involved in the various general formulas in section 2 are independent of each other, and any definition of these variables can be combined with each other in any way. That is, the definitions of any variable can be arbitrarily combined with the definitions of any other one or more variables to form different embodiments of this application. For example, any two or more of the following variables can be arbitrarily combined: Rs and R in section 2.1. L Section 2.2, B0; Section 2.3, B0 2.4. The structural formula in section 2.5, M1 and M2 in section 2.6, R0 in section 2.7, R1 and R2 in section 2.8, and Z0, L1, L2, L3, and L4 in section 2.9 are also included. The cationic lipid structures obtained after each combination scheme are shown in all the examples.

[0120] 2.11. Specific Structure

[0121] In one specific embodiment, the asymmetric cationic lipid is selected from any of the following structures:

[0122] The raw materials used in the preparation process can be obtained through purchase or synthesis. Raw materials that can be used to prepare asymmetric cationic lipids include, but are not limited to, the following series of compounds:

[0123] Series (a):

[0124] Series (b):

[0125] Series (c):

[0126] Series (d):

[0127] Series (e):

[0128] Series (f):

[0129] Series (g):

[0130] Series (h):

[0131] Series (k):

[0132] Series (m):

[0133] Series(s):

[0134] Series (p):

[0135] The aforementioned raw materials also include their salts, stereoisomers, deuterated derivatives, solvates, protected forms, deprotected forms, protonated forms, deprotonated forms, forms with altered leaving groups, and active ester forms. The altered leaving group form refers to the form in which one leaving group in the aforementioned raw material molecule is replaced by another leaving group, for example, -Br is replaced by -Cl, or -OTs are replaced by -OMs. The active ester form refers to the form in which the aforementioned raw material molecule is converted into a reactive ester, including but not limited to succinimidyl esters obtained from carboxylic acids, isothiocyanates obtained from amines, sulfonates or reactive carbonates obtained from alcohols, etc.

[0136] Asymmetric cationic lipids can be prepared through the following steps:

[0137] The first step is to synthesize the first lipid monomer;

[0138] The second step is to synthesize the second lipid monomer;

[0139] The third step is to directly couple the two monomers mentioned above to obtain the asymmetric cationic lipid shown in formula (1);

[0140] The spacer group contains carbonate bonds, urethane bonds, or thiourea bonds formed in any of the aforementioned steps.

[0141] Alternatively, asymmetric cationic lipids can be prepared by the following steps:

[0142] The first step is to synthesize the first lipid monomer;

[0143] The second step is to synthesize the second lipid monomer;

[0144] The third step involves coupling one of the lipid monomers with a bifunctional compound to obtain an intermediate.

[0145] The fourth step involves coupling the aforementioned intermediate with another lipid monomer to obtain the asymmetric cationic lipid shown in formula (1).

[0146] In this process, the carbonate bonds, urethane bonds, or thiourea bonds contained in the spacer groups are formed in the third or fourth step.

[0147] Alternatively, asymmetric cationic lipids can be prepared by the following steps:

[0148] The first step is to synthesize an asymmetric bifunctional compound;

[0149] The second step is to couple the aforementioned asymmetric bifunctional compound with a compound containing a hydrophobic segment to obtain the asymmetric cationic lipid shown in formula (1).

[0150] In this process, the carbonate bonds, urethane bonds, or thiourea bonds contained in the spacer groups, as well as the two different ionizable heads, are all formed in the first step, and the hydrophobic tail chain is formed in the second step.

[0151] Any step in the aforementioned preparation method may include one or more reactions. There are no particular limitations on the combination of different lipid monomers, as long as the compound shown in formula (1) can be obtained, and it is not limited to the specific combination in the examples.

[0152] 3. Lipid compositions, lipid pharmaceutical compositions and their formulations

[0153] One embodiment of this application:

[0154] A lipid composition comprising an asymmetric cationic lipid of formula (1).

[0155] In one specific embodiment, the lipid composition contains an asymmetric cationic lipid containing disulfide bonds as shown in formula (1), and further contains one or more of phospholipids, steroid lipids, polyethylene glycol-modified lipids, another cationic lipid, and anionic lipids; preferably, the lipid composition further contains any one of phospholipids, steroid lipids, and polyethylene glycol-modified lipids; more preferably, the lipid composition further contains any two of phospholipids, steroid lipids, and polyethylene glycol-modified lipids; even more preferably, the lipid composition further contains phospholipids, steroid lipids, and polyethylene glycol-modified lipids; most preferably, the lipid composition further contains phospholipids, steroid lipids, polyethylene glycol-modified lipids, and another cationic lipid; or the lipid composition further contains phospholipids, steroid lipids, polyethylene glycol-modified lipids, and anionic lipids. The aforementioned lipid composition includes the following cases:

[0156] Scenario 1: The lipid composition further contains phospholipids; or,

[0157] Scenario 2: The lipid composition further contains steroid lipids; or,

[0158] Scenario 3: The lipid composition further contains polyethylene glycol-modified lipids; or,

[0159] Scenario 4: The lipid composition further contains polyethylene glycol-modified lipids and phospholipids; or,

[0160] Scenario 5: The lipid composition further contains polyethylene glycol-modified lipids and steroid lipids; or,

[0161] Scenario 6: The lipid composition further contains phospholipids and steroid lipids; or,

[0162] Scenario 7: The lipid composition further contains polyethylene glycol-modified lipids, phospholipids, and steroid lipids; or,

[0163] Scenario 8: The lipid composition further contains phospholipids, steroid lipids, polyethylene glycol-modified lipids, and another cationic lipid; or,

[0164] Situation 9: The lipid composition further contains phospholipids, steroid lipids, polyethylene glycol-modified lipids, and anionic lipids;

[0165] In any of the aforementioned lipid compositions, the polyethylene glycolated lipid is selected from any one of non-targeted polyethylene glycolated lipids, targeted polyethylene glycolated lipids, and combinations thereof;

[0166] Preferably, the lipid composition is selected from Situation 7, wherein the cationic lipids account for 30% to 65% of the total lipids, preferably 35% to 55%; the phospholipids account for 2% to 15% of the total lipids, preferably 5% to 12%; the steroid lipids account for 25% to 50% of the total lipids, preferably 38% to 50%; and the polyethylene glycol-modified lipids account for 0.5% to 10% of the total lipids, preferably from 1% to 3%, wherein the targeted polyethylene glycol-modified lipids account for 0-0.5% of the total lipids.

[0167] In one specific embodiment, the phospholipid in the lipid composition is selected from 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), and 1,2-di-O-octadecyl-sn-glycerol-3-phosphocholine (18:0 Diether). PC), 1-oleoyl-2-cholesterolylhemisuccino-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16 Lyso PC), 1,2-dilinanoyl-sn-glycerol-3-phosphocholine, 1,2-disarachidanoyl-sn-glycerol-3-phosphocholine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphocholine), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphoethanolamine (4ME) 16.0PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG), palmitoyl The following are included in the following formulations: oleoylphosphatidylethanolamine (POPE), distearyl-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and combinations thereof.

[0168] In one specific embodiment, the steroid lipids in the lipid composition are selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatine, ursolic acid, α-tocopherol, and combinations thereof.

[0169] In one specific embodiment, the non-targeted polyethylene glycol-modified lipid in the lipid composition is selected from any one of polyethylene glycol-dispalmitoylphosphatidylcholine (PEG-DPPC), polyethylene glycol-dimyristylglycerol (PEG-DMG), polyethylene glycol-distearate phosphatidylethanolamine (PEG-DSPE), polyethylene glycol-dioleoylphosphatidylethanolamine (PEG-DOPE), polyethylene glycol-cholesterol (PEG-Chol), polyethylene glycol-diacylglycerol (PEG-DAG), polyethylene glycol-dialkoxypropyl (PEG-DAA), and combinations thereof; preferably. The non-targeted PEGylated lipids are selected from any one of polyethylene glycol 500-dispalmitoylphosphatidylcholine, polyethylene glycol 2000-dispalmitoylphosphatidylcholine, polyethylene glycol 500-disstearoylphosphatidylethanolamine, polyethylene glycol 2000-disstearoylphosphatidylethanolamine, polyethylene glycol 500-dioleoylphosphatidylethanolamine, polyethylene glycol 2000-dioleoylphosphatidylethanolamine, polyethylene glycol 500-dimyristoylglycerol, polyethylene glycol 2000-dimyristoylglycerol (PEG2k-DMG), and combinations thereof; or, the non-targeted PEGylated lipids are selected from...

[0170] any combination thereof, where n1 is an integer from 10 to 250.

[0171] In one specific embodiment, the targeted polyethylene glycol-modified lipid in the lipid composition is a folic acid or N-acetylgalactosamine-modified polyethylene glycol-modified lipid; preferably, the targeted polyethylene glycol-modified lipid is selected from...

[0172] any combination thereof, where n2 is an integer from 10 to 250.

[0173] In one specific embodiment, another cationic lipid in the lipid composition is selected from 1,2-dioleoyl-3-trimethylammonium propane (methyl sulfate) (DOTAP), 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTMA), 1-[2-(oleoyloxy)ethyl]-2-oleo-3-(2-hydroxyethyl)imidazoline chloride (DOTIM), 1,2-dioleoyl-3-dimethylamino-propane (DODMA), 2,3-dioleoyl-3-dimethylamino-propane, etc. 1,3-Di(tetradecanoyloxy)propyltrimethylazone chloride (DMTAP), bis(decyl)dimethylammonium chloride (DDAC), bis(decyl)dimethylammonium bromide (DDAB), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propyl-1-ammonium (DOBAQ), 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-di Ketone (cKK-E12), 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecane-2-ol) (C12-200), methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) (DLin-MC3-DMA), 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine Any one of (EPC), ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)(6-oxo-6-(undecapoxy)hexyl)amino]octanoate (heptadecane-9-yl) ester (SM-102), and ((2-(2-hydroxyethoxy)ethyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (DHA-1).

[0174] In one specific embodiment, the anionic lipid in the lipid composition is selected from any one of 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt (18:1PA), 1,2-dimyristoyl-sn-glycerol-3-phosphate sodium salt (14:0PA), bis(monooleoylglycerol)ammonium phosphate (18:1BMP), and cardiolipin (CL).

[0175] In one specific embodiment, the polyethylene glycol portion of any polyethylene glycol-modified lipid has a molecular weight of 0.1-5 kDa, preferably 0.5-2 kDa, and more preferably 500 Da, 1000 Da, or 2000 Da.

[0176] In one specific embodiment, the polyethylene glycolated lipid in the lipid composition is a combination of a non-targeted polyethylene glycolated lipid and a targeted polyethylene glycolated lipid.

[0177] One embodiment of this application:

[0178] A lipid pharmaceutical composition comprising any of the aforementioned lipid compositions, and further comprising one or more pharmaceutically active ingredients.

[0179] In one specific embodiment, the active pharmaceutical ingredient is a nucleic acid, a small molecule, an oligopeptide, a polypeptide, or a protein, preferably a nucleic acid, more preferably DNA, RNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir, or ribozyme, and most preferably mRNA or siRNA.

[0180] In one specific embodiment, the lipid drug composition is an LNP drug composition, an LPP drug composition, or a PNP drug composition, preferably an LNP drug composition, more preferably an LNP-nucleic acid drug composition, and most preferably an LNP-mRNA drug composition. Wherein, "LNP drug composition" is a drug composition in the form of lipid nanoparticles, "LPP drug composition" is a drug composition in the form of lipopolyplexes, and "PNP drug composition" is a drug composition in the form of polypeptide nanoparticles; wherein, "LNP-nucleic acid drug composition" is an LNP drug composition loaded with nucleic acids, and "LNP-mRNA drug composition" is an LNP drug composition loaded with mRNA.

[0181] In one specific embodiment, the lipid pharmaceutical composition is used as a therapeutic agent, preferably selected from any one of antitumor agents, antibiotics, antiviral agents, antifungal agents, antiparasitic agents, and vaccines.

[0182] In one specific embodiment, the active pharmaceutical ingredient in the lipid pharmaceutical composition includes, but is not limited to, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracycline, nitrogen mustard, thiotepa, chlorambucil, lactamase, mefenamic acid, carmustine, romustine, busulfan, dibromomannitol, mitomycin C, cisdichlorodiamine cycloplatin(II), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine, debucaine, chlorpromazine, propranolol, demoproxil, labetalol, clonidine, hydralazine, imipramine, and amitriptyline. Doxepin, phenytoin, diphenhydramine, chlorpheniramine, promethazine, gentamicin, ciprofloxacin, cefoxitin, miconazole, teconazole, econazole, isoconazole, butonazole, clotrimazole, itraconazole, nystatin, neftifine, amphotericin B, antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma eye drops, vitamins, sedatives, imaging agents, paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, colchicine, daunorubicin, dihydroxyanthraquinone, safflowerin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, puromycin, serotonin.

[0183] In one specific embodiment, the active pharmaceutical ingredient in the lipid pharmaceutical composition is a nucleic acid, and the N / P ratio is (0.1-100):1, preferably (0.2-30):1, more preferably (0.5-20):1.

[0184] In one specific embodiment, the lipid drug composition is an LNP-nucleic acid drug composition, the preparation of which includes the following steps:

[0185] (1) Dissolve the lipid component in an organic solvent to obtain an organic phase solution;

[0186] (2) Dissolve the nucleic acid drug in a buffer solution to obtain an aqueous solution;

[0187] (3) Mix the organic phase solution and the aqueous phase solution to form nanoparticles by combining the lipid component with the nucleic acid drug;

[0188] (4) Remove organic solvents and free molecules by ultrafiltration washing, filter with a sterile filter, and set aside;

[0189] The organic solvent is preferably any one or a mixture of more than one of methanol, ethanol, propanol, tert-butanol, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone; the buffer solution is preferably a citrate buffer solution; preferably, the concentration of the buffer solution is 5-80 mM and the pH is 2-6; more preferably, the concentration of the buffer solution is 10-50 mM and the pH is 3-5; the volume ratio of the organic phase solution to the aqueous phase solution is preferably 1:1-10, more preferably 1:2 or 1:3.

[0190] In one specific embodiment, the lipid pharmaceutical composition is an LNP pharmaceutical composition with a particle size of 1 to 1000 nm, preferably 20 to 500 nm, more preferably 60 to 200 nm, and most preferably 60 to 150 nm.

[0191] One embodiment of this application:

[0192] A lipid pharmaceutical composition formulation comprising any of the aforementioned lipid pharmaceutical compositions, and further comprising a working solution; wherein the working solution is preferably a pharmaceutically acceptable carrier, more preferably any one of deionized water, ultrapure water, phosphate buffer, and physiological saline, and more preferably phosphate buffer or physiological saline.

[0193] In one specific embodiment, the ratio of the lipid drug composition to the working solution in the lipid drug composition formulation is not particularly limited. Preferably, the ratio of lipid drug composition to working solution is 0.05-20 g: 100 mL, more preferably, the ratio of lipid drug composition to working solution is 0.1-10 g: 100 mL, and most preferably, the ratio of lipid drug composition to working solution is 0.2-5 g: 100 mL. 4. Detailed Implementation

[0194] The following specific examples further describe the preparation of asymmetric cationic lipids and lipid pharmaceutical compositions, as well as related bioactivity tests, and do not limit the scope of this application. Intermediates and final products can be purified by methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, supercritical extraction, and column chromatography. The structures in the following examples are derived from... 1 Its molecular weight was determined by H NMR and confirmed by mass spectrometry.

[0195] Example 1: Preparation of cationic lipid G-01

[0196] Step a: N-methylpiperazine (f15, 4.0 mmol, 0.40 g, 1.0 eq), K11 (4.8 mmol, 1.70 g, 1.2 eq; obtained by esterification of 5-hexen-1-ol and 2-hexylundecanoic acid, followed by epoxidation with m-chloroperoxybenzoic acid), and N,N-diisopropylethylamine (DIPEA, 8.0 mmol, 1.03 g, 2.0 eq) were dissolved in ethanol (EtOH, 20 mL) and reacted with the solution at 60 °C for 10 hours. After the reaction was complete, the mixture was diluted with dichloromethane and washed with saturated sodium chloride solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the lipid monomer M-01 (1.53 g, 84%).

[0197] Step b: 2-(methylamino)ethanol (m14, 4.0 mmol, 0.30 g, 1.0 eq), 6-bromohexyl-2-hexyldecanoate (b12, 4.8 mmol, 2.01 g, 1.2 eq; obtained by esterification of 2-hexyldecanoic acid and 6-bromohexanol), potassium carbonate (K2CO3, 12.0 mmol, 1.66 g, 3.0 eq), and potassium iodide (KI, 4.0 mmol, 0.66 g, 1.0 eq) were sequentially added to acetonitrile (ACN, 20 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-02 (1.27 g, 77%).

[0198] Step c: Dissolve M-01 (2.0 mmol, 0.91 g, 1.0 eq) in dichloromethane (DCM, 20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and N,N'-succinimide carbonate (DSC, 2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-02 (2.0 mmol, 0.83 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-01 (1.60 g, 89%). 1H NMR(400MHz, CDCl3)δ:4.86-4.76(m,1H,>CHOC(=O)O-),4.22(t,2H,-CH2OC(=O)O-),4.08(t,4H,-CH2OC(= O)-),2.72-2.31(m,22H,-CH2N<,>CHC(=O)O-,>NCH3),1.65-1.21(m,62H,-CH2-),0.88(t,12H,-CH2CH3). MS(ESI):m / z=894.5([M+H] + ).

[0199] Example 2: Preparation of cationic lipid G-02

[0200] Step a: Under an argon atmosphere, Boc-L-glutamic acid (p1-Boc, 8.0 mmol, 1.98 g, 1.0 eq), dodecanol (e1, 8.0 mmol, 1.49 g, 1.0 eq), and 4-dimethylaminopyridine (DMAP, 1.6 mmol, 0.20 g, 0.2 eq) were sequentially added to a flask containing 20 mL of dichloromethane. The mixture was stirred in an ice bath, and a solution (25 mL) of N,N'-dicyclohexylcarbodiimide (DCC, 12.0 mmol, 2.48 g, 1.5 eq) in dichloromethane was slowly added dropwise. The ice bath was removed, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain intermediate INT2-1 (2.81 g, 85%).

[0201] Step b: Dissolve INT2-1 (6.0 mmol, 2.49 g, 1.0 eq) in dichloromethane (30 mL), and then add N',N'-dimethylpropane-1,3-diamine (a4, 7.2 mmol, 0.74 g, 1.2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl, 9.0 mmol, 1.73 g, 1.5 eq), 1-hydroxybenzotriazole (HOBt, 9.0 mmol, 1.22 g, 1.5 eq), and triethylamine (TEA, 18.0 mmol, 1.82 g, 3.0 eq) sequentially. Stir the reaction mixture overnight at room temperature. After the reaction is complete, dilute with dichloromethane (30 mL), and then wash successively with 1M HCl (30 mL * 2), saturated saline solution (30 mL * 2), and saturated sodium bicarbonate solution (30 mL * 2). The organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain the lipid monomer M-03-Boc (2.37 g, 79%).

[0202] Step c: M-03-Boc (4.0 mmol, 2.00 g, 1.0 eq) was dissolved in a trifluoroacetic acid / dichloromethane mixture (TFA / DCM, 1:1 v / v; 20 mL), and the mixture was stirred for 3 hours to remove Boc. After the reaction was complete, the mixture was washed with purified water (10 mL * 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the lipid monomer M-03 (1.46 g, 91%), which was used for the next reaction without purification.

[0203] Step d: M-02 (2.0 mmol, 0.83 g, 1.0 eq) was dissolved in dichloromethane (20 mL), and DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq) were added. The mixture was stirred overnight at room temperature. M-03 (2.0 mmol, 0.80 g, 1.0 eq) was added, and the mixture was reacted at room temperature for 3 hours. After the reaction was complete, the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain asymmetric cationic lipid G-02 (1.41 g, 84%). 1 H NMR (400MHz, CDCl3) δ: 4.21-4.12(m,3H,Glu-α-CH<,-CH2OC(=O)NH-), 4.10-4.01(m,4H,-CH2OC(=O)-), 3. 40-3.30(m,2H,-C(=O)NHCH2-),2.61(t,2H,>NCH2CH2OC(=O)NH-),2.47-2.35(m,6H,Glu-γ-CH2-,-CH2N<), 2.30-2.23(m,9H,-N(CH3)-),2.19-2.13(m,2H,Glu-β-CH2-),1.96-1.87(m,1H,>CHC(=O)O-)1.70-1.56(m, 8H,-CH2CH<,-CH2CH2OC(=O)-,-C(=O)NHCH2CH2-), 1.50-1.25(m,46H,-CH2-), 0.92-0.84(m,9H,-CH2CH3). MS(ESI):m / z=839.5([M+H] + ).

[0204] Example 3: Preparation of cationic lipid G-03

[0205] Step a: 1-(2-N-Boc-aminoethyl)piperazine (m8-Boc, 4.0 mmol, 0.92 g, 1.0 eq), heptadecano-9-yl 8-bromooctanoate (b9, 4.8 mmol, 2.22 g, 1.2 eq; obtained by esterification of 8-bromooctanoic acid and 9-heptadecanoyl alcohol), potassium carbonate (12.0 mmol, 1.66 g, 3.0 eq), and potassium iodide (4.0 mmol, 0.66 g, 1.0 eq) were sequentially added to acetonitrile (30 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-04-Boc (2.22 g, 91%).

[0206] Step b: 2-(methylamino)ethanol (m14, 4.0 mmol, 0.30 g, 1.0 eq), undecyl 6-bromohexanoate (b3, 4.8 mmol, 1.68 g, 1.2 eq; obtained by esterification of 6-bromohexanoate and undecyl alcohol), potassium carbonate (12.0 mmol, 1.66 g, 3.0 eq), and potassium iodide (4.0 mmol, 0.66 g, 1.0 eq) were sequentially added to acetonitrile (30 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-05 (1.18 g, 86%).

[0207] Step c: Dissolve M-04-Boc (3.0 mmol, 1.83 g, 1.0 eq) in a TFA / DCM mixture (1:1 v / v; 20 mL) and stir for 3 hours to remove Boc. After the reaction, wash with purified water (10 mL * 2). Dry the organic phase with anhydrous sodium sulfate and filter. Concentrate the filtrate under reduced pressure to obtain lipid monomer M-04 (1.43 g, 93%), which is used in the next step without purification.

[0208] Step d: M-05 (2.0 mmol, 0.69 g, 1.0 eq) was dissolved in dichloromethane (20 mL), and DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq) were added. The mixture was stirred overnight at room temperature. M-04 (2.0 mmol, 1.02 g, 1.0 eq) was added, and the mixture was reacted at room temperature for 3 hours. After the reaction was complete, the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain asymmetric cationic lipid G-03 (1.44 g, 82%). 1H NMR(400MHz, CDCl3)δ:4.86-4.80(m,1H,>CH-),4.23-4.03(m,4H,-CH2OC(=O)NH-,-C(=O)OCH2-),3.32-3.21(m,2H,-CH2NHC(=O)O-),2.71-2.4 0(m,16H,-CH2N<),2.33(s,3H,>NCH3),2.30(t,2H,-CH2C(=O)O-),2.28(t,2H,-CH2C(=O)O-),1.64-1.23(m,62H,-CH2-),0.88(t,9H,-CH2CH3). MS(ESI):m / z=879.5([M+H] + ).

[0209] Example 4: Preparation of cationic lipid G-04

[0210] Step a: 3-Amino-1-propanol (m34, 6.0 mmol, 0.45 g, 1.2 eq) and K7 (5.0 mmol, 1.91 g, 1.0 eq; obtained by esterification of 6-heptenoic acid and 9-heptadecyl alcohol, followed by epoxidation with m-chloroperoxybenzoic acid) were dissolved in ethanol (25 mL) and stirred overnight at room temperature. After the reaction was complete, the reaction solution was concentrated. The residue was purified by column chromatography to give intermediate INT4-1 (1.88 g, 82%).

[0211] Step b: INT4-1 (4.0 mmol, 1.83 g, 1.0 eq), 1-(2-chloroethyl)pyrrolidine hydrochloride (g1-HCl, 4.8 mmol, 0.82 g, 1.2 eq), potassium carbonate (16.8 mmol, 2.32 g, 4.2 eq), and potassium iodide (5.6 mmol, 0.93 g, 1.4 eq) were added sequentially to acetonitrile (30 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-06 (1.75 g, 79%).

[0212] Step c: 4-Piperidinol (m16, 3.0 mmol, 0.39 g, 1.0 eq), b3 (3.6 mmol, 1.26 g, 1.2 eq), potassium carbonate (9.0 mmol, 1.24 g, 3.0 eq), and potassium iodide (3.0 mmol, 0.50 g, 1.0 eq) were sequentially added to acetonitrile (15 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-07 (1.09 g, 91%).

[0213] Step d: M-06 (2.0 mmol, 1.11 g, 1.0 eq) was dissolved in dichloromethane (20 mL), and DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq) were added. The mixture was stirred overnight at room temperature. M-07 (2.0 mmol, 0.80 g, 1.0 eq) was added, and the mixture was reacted at room temperature for 3 hours. After the reaction was complete, the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain asymmetric cationic lipid G-04 (1.64 g, 84%). 1 H NMR(400MHz, CDCl3)δ:4.86-4.80(m,1H,>CHOC(=O)-),4.17-4.11(m,4H,-CH2OC(=O)O CH2-),4.05(t,2H,-CH2OC(=O)-),3.65-3.60(m,1H,-CH(OH)-),2.98-2.89(m,2H,>NCH a H b -),2.69-2.36(m,14H,>NCH2-),2.30(t,2H,-OC(=O)CH2-),2.28(t,2H,-OC(=O)CH2-),1.98-1.66(m,6H,>NCH a H b -,-CH2-),1.64-1.22(m,67H,-CH2-,>CH-),0.88(t,9H,-CH2CH3). MS(ESI):m / z=978.6([M+H] + ).

[0214] Example 5: Preparation of cationic lipid G-05

[0215] Step a: 3-(methylamino)-1-propanol (m23, 4.0 mmol, 0.36 g, 1.0 eq), b12 (4.8 mmol, 2.01 g, 1.2 eq), potassium carbonate (12.0 mmol, 1.66 g, 3.0 eq), and potassium iodide (4.0 mmol, 0.66 g, 1.0 eq) were sequentially added to acetonitrile (25 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-08 (1.42 g, 83%).

[0216] Step b: N-hydroxyethylpiperazine (m7, 3.0 mmol, 0.39 g, 1.0 eq), b12 (3.6 mmol, 1.51 g, 1.2 eq), potassium carbonate (9.0 mmol, 1.24 g, 3.0 eq), and potassium iodide (3.0 mmol, 0.50 g, 1.0 eq) were sequentially added to acetonitrile (20 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-09 (1.04 g, 74%).

[0217] Step c: Dissolve M-08 (2.0 mmol, 0.86 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-09 (2.0 mmol, 0.94 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-05 (1.51 g, 82%). 4.25(t,2H,-CH2OC(=O)O-),4.18(t,2H,-CH2OC(=O)O-),4.06(t,4H,-C(=O)OCH2-),2.6 7(t,2H,>NCH2CH2OC(=O)O-),2.64-2.35(m,10H,>NCH2CH2N<,>CH-),2.35-2.27(m,6H,>N CH2-),2.20(s,3H,>NCH3),1.87-1.80(m,2H,>NCH2CH2CH2OC(=O)O-),1.66-1.53(m,8H, -CH2CH<),1.52-1.23(m,56H,-CH2-),0.87(t,12H,-CH2CH3).MS(ESI):m / z=922.6([M+H] + ).

[0218] Example 6: Preparation of cationic lipid G-06

[0219] Step a: Under an argon atmosphere, p1-Boc (6.0 mmol, 1.48 g, 1.0 eq), oleyl alcohol (e11, 6.0 mmol, 1.61 g, 1.0 eq), and DMAP (1.2 mmol, 0.15 g, 0.2 eq) were sequentially added to a flask containing 20 mL of dichloromethane. The mixture was stirred in an ice bath, and a solution of DCC (9.0 mmol, 1.86 g, 1.5 eq) in dichloromethane (20 mL) was slowly added dropwise. The ice bath was removed, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain intermediate INT10-1 (2.67 g, 89%).

[0220] Step b: Dissolve INT10-1 (5.0 mmol, 2.49 g, 1.0 eq) in dichloromethane (30 mL), and then add a4 (6.0 mmol, 0.61 g, 1.2 eq), EDC HCl (7.5 mmol, 1.44 g, 1.5 eq), HOBt (7.5 mmol, 1.01 g, 1.5 eq), and TEA (15.0 mmol, 1.52 g, 3.0 eq) sequentially. Stir the reaction mixture overnight at room temperature. After the reaction is complete, dilute with dichloromethane (30 mL), and then wash sequentially with 1M HCl (30 mL * 2), saturated saline solution (30 mL * 2), and saturated sodium bicarbonate solution (30 mL * 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain the lipid monomer M-10-Boc (2.31 g, 79%).

[0221] Step c: 4-Hydroxymethylpiperidine (m6, 3.0 mmol, 0.35 g, 1.0 eq), e11-Ms (3.6 mmol, 1.25 g, 1.2 eq; obtained by methanesulfonation of e11), potassium carbonate (9.0 mmol, 1.24 g, 3.0 eq), and potassium iodide (3.0 mmol, 0.50 g, 1.0 eq) were sequentially added to acetonitrile (15 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-11 (0.93 g, 85%).

[0222] Step d: M-10-Boc (3.0 mmol, 1.75 g, 1.0 eq) was dissolved in a TFA / DCM mixture (1:1 v / v; 20 mL), and the mixture was stirred for 3 hours to remove Boc. After the reaction was complete, the mixture was washed with purified water (10 mL * 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the lipid monomer M-10 (1.30 g, 90%), which was used for the next reaction without purification.

[0223] Step e: Dissolve M-11 (2.0 mmol, 0.73 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-10 (2.0 mmol, 0.96 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-06 (1.39 g, 80%).1 H NMR(400MHz, CDCl3)δ:5.39-5.29(m,4H,-CH2-CH=CH-CH2-),4.19-4.10(m,1H,Glu-α-CH<),4.06 (t,2H,-CH2OC(=O)-),3.91(d,2H,-CH2OC(=O)NH-),3.35(q,2H,-C(=O)NHCH2-),2.96(d,2H,>NCH a H b -),2.53-2.30(m,6H,Glu-γ-CH2-,-CH2N<),2.24(s,6H,-N(CH3)2),2.18-2.11(m,1H,Glu-β-CH a H b -),2.04-1.91(m,12H,-CH2-CH=CH-CH2-,>NCH a H b -,Glu-β-CH a H b -,>NCH2CH2CH<),1.75-1.56(m,8H,-CH2CH2OC(=O)-,>NCH2CH2CH<,-C(=O)NHCH2CH2CH2N<),1.54-1.26(m,46H,-CH2-),0.88(t,6H,-CH2CH3). MS(ESI):m / z=873.5([M+H] + ).

[0224] Example 7: Preparation of cationic lipid G-07

[0225] Step a: Under an argon atmosphere, p1-Boc (6.0 mmol, 1.48 g, 1.0 eq), e13 (6.0 mmol, 1.63 g, 1.0 eq; obtained by esterification of lauric acid and 1,4-butanediol), and DMAP (1.2 mmol, 0.15 g, 0.2 eq) were sequentially added to a flask containing 20 mL of dichloromethane. The mixture was stirred in an ice bath, and a solution of DCC (9.0 mmol, 1.86 g, 1.5 eq) in dichloromethane (20 mL) was slowly added dropwise. The ice bath was removed, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain intermediate INT7-1 (2.50 g, 83%).

[0226] Step b: Under an argon atmosphere, INT7-1 (4.0 mmol, 2.01 g, 1.0 eq), 3-dimethylamino-1-propanol (a25, 4.8 mmol, 0.50 g, 1.2 eq), and DMAP (0.8 mmol, 0.10 g, 0.2 eq) were sequentially added to a flask containing 20 mL of dichloromethane. The mixture was stirred in an ice bath, and a solution of DCC (6.0 mmol, 1.24 g, 1.5 eq) in dichloromethane (15 mL) was slowly added dropwise. The ice bath was removed, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain the lipid monomer M-12-Boc (2.05 g, 87%).

[0227] Step c: 2-(methylamino)ethanol (m14, 3.0 mmol, 0.23 g, 1.0 eq), b9 (3.6 mmol, 1.66 g, 1.2 eq), potassium carbonate (9.0 mmol, 1.24 g, 3.0 eq), and potassium iodide (3.0 mmol, 0.50 g, 1.0 eq) were sequentially added to acetonitrile (20 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-13 (1.17 g, 86%).

[0228] Step d: M-12-Boc (3.0 mmol, 1.76 g, 1.0 eq) was dissolved in a TFA / DCM mixture (1:1 v / v; 20 mL), and the mixture was stirred for 3 hours to remove Boc. After the reaction was complete, the mixture was washed with purified water (10 mL * 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the lipid monomer M-12 (1.34 g, 92%), which was used for the next reaction without purification.

[0229] Step e: Dissolve M-13 (2.0 mmol, 0.91 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-12 (2.0 mmol, 0.97 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-07 (1.41 g, 73%). 1H NMR(400MHz, CDCl3)δ:4.88-4.83(m,1H,>CH-),4.42(q,1H,Glu-α-CH<),4.14(t,2H, -C(=O)OCH2-),4.12(t,2H,-CH2OC(=O)NH-),4.06-4.03(m,4H,-CH2C(=O)O-),2.55(t ,2H,-CH2CH2OC(=O)NH-),2.47-2.31(m,11H,Glu-γ-CH2-,-N(CH3)-,-CH2C(=O)O-,-C H2N<),2.28(t,2H,-CH2C(=O)O-),2.26(s,6H,-N(CH3)2),2.25-2.19(m,1H,Glu-β-CH a H b -), 2.04-1.96(m,1H,Glu-β-CH a H b -),1.80-1.23(m,62H,-CH2-),0.88(t,9H,-CH2CH3). MS(ESI):m / z=968.5([M+H] + ).

[0230] Example 8: Preparation of cationic lipid G-08

[0231] Step a: Under an argon atmosphere, p1-Boc (8.0 mmol, 1.98 g, 1.0 eq), linolenic acid (e12, 8.0 mmol, 2.13 g, 1.0 eq), and DMAP (1.6 mmol, 0.20 g, 0.2 eq) were sequentially added to a flask containing 20 mL of dichloromethane. The mixture was stirred in an ice bath, and a solution of DCC (12.0 mmol, 2.48 g, 1.5 eq) in dichloromethane (25 mL) was slowly added dropwise. The ice bath was removed, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain intermediate INT8-1 (3.53 g, 89%).

[0232] Step b: Dissolve INT8-1 (6.0 mmol, 2.97 g, 1.0 eq) in dichloromethane (30 mL), and then add (1-methylpiperidin-4-yl)methylamine (a9, 7.2 mmol, 0.92 g, 1.2 eq), EDC HCl (9.0 mmol, 1.73 g, 1.5 eq), HOBt (9.0 mmol, 1.22 g, 1.5 eq), and TEA (18.0 mmol, 1.82 g, 3.0 eq) sequentially. Stir the reaction mixture overnight at room temperature. After the reaction is complete, dilute with dichloromethane (30 mL), and then wash sequentially with 1M HCl (30 mL * 2), saturated saline solution (30 mL * 2), and saturated sodium bicarbonate solution (30 mL * 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain the lipid monomer M-14-Boc (2.74 g, 75%).

[0233] Step c: Under an argon atmosphere, (2,2-dimethyl-1,3-dioxacyclopentan-4-yl)methanol (p12, 5.0 mmol, 0.66 g, 1.0 eq), linoleic acid (c7, 6.0 mmol, 1.68 g, 1.2 eq), and DMAP (1.0 mmol, 0.12 g, 0.2 eq) were sequentially added to a flask containing 20 mL of dichloromethane. The mixture was stirred in an ice bath, and a solution of DCC (7.5 mmol, 1.55 g, 1.5 eq) in dichloromethane (15 mL) was slowly added dropwise. The ice bath was removed, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give intermediate INT8-2 (1.74 g, 88%).

[0234] Step d: Dissolve INT8-2 (4.0 mmol, 1.58 g, 1.0 eq) in methanol (20 mL), add Amberlyst-15 (H form; 0.63 g), and stir at room temperature for 16 hours. After the reaction is complete, filter to remove Amberlyst-15. Concentrate the filtrate under reduced pressure, and purify the residue by column chromatography to obtain the corresponding intermediate INT8-3 (1.35 g, 95%).

[0235] Step e: Under an argon atmosphere, INT8-3 (3.0 mmol, 1.06 g, 1.0 eq), 4-(dimethylamino)butyrate (d9-HCl, 3.6 mmol, 0.60 g, 1.2 eq), DMAP (0.6 mmol, 0.07 g, 0.2 eq), and DIPEA (4.5 mmol, 0.58 g, 1.5 eq) were sequentially added to a flask containing 10 mL of dichloromethane. The mixture was stirred in an ice bath, and a solution of DCC (4.5 mmol, 0.93 g, 1.5 eq) in dichloromethane (10 mL) was slowly added dropwise. The ice bath was removed, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain lipid monomer M-15 (1.17 g, 83%).

[0236] Step f: M-14-Boc (3.0 mmol, 1.82 g, 1.0 eq) was dissolved in a TFA / DCM mixture (1:1 v / v; 20 mL), and the mixture was stirred for 3 hours to remove Boc. After the reaction was complete, the mixture was washed with purified water (10 mL * 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the lipid monomer M-14 (1.45 g, 96%), which was used for the next reaction without purification.

[0237] Step g: Dissolve M-15 (2.0 mmol, 0.94 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-14 (2.0 mmol, 1.01 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-08 (1.58 g, 79%). 1 H NMR(400MHz, CDCl3)δ:5.49-5.27(m,8H,-CH=CH-),5.15(p,1H,>CHOC(=O)NH-),4.32-4.13(m,5H,-CH2O C(=O)-,Glu-α-CH<),4.06(t,2H,-C(=O)OCH2-),3.10-2.99(m,2H,-C(=O)NHCH2-),2.93-2.74(m,6H,-CH a H b N(CH3)-,-CH=CH-CH2-CH=CH-),2.51(dt,1H,Glu-γ-CH a Hb -),2.37(dt,1H,Glu-γ-CH a H b -),2.34(t,2H,-CH2N(CH3)2),2.31(t,2H,-CH2C(=O)O-),2.29(s,3H,>NCH3),2.28(t,2H,-CH2C(=O)O- ),2.22(s,6H,-N(CH3)2),2.19-2.12(m,1H,-C(=O)NHCH2CH<),2.12-1.98(m,9H,-CH2-CH=CH-,Glu-β-CH a H b -), 1.97-1.87(m,3H,-CH) a H b N(CH3)-,Glu-β-CH a H b ),1.66-1.22(m,40H,-CH2-),0.88(t,6H,-CH2CH3). MS(ESI):m / z=999.5([M+H] + ).

[0238] Example 9: Preparation of cationic lipid G-09

[0239] Step a: Under an argon atmosphere, glycidyl 4-toluenesulfonic acid (p11, 6.0 mmol, 1.37 g, 1.0 eq) and e14 (7.2 mmol, 2.57 g, 1.2 eq; obtained by esterification of 2-hexyldecanoic acid and 1,6-hexanediol) were dissolved in anhydrous dichloromethane (40 mL), and boron trifluoride diethyl ether (1.2 mmol, 0.17 g, 0.2 eq) was added. The reaction was stirred overnight at room temperature. After the reaction was completed, the reaction solution was concentrated, and the residue was purified by column chromatography to obtain intermediate INT9-1 (2.90 g, 83%).

[0240] Step b: INT9-1 (4.0 mmol, 2.34 g, 1.0 eq), f15 (4.8 mmol, 0.48 g, 1.2 eq), potassium carbonate (12.0 mmol, 1.66 g, 3.0 eq), and potassium iodide (4.0 mmol, 0.66 g, 1.0 eq) were added sequentially to acetonitrile (20 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-16 (1.54 g, 75%).

[0241] Step c: Dissolve N'-(3-aminopropyl)-N'-methylpropane-1,3-diamine (m31, 3.0 mmol, 0.44 g, 1.0 eq) in dichloromethane (15 mL), and then add c6 (3.0 mmol, 1.15 g, 1.0 eq; obtained by esterification of 9-heptadecyl alcohol and adipic acid), EDC HCl (4.5 mmol, 0.86 g, 1.5 eq), HOBt (4.5 mmol, 0.61 g, 1.5 eq), and TEA (9.0 mmol, 0.91 g, 3.0 eq) sequentially. Stir the reaction mixture overnight at room temperature. After the reaction is complete, dilute with dichloromethane (15 mL), and then wash successively with 1M HCl (15 mL x 2), saturated saline solution (15 mL x 2), and saturated sodium bicarbonate solution (15 mL x 2). The organic phase was dried with anhydrous magnesium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain lipid monomer M-17 (1.29 g, 84%).

[0242] Step d: M-16 (2.0 mmol, 1.03 g, 1.0 eq) was dissolved in dichloromethane (20 mL), and DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq) were added. The mixture was stirred overnight at room temperature. M-17 (2.0 mmol, 1.02 g, 1.0 eq) was added, and the mixture was reacted at room temperature for 3 hours. After the reaction was complete, the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain asymmetric cationic lipid G-09 (1.64 g, 78%). 1 H NMR(400MHz, CDCl3)δ:4.93-4.87(m,1H,-OCH2CH<),4.86-4.81(m,1H,>CH-),4.07(t,2H, -CH2OC(=O)-),3.57-3.39(m,4H,-CH2OCH2-),3.28(q,2H,-C(=O)NHCH2-),3.20-3.09(m,2 H,-CH2NHC(=O)O-),2.71-2.32(m,21H,-CH2N<,>CHC(=O)O-,-N(CH3)-),2.30(t,2H,-OC( =O)CH2-), 2.12(t,2H,-CH2C(=O)NH-), 1.68-1.20(m,68H,-CH2-), 0.87(t,12H,-CH2CH3). MS(ESI):m / z=1050.7([M+H] + ).

[0243] Example 10: Preparation of cationic lipid G-10

[0244] Step a: Under an argon atmosphere, p11 (6.0 mmol, 1.37 g, 1.0 eq) and e13 (7.2 mmol, 1.96 g, 1.2 eq) were dissolved in anhydrous dichloromethane (30 mL), and boron trifluoride diethyl ether (BF3OEt2, 1.2 mmol, 0.17 g, 0.2 eq) was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by column chromatography to obtain intermediate INT10-1 (2.61 g, 87%).

[0245] Step b: INT10-1 (4.0 mmol, 2.00 g, 1.0 eq), N,N',N'-trimethylethane-1,2-diamine (f1, 4.8 mmol, 0.49 g, 1.2 eq), potassium carbonate (12.0 mmol, 1.66 g, 3.0 eq), and potassium iodide (4.0 mmol, 0.66 g, 1.0 eq) were sequentially added to acetonitrile (20 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-18 (1.41 g, 82%).

[0246] Step c: M16 (4.0 mmol, 0.52 g, 1.0 eq), B9 (4.8 mmol, 2.22 g, 1.2 eq), potassium carbonate (12.0 mmol, 1.66 g, 3.0 eq), and potassium iodide (4.0 mmol, 0.66 g, 1.0 eq) were added sequentially to acetonitrile (25 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-19 (1.80 g, 88%).

[0247] Step d: M-18 (2.0 mmol, 0.86 g, 1.0 eq) was dissolved in dichloromethane (20 mL), and DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq) were added. The mixture was stirred overnight at room temperature. M-19 (2.0 mmol, 1.02 g, 1.0 eq) was added, and the mixture was reacted at room temperature for 3 hours. After the reaction was complete, the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain asymmetric cationic lipid G-10 (1.75 g, 91%). 1H NMR(400MHz, CDCl3)δ:5.16-5.09(m,1H,-OCH2CH<),4.85-4.80(m,1H,>CHOC(=O)-),4.14(t,2H, -CH2OC(=O)O-),4.05(t,2H,-CH2OC(=O)-),3.57-3.40(m,4H,-CH2OCH2-),2.98-2.89(m,2H,>NCH a H b -),2.69-2.40(m,8H,-CH2N<),2.33(s,3H,-N(CH3)-),2.30(t,2H,-OC(=O)CH 2-),2.28(t,2H,-OC(=O)CH2-),2.24(s,6H,-N(CH3)2),1.98-1.86(m,2H,>NCH a H b -),1.65-1.21(m,67H,-CH2-,>CH-),0.89(t,9H,-CH2CH3). MS(ESI):m / z=966.6([M+H] + ).

[0248] Example 11: Preparation of cationic lipid G-11

[0249] Step a: Under an argon atmosphere, INT8-3 (3.0 mmol, 1.06 g, 1.0 eq), 1-methylpiperidine-4-carboxylic acid (d10, 3.6 mmol, 0.52 g, 1.2 eq), and DMAP (0.6 mmol, 0.07 g, 0.2 eq) were sequentially added to a flask containing 10 mL of dichloromethane. The mixture was stirred in an ice bath, and a solution of DCC (4.5 mmol, 0.93 g, 1.5 eq) in dichloromethane (10 mL) was slowly added dropwise. The ice bath was removed, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain lipid monomer M-20 (1.23 g, 85%).

[0250] Step b: M6 (3.0 mmol, 0.35 g, 1.0 eq), E12-Ms (3.6 mmol, 1.24 g, 1.2 eq; obtained by methanesulfonation of E12), potassium carbonate (9.0 mmol, 1.24 g, 3.0 eq), and potassium iodide (3.0 mmol, 0.50 g, 1.0 eq) were sequentially added to acetonitrile (15 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-21 (0.81 g, 74%).

[0251] Step c: Dissolve M-20 (2.0 mmol, 0.96 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-21 (2.0 mmol, 0.73 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-11 (1.49 g, 86%). 1 H NMR(400MHz, CDCl3)δ:5.36-5.19(m,8H,-CH=CH-),5.06-5.01(m,1H,>CHOC(=O)O-),4.44-4.36(m,1H,>CHCH a H b OC(=O)-),4.30-4.19(m,1H,>CHCH a H b OC(=O)-),4.15-4.02(m,3H,>CHCH a H b OC(=O)-,-CH2OC(=O)O-),3.98-3.84(m,1H,>CHCH a H b OC(=O)-),3.70-3.56(m,3H,>NCH a H b -,-N(CH3)CH a H b -), 3.42-3.34(m,1H,-N(CH3)CH a H b -),2.91-2.86(m,2H,>NCH2-),2.74-2.68(m,6H,-CH=CH-CH2-CH=CH-,>NCHa H b -), 2.62-2.53(m,2H,-N(CH3)CH a H b -),2.47-2.39(m,1H,-OC(=O)CH<),2.29-2.24(t,2H,-OC(=O)CH2-),2.12-2.07(m,2H,-CH a H b CH<),2.02-1.93(m,16H,>NCH3,-CH<,-CH2CH<,-CH2-CH=CH-),1.72-1.64(m,2H,-CH a H b CH<),1.57-1.51(m,2H,-CH2CH2N<),1.31-1.19(m,32H,-CH2-),0.82(t,6H,-CH2CH3). MS(ESI):m / z=869.4([M+H] + ).

[0252] Example 12: Preparation of cationic lipid G-12

[0253] Step a: Under an argon atmosphere, p11 (6.0 mmol, 1.37 g, 1.0 eq) and e15 (7.2 mmol, 2.37 g, 1.2 eq; obtained by esterification of 1,4-butanediol and 2-hexyldecanoic acid) were dissolved in anhydrous dichloromethane (30 mL), and boron trifluoride diethyl ether (BF3OEt2, 1.2 mmol, 0.17 g, 0.2 eq) was added. The reaction was stirred overnight at room temperature. After the reaction was completed, the reaction solution was concentrated, and the residue was purified by column chromatography to obtain intermediate INT12-1 (2.60 g, 78%).

[0254] Step b: INT12-1 (4.0 mmol, 2.23 g, 1.0 eq), f1 (4.8 mmol, 0.49 g, 1.2 eq), potassium carbonate (12.0 mmol, 1.66 g, 3.0 eq), and potassium iodide (4.0 mmol, 0.66 g, 1.0 eq) were added sequentially to acetonitrile (20 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-22 (1.89 g, 81%).

[0255] Step c: Dissolve M-22 (2.0 mmol, 1.17 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-01 (2.0 mmol, 0.91 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-12 (1.66 g, 86%). 1 H NMR(400MHz, CDCl3)δ:5.11-5.04(m,1H,-OCH2CH<),4.88-4.79(m,1H,>CHCH2N<),4.07(t,4H,-CH2OC(=O)-),3.58-3.36(m,4H,-CH2 OCH2-),2.74-2.30(m,24H,-CH2N<,>CHC(=O)O-,>NCH3),2.25(s,6H,-N(CH3)2),1.67-1.23(m,58H,-CH2-),0.89(t,12H,-CH2CH3). MS(ESI):m / z=967.6([M+H] + ).

[0256] Example 13: Preparation of cationic lipid G-13

[0257] Step a: N,N-Dimethylethylenediamine (a1, 4.0 mmol, 0.35 g, 1.0 eq) and DIPEA (8.0 mmol, 1.03 g, 2.0 eq) were dissolved in dichloromethane (20 mL), and b3 (4.8 mmol, 1.68 g, 1.2 eq) was added with stirring. The reaction was carried out overnight at room temperature. After the reaction was completed, the reaction was quenched with deionized water (10 mL), and the mixture was allowed to stand to separate into layers. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to give intermediate INT13-1 (1.27 g, 89%).

[0258] Step b: Dissolve INT13-1 (3.0 mmol, 1.07 g, 1.0 eq) and DIPEA (6.0 mmol, 0.78 g, 2.0 eq) in dichloromethane (15 mL), and add 2-bromoethanol (S-1, 3.6 mmol, 0.45 g, 1.2 eq) with stirring. React overnight at room temperature. After the reaction is complete, quench the reaction with deionized water (5 mL) and allow to stand for phase separation. Wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate. Purify the residue by column chromatography to obtain lipid monomer M-23 (1.10 g, 92%).

[0259] Step c: Dissolve M-23 (2.0 mmol, 0.80 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-13 (2.0 mmol, 0.91 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-13 (1.53 g, 87%). 1 H NMR(400MHz, CDCl3)δ:4.84-4.77(m,1H,>CH-),4.26(t,2H,-CH2OC(=O)O-),4 .20(t,2H,-CH2OC(=O)O-),4.05(t,2H,-CH2OC(=O)-),2.70-2.35(m,12H,-CH 2N<),2.33(s,3H,>NCH3),2.30(t,2H,-CH2C(=O)O-),2.27(t,2H,-CH2C(=O)O -),2.23(s,6H,-N(CH3)2),1.65-1.18(m,62H,-CH2-),0.87(t,9H,-CH2CH3). MS(ESI): m / z = 882.6([M+H]) + ).

[0260] Example 14: Preparation of cationic lipid G-14

[0261] Step a: Dissolve a1 (4.0 mmol, 0.35 g, 1.0 eq) and DIPEA (8.0 mmol, 1.03 g, 2.0 eq) in dichloromethane (30 mL), and add b12 (4.8 mmol, 2.01 g, 1.2 eq) with stirring. React overnight at room temperature. After the reaction is complete, quench the reaction with deionized water (10 mL) and allow to stand for phase separation. Wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate. Purify the residue by column chromatography to obtain intermediate INT14-1 (1.55 g, 91%).

[0262] Step b: Dissolve INT14-1 (3.0 mmol, 1.28 g, 1.0 eq) and DIPEA (6.0 mmol, 0.78 g, 2.0 eq) in dichloromethane (15 mL), and add S-1 (3.6 mmol, 0.45 g, 1.2 eq) with stirring. React overnight at room temperature. After the reaction is complete, quench the reaction with deionized water (5 mL) and allow to stand for phase separation. Wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate. Purify the residue by column chromatography to obtain lipid monomer M-24 (1.26 g, 89%).

[0263] Step c: Dissolve M-24 (2.0 mmol, 0.94 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-02 (2.0 mmol, 0.83 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-14 (1.64 g, 90%). 1 H NMR(400MHz, CDCl3)δ:4.26(t,2H,-CH2OC(=O)O-),4.21(t,2H,-CH2OC(=O)O-),4.08(t,4H,-CH2OC(=O)-),2.68- 2.31(m,17H,-CH2N<,-N(CH3)-,>CH-), 2.22(s,6H,-N(CH3)2), 1.66-1.23(m,64H,-CH2-), 0.88(t,12H,-CH2CH3). MS(ESI):m / z=910.6([M+H] + ).

[0264] Example 15: Preparation of cationic lipid G-15

[0265] M-24 (2.0 mmol, 0.94 g, 1.0 eq) was dissolved in dichloromethane (20 mL), and DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq) were added. The mixture was stirred overnight at room temperature. M-09 (2.0 mmol, 0.94 g, 1.0 eq) was then added, and the mixture was reacted at room temperature for 3 hours. After the reaction was complete, the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain the asymmetric cationic lipid G-15 (1.69 g, 88%). 1 H NMR(400MHz, CDCl3)δ:4.30(t,2H,-CH2OC(=O)O-),4.23(t,2H,-CH2OC(=O)O-),4.10(t,4H,-C(=O)OCH2-),2.69 -2.30(m,22H,-CH2N<,>CHC(=O)O-),2.24(s,6H,-N(CH3)2),1.67-1.23(m,64H,-CH2-),0.89(t,12H,-CH2CH3). MS(ESI):m / z=965.6([M+H] + ).

[0266] Example 16: Preparation of cationic lipid G-16

[0267] Step a: Dissolve p1-Boc (6.0 mmol, 1.48 g, 1.0 eq) in dichloromethane (20 mL), and then add h8 (6.0 mmol, 2.13 g, 1.0 eq; obtained by amidation reaction of 2-hexyldecanoic acid and hexamethylenediamine), EDC HCl (9.0 mmol, 1.73 g, 1.5 eq), HOBt (9.0 mmol, 1.22 g, 1.5 eq), and TEA (18.0 mmol, 1.82 g, 3.0 eq) sequentially. Stir the reaction overnight at room temperature. After the reaction is complete, dilute with dichloromethane (20 mL), and then wash successively with 1M HCl (20 mL x 2), saturated brine (20 mL x 2), and saturated sodium bicarbonate solution (20 mL x 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain intermediate INT16-1 (3.15 g, 90%).

[0268] Step b: Dissolve INT16-1 (4.0 mmol, 2.34 g, 1.0 eq) in dichloromethane (20 mL), then add a4 (4.8 mmol, 0.49 g, 1.2 eq), EDC HCl (6.0 mmol, 1.15 g, 1.5 eq), HOBt (6.0 mmol, 0.81 g, 1.5 eq), and TEA (12.0 mmol, 1.21 g, 3.0 eq) sequentially. Stir the reaction mixture overnight at room temperature. After the reaction is complete, dilute with dichloromethane (25 mL), and wash successively with 1 M HCl (25 mL x 2), saturated saline solution (25 mL x 2), and saturated sodium bicarbonate solution (25 mL x 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain the lipid monomer M-25-Boc (2.18 g, 82%).

[0269] Step c: M6 (3.0 mmol, 0.35 g, 1.0 eq), B12 (3.6 mmol, 1.51 g, 1.2 eq), potassium carbonate (9.0 mmol, 1.24 g, 3.0 eq), and potassium iodide (3.0 mmol, 0.50 g, 1.0 eq) were added sequentially to acetonitrile (15 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-26 (1.21 g, 89%).

[0270] Step d: M-25-Boc (3.0 mmol, 2.00 g, 1.0 eq) was dissolved in a TFA / DCM mixture (1:1 v / v; 20 mL), and the mixture was stirred for 3 hours to remove Boc. After the reaction was complete, the mixture was washed with purified water (10 mL * 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the lipid monomer M-25 (1.60 g, 94%), which was used for the next reaction without purification.

[0271] Step e: Dissolve M-26 (2.0 mmol, 0.91 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-25 (2.0 mmol, 1.14 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-16 (1.54 g, 73%). 1H NMR(400MHz, CDCl3)δ:4.23-4.16(m,1H,Glu-α-CH<),4.09(t,2H,-C(=O)OCH2-),3. 93(d,2H,-CH2OC(=O)NH-),3.28-3.17(m,6H,-C(=O)NHCH2-),2.95-2.90(m,2H,>NCH a H b -),2.43-1.96(m,16H,>NCH2-,>CHC(=O)O-,Glu-γ-CH2-,>CHC(=O)NH-,-N(CH3)2,>NCH a H b -),1.72-1.21(m,73H,Glu-β-CH2-,-CH2-,>CH-),0.88(t,12H,-CH2CH3). MS(ESI):m / z=1047.7([M+H] + ).

[0272] Example 17: Preparation of cationic lipid G-17

[0273] Step a: Under an argon atmosphere, p12 (5.0 mmol, 0.66 g, 1.0 eq), oleic acid (c8, 6.0 mmol, 1.69 g, 1.2 eq), and DMAP (1.0 mmol, 0.12 g, 0.2 eq) were sequentially added to a flask containing 20 mL of dichloromethane. The mixture was stirred in an ice bath, and a solution of DCC (7.5 mmol, 1.55 g, 1.5 eq) in dichloromethane (15 mL) was slowly added dropwise. The ice bath was removed, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain intermediate INT17-1 (1.67 g, 84%).

[0274] Step b: Dissolve INT17-1 (4.0 mmol, 1.59 g, 1.0 eq) in methanol (20 mL), add Amberlyst-15 (H form; 0.63 g), and stir at room temperature for 16 hours. After the reaction is complete, filter to remove Amberlyst-15. Concentrate the filtrate under reduced pressure, and purify the residue by column chromatography to obtain the corresponding intermediate INT17-2 (1.29 g, 90%).

[0275] Step c: Under an argon atmosphere, INT17-2 (3.0 mmol, 1.07 g, 1.0 eq), d10 (3.6 mmol, 0.52 g, 1.2 eq), DMAP (0.6 mmol, 0.07 g, 0.2 eq), and DIPEA (4.5 mmol, 0.58 g, 1.5 eq) were sequentially added to a flask containing 10 mL of dichloromethane. The mixture was stirred in an ice bath, and a solution of DCC (4.5 mmol, 0.93 g, 1.5 eq) in dichloromethane (10 mL) was slowly added dropwise. The ice bath was removed, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain lipid monomer M-27 (1.25 g, 86%).

[0276] Step d: Dissolve m31 (3.0 mmol, 0.44 g, 1.0 eq) in dichloromethane (15 mL), then add c8 (3.0 mmol, 0.85 g, 1.0 eq), EDC HCl (4.5 mmol, 0.86 g, 1.5 eq), HOBt (4.5 mmol, 0.61 g, 1.5 eq), and TEA (9.0 mmol, 0.91 g, 3.0 eq) sequentially. Stir the reaction mixture overnight at room temperature. After the reaction is complete, dilute with dichloromethane (15 mL), then wash sequentially with 1M HCl (15 mL x 2), saturated saline solution (15 mL x 2), and saturated sodium bicarbonate solution (15 mL x 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain lipid monomer M-28 (1.08 g, 88%).

[0277] Step e: Dissolve M-27 (2.0 mmol, 0.96 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-28 (2.0 mmol, 0.82 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-17 (1.46 g, 80%). 1H NMR(400MHz, CDCl3)δ:5.38-5.32(m,4H,-CH2-CH=CH-CH2-),5.13(p,1H,>CHOC(=O)NH-),4.33-4.12(m, 4H,-CH2OC(=O)-),3.24(q,2H,-C(=O)NHCH2-),3.20-3.11(m,2H,-CH2NHC(=O)O-),2.87-2.77(m,2H,>NH a H b -),2.41-2.19(m,13H,>NCH2-,>NCH3,-OC(=O)CH<,-OC(=O)CH2-),2.13(t,2H,-CH2C(=O)NH-),2.02-1.96(m,14H,-CH2-CH=CH-CH2-,>NH a H b -,>CHCH2-),1.73-1.23(m,48H,-CH2-),0.88(t,6H,-CH2CH3). MS(ESI):m / z=917.5([M+H] + ).

[0278] Example 18: Preparation of cationic lipid G-18

[0279] Step a: F15 (4.0 mmol, 0.40 g, 1.0 eq), K7 (4.8 mmol, 1.84 g, 1.2 eq), and DIPEA (8.0 mmol, 1.03 g, 2.0 eq) were dissolved in ethanol (20 mL) and reacted at 60 °C with stirring for 10 hours. After the reaction was complete, the mixture was diluted with dichloromethane and washed with saturated sodium chloride solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain lipid monomer M-29 (1.61 g, 83%).

[0280] Step b: Dissolve M-29 (2.0 mmol, 0.97 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-05 (2.0 mmol, 0.69 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-18 (1.33 g, 78%). 1H NMR(400MHz, CDCl3)δ:4.89-4.78(m,2H,>CH-),4.23(t,2H,>NCH2CH2OC(=O)O-),4.05(t,2H,-CH2OC(=O)-),2.73-2.2 9(m,22H,-CH2N<,>NCH3,-CH2C(=O)O-), 2.28(t,2H,-CH2C(=O)O-), 1.64-1.23(m,58H,-CH2-), 0.88(t,9H,-CH2CH3). MS(ESI):m / z=852.5([M+H] + ).

[0281] Example 19: Preparation of cationic lipid G-19

[0282] Step a: Under an argon atmosphere, p1-Boc (6.0 mmol, 1.48 g, 1.0 eq), e15 (6.0 mmol, 1.97 g, 1.0 eq), and DMAP (1.2 mmol, 0.15 g, 0.2 eq) were sequentially added to a flask containing 20 mL of dichloromethane. The mixture was stirred in an ice bath, and a solution of DCC (9.0 mmol, 1.86 g, 1.5 eq) in dichloromethane (20 mL) was slowly added dropwise. The ice bath was removed, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give intermediate INT19-1 (2.80 g, 84%).

[0283] Step b: Under an argon atmosphere, INT19-1 (4.0 mmol, 2.23 g, 1.0 eq), 3-piperidin-1-ylpropanol (a27, 4.8 mmol, 0.69 g, 1.2 eq), and DMAP (0.8 mmol, 0.10 g, 0.2 eq) were sequentially added to a flask containing 20 mL of dichloromethane. The mixture was stirred in an ice bath, and a solution of DCC (6.0 mmol, 1.24 g, 1.5 eq) in dichloromethane (15 mL) was slowly added dropwise. The ice bath was removed, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain the lipid monomer M-30-Boc (2.48 g, 91%).

[0284] Step c: Dissolve M-30-Boc (3.0 mmol, 2.05 g, 1.0 eq) in a TFA / DCM mixture (1:1 v / v; 20 mL) and stir for 3 hours to remove Boc. After the reaction, wash with purified water (10 mL * 2). Dry the organic phase with anhydrous sodium sulfate and filter. Concentrate the filtrate under reduced pressure to obtain lipid monomer M-30 (1.63 g, 93%), which is used in the next step without purification.

[0285] Step d: M-26 (2.0 mmol, 0.91 g, 1.0 eq) was dissolved in dichloromethane (20 mL), and DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq) were added. The mixture was stirred overnight at room temperature. M-30 (2.0 mmol, 1.17 g, 1.0 eq) was added, and the mixture was reacted at room temperature for 3 hours. After the reaction was complete, the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain asymmetric cationic lipid G-19 (1.77 g, 83%). 1 H NMR(400MHz, CDCl3)δ:4.44(q,1H,Glu-α-CH<),4.12-4.17(m,6H,-CH2OC(=O)-) ,4.05(t,2H,-CH2OC(=O)-),3.92(d,2H,-CH2OC(=O)NH-),2.96-2.90(m,2H,>NCH a H b -),2.46-2.32(m,12H,Glu-γ-CH2-,>CHC(=O)O-,-CH2N<),2.25-2.18(m,1H,Glu-β-CH a H b -), 2.05-1.97(m,1H,Glu-β-CH a H b -), 1.97-1.90(m,2H,>NCH a H b -),1.72-1.23(m,73H,-CH2-,>CH-),0.88(t,12H,-CH2CH3). MS(ESI):m / z=1062.6([M+H] + ).

[0286] Example 20: Preparation of cationic lipid G-20

[0287] Step a: m8-Boc (4.0 mmol, 0.92 g, 1.0 eq), b3 (4.8 mmol, 1.68 g, 1.2 eq), potassium carbonate (12.0 mmol, 1.66 g, 3.0 eq), and potassium iodide (4.0 mmol, 0.66 g, 1.0 eq) were added sequentially to acetonitrile (30 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-31-Boc (1.63 g, 82%).

[0288] Step b: M-31-Boc (3.0 mmol, 1.49 g, 1.0 eq) was dissolved in a TFA / DCM mixture (1:1 v / v; 20 mL), and the mixture was stirred for 3 hours to remove Boc. After the reaction was complete, the mixture was washed with purified water (10 mL * 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the lipid monomer M-31 (1.14 g, 96%), which was used for the next reaction without purification.

[0289] Step c: Dissolve M-29 (2.0 mmol, 0.97 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-31 (2.0 mmol, 0.80 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-20 (1.40 g, 77%). 1 H NMR(400MHz, CDCl3)δ:4.85-4.75(m,2H,>CHOC(=O)O-,>CHOC(=O)-), 4.05(t,2H,-CH2OC(=O)-), 3.31-3.20(m,2H,-CH2NHC(=O)O-), 2.72-2.36(m,22H,-CH2N<), 2.33(s,3H,>NCH3), 2.32-2.26(m,4H,-CH2C(=O)O-), 1.66-1.22(m,58H,-CH2-), 0.87(t,9H,-CH2CH3). MS(ESI):m / z=906.6([M+H] + ).

[0290] Example 21: Preparation of cationic lipid G-21

[0291] Step a: Dissolve INT2-1 (6.0 mmol, 2.49 g, 1.0 eq) in dichloromethane (30 mL), and then add 3-dimethylamino-1-propanol (a35, 7.2 mmol, 0.74 g, 1.2 eq), EDC HCl (9.0 mmol, 1.73 g, 1.5 eq), HOBt (9.0 mmol, 1.22 g, 1.5 eq), and TEA (18.0 mmol, 1.82 g, 3.0 eq) sequentially. Stir the reaction mixture overnight at room temperature. After the reaction is complete, dilute with dichloromethane (30 mL), and then wash sequentially with 1M HCl (30 mL * 2), saturated saline solution (30 mL * 2), and saturated sodium bicarbonate solution (30 mL * 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain the lipid monomer M-32-Boc (2.44 g, 81%).

[0292] Step b: Ethanolamine (m47, 4.0 mmol, 0.24 g, 1.0 eq) and DIPEA (8.0 mmol, 1.03 g, 2.0 eq) were dissolved in dichloromethane (15 mL), and b3 (4.8 mmol, 1.68 g, 1.2 eq) was added with stirring. The reaction was carried out overnight at room temperature. After the reaction was completed, the reaction was quenched with deionized water (5 mL), and the mixture was allowed to stand to separate into layers. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography to give intermediate INT21-1 (1.18 g, 90%).

[0293] Step c: Dissolve INT21-1 (3.0 mmol, 0.99 g, 1.0 eq) and DIPEA (6.0 mmol, 0.78 g, 2.0 eq) in dichloromethane (20 mL), and add b9 (3.6 mmol, 1.66 g, 1.2 eq) with stirring. React overnight at room temperature. After the reaction is complete, quench the reaction with deionized water (5 mL) and allow to stand for phase separation. Wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate. Purify the residue by column chromatography to obtain lipid monomer M-33 (1.83 g, 86%).

[0294] Step d: M-32-Boc (3.0 mmol, 1.50 g, 1.0 eq) was dissolved in a TFA / DCM mixture (1:1 v / v; 20 mL), and the mixture was stirred for 3 hours to remove Boc. After the reaction was complete, the mixture was washed with purified water (10 mL * 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the lipid monomer M-32 (1.09 g, 91%), which was used for the next reaction without purification.

[0295] Step e: Dissolve M-33 (2.0 mmol, 1.42 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-32 (2.0 mmol, 0.80 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-21 (1.77 g, 78%). 1 H NMR(400MHz, CDCl3)δ:4.88-4.82(m,1H,>CH-),4.45(q,1H,Glu-α-CH<),4.12(t,2H,-C(=O)OCH2-),4.10(t,2H,-CH2OC(=O)NH-),4.06(t,4H,-CH 2C(=O)O-),2.74-2.30(m,12H,Glu-γ-CH2-,-CH2N<,-CH2C(=O)O-),2.27(t,2H,-CH2C(=O)O-),2.25(s,6H,-N(CH3)2),2.24-2.18(m,1H,Glu-β-CH a H b -), 2.04-1.97(m,1H,Glu-β-CH a H b -),1.77-1.19(m,84H,-CH2-),0.88(t,12H,-CH2CH3). MS(ESI):m / z=1136.7([M+H] + ).

[0296] Example 22: Preparation of cationic lipid G-22

[0297] Step a: M47 (3.0 mmol, 0.18 g, 1.0 eq), E12-Ms (7.2 mmol, 2.48 g, 2.4 eq), potassium carbonate (18.0 mmol, 2.49 g, 6.0 eq), and potassium iodide (6.0 mmol, 1.00 g, 2.0 eq) were added sequentially to acetonitrile (25 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-34 (1.16 g, 69%).

[0298] Step b: Dissolve M-15 (2.0 mmol, 0.94 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-34 (2.0 mmol, 1.12 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-22 (1.85 g, 88%). 1 H NMR(400MHz, CDCl3)δ:5.50-5.26(m,12H,-CH=CH-),5.25-5.20(m,1H,>CHOC(=O)O-),4.34-4.26(m,2H ,>CHCH2OC(=O)-),4.22(t,2H,-CH2OC(=O)O-),4.04(d,2H,>CHCH2OC(=O)-),2.86-2.74(m,6H,-CH=CH -CH2-CH=CH-),2.74-2.34(m,8H,-CH2N<),2.30(t,2H,-CH2C(=O)O-),2.26(t,2H,-CH2C(=O)O-),2.22 (s,6H,-N(CH3)2),2.11-1.98(m,12H,-CH2-CH=CH-),1.63-1.22(m,54H,-CH2-),0.88(t,9H,-CH2CH3). MS(ESI):m / z=1051.7([M+H] + ).

[0299] Example 23: Preparation of cationic lipid G-23

[0300] Step a: M47 (3.0 mmol, 0.18 g, 1.0 eq), B12 (7.2 mmol, 3.02 g, 2.4 eq), potassium carbonate (18.0 mmol, 2.49 g, 6.0 eq), and potassium iodide (6.0 mmol, 1.00 g, 2.0 eq) were added sequentially to acetonitrile (30 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-35 (1.65 g, 75%).

[0301] Step b: Dissolve M-35 (2.0 mmol, 1.48 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-02 (2.0 mmol, 0.83 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-23 (1.94 g, 82%). 1 H NMR(400MHz, CDCl3)δ:4.32-4.10(m,4H,-CH2OC(=O)O-),4.08(t,6H,-CH2OC(=O)-),2.71-2.40(m ,10H,-CH2N<),2.38-2.30(m,6H,>NCH3,>CH-),1.65-1.21(m,96H,-CH2-),0.88(t,18H,-CH2CH3). MS(ESI):m / z=1177.8([M+H] + ).

[0302] Example 24: Preparation of cationic lipid G-24

[0303] Step a: M47 (3.0 mmol, 0.18 g, 1.0 eq), B3 (7.2 mmol, 2.52 g, 2.4 eq), potassium carbonate (18.0 mmol, 2.49 g, 6.0 eq), and potassium iodide (6.0 mmol, 1.00 g, 2.0 eq) were added sequentially to acetonitrile (30 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-36 (1.56 g, 87%).

[0304] Step b: Dissolve M-36 (2.0 mmol, 1.20 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-19 (2.0 mmol, 1.02 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-24 (1.90 g, 84%). 1H NMR(400MHz, CDCl3)δ:4.87-4.80(m,1H,>CHOC(=O)-),4.21(t,2H,-CH2OC(=O)O- ),4.14(t,2H,-CH2OC(=O)O-),4.05(t,4H,-CH2OC(=O)-),2.97-2.87(m,2H,>NCH a H b -),2.71-2.40(m,8H,-CH2N<),2.34-2.25(m,6H,-OC(=O)CH2-),1.98-1.89(m,2H,>NCH a H b -),1.64-1.23(m,93H,-CH2-,>CH-),0.89(t,12H,-CH2CH3). MS(ESI):m / z=1133.8([M+H] + ).

[0305] Example 25: Preparation of cationic lipid G-25

[0306] Step a: M34 (3.0 mmol, 0.23 g, 1.0 eq), 1-bromotetradecane (b1, 7.2 mmol, 2.00 g, 2.4 eq), potassium carbonate (18.0 mmol, 2.49 g, 6.0 eq), and potassium iodide (6.0 mmol, 1.00 g, 2.0 eq) were added sequentially to acetonitrile (30 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-37 (1.15 g, 82%).

[0307] Step b: Dissolve M-37 (2.0 mmol, 0.94 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-04 (2.0 mmol, 1.02 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain asymmetric cationic lipid G-25 (1.61 g, 80%). 1H NMR(400MHz, CDCl3)δ:4.89-4.82(m,1H,>CH-),4.11(t,2H,-CH2OC(=O)NH-),3.36-3.24(m,2H,-CH2NHC(=O)O -),2.67-2.38(m,18H,-CH2N<),2.29(t,2H,-CH2C(=O)O-),1.65-1.21(m,88H,-CH2-),0.88(t,12H,-CH2CH3). MS(ESI):m / z=1003.8([M+H] + ).

[0308] Example 26: Preparation of cationic lipid G-26

[0309] Step a: Under an argon atmosphere, p1-Boc (6.0 mmol, 1.48 g, 1.0 eq), e17 (6.0 mmol, 3.17 g, 1.0 eq; synthesized according to the method in US20150203446A1), and DMAP (1.2 mmol, 0.15 g, 0.2 eq) were sequentially added to a flask containing 30 mL of dichloromethane. The mixture was stirred in an ice bath, and a solution of DCC (9.0 mmol, 1.86 g, 1.5 eq) in dichloromethane (20 mL) was slowly added dropwise. The ice bath was removed, and the reaction was stirred at room temperature for 24 h. After the reaction was complete, the precipitate was removed by filtration. The filtrate was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to give intermediate INT26-1 (4.00 g, 88%).

[0310] Step b: Dissolve INT26-1 (4.0 mmol, 3.03 g, 1.0 eq) in dichloromethane (30 mL), and then add a4 (4.8 mmol, 0.49 g, 1.2 eq), EDC HCl (6.0 mmol, 1.15 g, 1.5 eq), HOBt (6.0 mmol, 0.81 g, 1.5 eq), and TEA (12.0 mmol, 1.21 g, 3.0 eq) sequentially. Stir the reaction mixture overnight at room temperature. After the reaction is complete, dilute with dichloromethane (30 mL), and then wash sequentially with 1M HCl (30 mL * 2), saturated saline solution (30 mL * 2), and saturated sodium bicarbonate solution (30 mL * 2). Dry the organic phase with anhydrous magnesium sulfate, filter, concentrate, and purify the residue by column chromatography to obtain the lipid monomer M-38-Boc (2.88 g, 85%).

[0311] Step c: M-38-Boc (3.0 mmol, 2.53 g, 1.0 eq) was dissolved in a TFA / DCM mixture (1:1 v / v; 30 mL), and the mixture was stirred for 3 hours to remove Boc. After the reaction was complete, the mixture was washed with purified water (15 mL * 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the lipid monomer M-38 (2.06 g, 93%), which was used for the next reaction without purification.

[0312] Step d: M-20 (2.0 mmol, 0.96 g, 1.0 eq) was dissolved in dichloromethane (20 mL), and DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq) were added. The mixture was stirred overnight at room temperature. M-38 (2.0 mmol, 1.48 g, 1.0 eq) was added, and the mixture was reacted at room temperature for 3 hours. After the reaction was complete, the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography to obtain asymmetric cationic lipid G-26 (1.84 g, 74%). 1 H NMR(400MHz, CDCl3)δ:5.47-5.25(m,12H,-CH=CH-),5.12(p,1H,>CHOC(=O)NH-),4.88-4.80(m,1H,>CHOC(= O)-),4.31-4.12(m,5H,-CH2OC(=O)-,Glu-α-CH<),3.25-3.17(m,2H,-C(=O)NHCH2-),2.87-2.71(m,8H,>NH a H b -,-CH=CH-CH2-CH=CH-),2.50(dt,1H,Glu-γ-CH a H b -),2.38(t,2H,-CH2N(CH3)2),2.36(dt,1H,Glu-γ-CH a H b -),2.34(s,3H,>NCH3),2.32-2.18(m,3H,-CH2C(=O)O-,>CHC(=O)O-),2.23(s,6H,-N(CH3)2),2.11-1.69(m,20H,Glu-β-CH2-,>NH a H b -,>CHCH2-,-CH2-CH=CH-),1.66-1.24(m,58H,-CH2-),0.88(t,9H,-CH2CH3). MS(ESI):m / z=1247.8([M+H] + ).

[0313] Example 27: Preparation of cationic lipid G-27

[0314] Step a: Under an argon atmosphere, p11 (4.0 mmol, 0.91 g, 1.0 eq) and e15 (4.8 mmol, 1.58 g, 1.2 eq) were dissolved in anhydrous dichloromethane (20 mL), and boron trifluoride diethyl ether (0.8 mmol, 0.11 g, 0.2 eq) was added. The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by column chromatography to obtain intermediate INT27-1 (1.95 g, 88%).

[0315] Step b: INT27-1 (3.0 mmol, 1.67 g, 1.0 eq), m49-TBS (3.6 mmol, 0.89 g, 1.2 eq; obtained by monoalkylation of N,N'-dimethylethylenediamine with bromoethanol followed by protection of the hydroxyl group of the product), potassium carbonate (9.0 mmol, 1.24 g, 3.0 eq), and potassium iodide (3.0 mmol, 0.50 g, 1.0 eq) were sequentially added to acetonitrile (20 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-39 (1.56 g, 82%).

[0316] Step c: Dissolve M-24 (2.0 mmol, 0.94 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-39 (2.0 mmol, 1.26 g, 1.0 eq), and react for 3 hours at room temperature. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate. Dissolve the residue in tetrahydrofuran (THF, 15 mL), then add a 1 M tetrabutylammonium fluoride tetrahydrofuran solution (TBAF / THF, 5 mL), stir overnight, and remove TBS protection. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was dissolved in dichloromethane (15 mL), washed with saturated ammonium chloride solution, dried with anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain asymmetric cationic lipid G-27 (1.57 g, 77%). 1H NMR(400MHz, CDCl3)δ:5.12-5.06(m,1H,-OCH2CH<),4.25(t,2H,-CH2OC(=O)O-),4.07(t,4H,-CH2OC(=O)-),3.65-3.39(m,6H,-CH2OCH2- ,-CH2OH),2.68-2.29(m,24H,-CH2N<,>CHC(=O)O-,-N(CH3)-),2.23(s,6H,-N(CH3)2),1.66-1.20(m,60H,-CH2-),0.88(t,12H,-CH2CH3). MS(ESI):m / z=1013.7([M+H] + ).

[0317] Example 28: Preparation of cationic lipid G-28

[0318] Step a: 2-(methylamino)ethylcarbamate tert-butyl ester (m1-Boc, 4.0 mmol, 0.70 g, 1.0 eq), b3 (4.8 mmol, 1.68 g, 1.2 eq), potassium carbonate (12.0 mmol, 1.66 g, 3.0 eq), and potassium iodide (4.0 mmol, 0.66 g, 1.0 eq) were sequentially added to acetonitrile (20 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-40-Boc (1.47 g, 83%).

[0319] Step b: Dissolve 2-(tert-butyldimethylsiloxy)ethylamine (m47-TBS, 4.0 mmol, 0.70 g, 1.0 eq) and DIPEA (8.0 mmol, 1.03 g, 2.0 eq) in dichloromethane (25 mL), and add b9 (4.8 mmol, 2.22 g, 1.2 eq) with stirring. React overnight at room temperature. After the reaction is complete, quench the reaction with deionized water (5 mL), and allow to stand for phase separation. Wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, filter, and concentrate. Purify the residue by column chromatography to obtain intermediate INT28-1 (2.02 g, 91%).

[0320] Step c: INT28-1 (3.0 mmol, 1.67 g, 1.0 eq), 3-bromopropanol (S-2, 3.6 mmol, 0.50 g, 1.2 eq), potassium carbonate (9.0 mmol, 1.24 g, 3.0 eq), and potassium iodide (3.0 mmol, 0.50 g, 1.0 eq) were added sequentially to acetonitrile (20 mL), and the mixture was stirred overnight at 85 °C. After the reaction was complete, the mixture was cooled to room temperature. The reaction mixture was filtered through diatomaceous earth, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the lipid monomer M-41 (1.48 g, 80%).

[0321] Step d: M-40-Boc (3.0 mmol, 1.33 g, 1.0 eq) was dissolved in a TFA / DCM mixture (1:1 v / v; 20 mL), and the mixture was stirred for 3 hours to remove Boc. After the reaction was complete, the mixture was washed with purified water (10 mL * 2). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain the lipid monomer M-40 (0.99 g, 96%), which was used for the next reaction without purification.

[0322] Step e: Dissolve M-41 (2.0 mmol, 1.23 g, 1.0 eq) in dichloromethane (20 mL), add DIPEA (6.0 mmol, 0.78 g, 3.0 eq) and DSC (2.4 mmol, 0.61 g, 1.2 eq), and stir overnight at room temperature. Add M-40 (2.0 mmol, 0.69 g, 1.0 eq), and react at room temperature for 3 hours. After the reaction is complete, wash with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate. Dissolve the residue in tetrahydrofuran (15 mL), then add 1 M TBAF / THF solution (5 mL), stir overnight, and remove TBS protection. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was dissolved in dichloromethane (15 mL), washed with saturated ammonium chloride solution, dried with anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain asymmetric cationic lipid G-28 (1.52 g, 88%). 1H NMR(400MHz, CDCl3)δ:4.89-4.82(m,1H,>CHOC(=O)-),4.13(t,2H,-CH2OC(=O)NH-),4.06(t,2H,-CH2OC(=O)-),3.54(t,2H,-CH2OH),3.32-3.15(m,2H, -CH2NHC(=O)O-),2.57(t,2H,-CH2N<),2.48-2.24(m,12H,-CH2N<,-CH2C(=O )O-),2.21(s,3H,>NCH3),1.67-1.23(m,64H,-CH2-),0.88(t,9H,-CH2CH3). MS(ESI):m / z=868.5([M+H] + ).

[0323] Example 29: Preparation of symmetrical cationic lipids C-01, C-02, C-03 and C-04 (Comparative Example)

[0324] Referring to the aforementioned preparation method for G-15, symmetrical cationic lipids C-01 and C-02 were synthesized using two identical lipid monomers (M-09 or M-24), with the following structures:

[0325] Referring to the aforementioned preparation method for G-18, symmetrical cationic lipids C-03 and C-04 were synthesized using two identical lipid monomers (M-05 or M-29), with the following structures:

[0326] Example 30: Preparation and Characterization of Lipid Nanoparticles

[0327] A certain amount of lipid stock solution was transferred, and cationic lipids, DSPC, cholesterol, and PEG2k-DMG were dissolved in ethanol at a molar ratio of 50:10:38:1.5 to obtain an ethanol phase solution. Fluc-mRNA was added to 10–50 mM citrate buffer (pH = 4) to obtain an aqueous phase solution. The ethanol and aqueous phase solutions were mixed (1:3 v / v), and the external solution was replaced with PBS(-) by ultrafiltration using a centrifuge to obtain a nanoparticle solution. This solution was diluted to an appropriate concentration with PBS(-) before use. In the series of LNP-mRNAs prepared in this example, the cationic lipids included the asymmetric cationic lipids prepared in the previous examples, as well as the symmetric cationic lipids C-01, C-02, C-03, and C-04. Fluc-mRNA can express firefly luciferase, reacting with luciferin substrates to produce yellow-green fluorescence with a wavelength of 550–570 nm.

[0328] According to the literature (Hassett et al., J. Controlled Release 2021, 335, 237-246), LNP formulations encapsulating nucleic acid drugs exhibit better efficacy when the particle size is between 60 and 150 nm. In this embodiment, the particle size of LNP-mRNA was determined by dynamic light scattering (DLS), and the results are summarized in Table 1. The measured LNP-mRNA showed high size uniformity, with a PDI of less than 0.3 for all samples. The experimental results show that the LNP-mRNA prepared using the asymmetric cationic lipids of this application has a particle size of 70-90 nm, which is within the particle size range that can achieve good efficacy.

[0329] LNP-mRNA was ultracentrifuged (4℃, 60,000 rpm, 1 h) using an ultracentrifuge. The concentration of unencapsulated Fluc-mRNA in the supernatant was detected using a nucleic acid quantification instrument. The encapsulation efficiency of LNP for Fluc-mRNA was calculated, and the results are summarized in Table 1. The experimental results show that the lipid nanoparticles prepared using the asymmetric cationic lipids of this application have an encapsulation efficiency greater than 87%, indicating that the LNP of this application has a high encapsulation efficiency for nucleic acid drugs.

[0330] Table 1 - Summary of formulations of various lipid compositions and the particle size and encapsulation efficiency of the LNP-mRNA prepared from them.

[0331] Example 31: Serum stability evaluation

[0332] The LNP-mRNA prepared in Example 30 was added to a culture medium containing 10% fetal bovine serum (FBS) and stirred at 37°C. Samples were taken periodically to measure the particle size change of the LNP-mRNA. The experimental results showed that the particle size of the lipid nanoparticles prepared using the asymmetric cationic lipids of this application did not change significantly within 7 days.

[0333] Example 32: Cytotoxicity Evaluation

[0334] Prepare DMEM high-glucose complete medium (containing 10% FBS). Prepare working solutions of the sample (LNP-mRNA prepared in Example 30) using complete medium at concentrations of 0.1, 0.15, 0.2, 0.25, and 0.3 μg / 100 μL, and store for later use. Take 293T cells in logarithmic growth phase and administer at a rate of 7 × 10⁻⁶ cells / year. 3100 μL / well was seeded into 96-well plates. Both the control and sample groups had three replicates. After incubation for 24 h in a 5% CO2, 37°C incubator, the original culture medium was retained. The control group received 100 μL / well of complete culture medium, and the sample group received 100 μL / well of working solution. After another 24 h of incubation, 100 μL / well of medium containing 10% CCK-8 was added, and the plates were incubated for 2 h in a 5% CO2, 37°C incubator. The absorbance at 450 nm was measured using a microplate reader. The relative viability of the cells was calculated using the following formula:

[0335] Relative activity % = (sample absorbance value - background absorbance value) / (control group absorbance value - background absorbance value) × 100%; where the background absorbance value is the absorbance with only CCK-8 reagent and culture medium added.

[0336] Experimental results showed that the lipid drug nanoparticles prepared using the asymmetric cationic lipids of this application did not produce significant cytotoxicity at any of the five concentration gradients, and the cell survival rate was greater than 95%. Figure 1 shows the cytotoxicity test results of L-11.

[0337] Example 33: Evaluation of in vitro transfection effect

[0338] The LNP-mRNA prepared in Example 30 was added to the culture medium to prepare the required dose. Using 293T cells as a cell model, a seeding density of 6000 cells / well was established, and the cell suspension (100 μL / well) was seeded into 96-well plates with black borders and transparent bottoms. After seeding, the cells were incubated in a cell culture incubator for 24 h, and then 0.2 μg of Fluc-mRNA was administered per well. A positive control group was set up containing LNP-mRNAs containing C-01, C-02, C-03, and C-04, and an experimental group was set up containing LNP-mRNAs containing asymmetric cationic lipids from Examples 1 to 28. A blank control group was also set up, with the corresponding dose of free Fluc-mRNA added. 24 hours after transfection, the old culture medium was removed and replaced with a new culture medium containing D-fluorescein sodium (1.5 mg / mL) substrate. After incubation for 5 minutes, bioluminescence was detected using a microplate reader; stronger fluorescence indicated a greater amount of Fluc-mRNA that had entered the cytoplasm and completed translation. The experimental results are shown in Table 2, where the relative fluorescence intensity is the ratio of the fluorescence intensity of each group to that of the blank control group. The LNP-mRNA drug compositions prepared in this application all exhibit excellent in vitro transfection effects, meaning that the LNPs in the experimental groups are all effective nucleic acid delivery vectors. For example, the transfection effect of the experimental group containing G-15 was significantly better than that of the positive control group containing C-01 or C-02; the transfection effect of the experimental group containing G-18 was significantly better than that of the positive control group containing C-03 or C-04. This may be because the asymmetric cationic lipids of this application contain two different ionizable moieties. When the carbonate bond breaks in the cell, it initially degrades into two separate cationic lipid products with different ionizable groups. These independent cationic lipid products tend to redistribute, allowing the nucleic acid drug to more effectively escape from the endosome and improving drug delivery efficiency.

[0339] Table 2: Cell transfection test results

[0340] Example 34: Evaluation of in vivo transfection effect

[0341] L-11 was administered to 6-8 week old female BALB / c mice via tail vein injection at a dose of 10 μg / mouse, and in vivo fluorescence imaging was performed at 6, 12, and 24 hours after administration. After imaging at the last time point, the mice were euthanized, and imaging of major organs (heart, liver, spleen, lungs, and kidneys) was performed. 0.2 mL of D-fluorescein sodium (15 mg / mL) was injected intraperitoneally 10-15 min before imaging. The experimental results are shown in Figure 2. The lipid drug nanoparticles prepared from the asymmetric cationic lipids of this application can achieve highly efficient in vivo delivery of nucleic acid drugs.

Claims

1. An asymmetric cationic lipid, the structure of which is shown in formula (1): Or its salts, tautomers, stereoisomers, isotopic substitutes, or solvates; wherein... Each X is independently O, S, or NH; M1 and M2 are each selected independently. Any one of the following; Q is CH or N; p is 0, 1 or 2; Indicates a substituted or unsubstituted nitrogen heterocycle; either M1 or M2 is connected to R1 or R2; M1 and M2 have different specific structures; Each R M Independently for R S Or R L And it is bonded to any cyclic atom of a nitrogen atom or a nitrogen heterocycle; each R S Independently for C 1-5 Alkyl or C 1-5 Hydroxyalkyl; each R L Independently for substituted or unsubstituted C 6-40 hydrocarbon group, and the C 6-40 The skeleton of the hydrocarbon group may optionally be interrupted by one or two Z0s; L1, L2, L3, and L4 are each independently -(CH2). t -Z0-(CH2) t -; Each t is an independent integer from 0 to 6; R1 and R2 are each independent of each other. Each R0 is independently a substituted or unsubstituted C 1-40 hydrocarbon group, C 1-40 Heteroalkyl groups or fat-soluble vitamin residues; q is an integer from 0 to 3; each B0 is an independent linking bond, C 1-21 Alkylene or -(CH2) k -P0-(CH2) k - and any B0 that is a connecting bond is not simultaneously connected to two Z0; each k is an independent integer from 0 to 10; P0 is ortho-phenylene, meta-phenylene, or para-phenylene; the C 1-21 The alkylene group may be substituted or unsubstituted; Each Z0 is independently one of the following: a linker bond, -Q-, -QQ-, -C(=Q)-, -QC(=Q)-, -C(=Q)Q-, and -QC(=Q)Q-; Q is independently O, S, or NR' each time it appears; R' is a hydrogen atom or C. 1-4 alkyl.

2. The asymmetric cationic lipid according to claim 1, characterized in that, Each R S Independently -CH3, -CH2CH3 or -CH2CH2OH, preferably -CH3; each R L C is independently substituted or unsubstituted with a hydroxyl group 6-40 Alkyl, C 6-40 alkenyl or C 6-40 Alkyne group, and the C 6-40 Alkyl, C 6-40 alkenyl or C 6-40 The alkynyl skeleton is optionally interrupted by a Z0; preferably, each R L Independently selected from any of the following structures: Where each tm is an independent integer from 0 to 20, and each R m Independently linear or branched C 1-20 Alkyl, C 3-20 alkenyl or C 3-20 alkynyl group; the C 3-20 The alkenyl group contains one or two carbon-carbon double bonds; the C 3-20 The alkynyl group contains one or two carbon-carbon triple bonds.

3. The asymmetric cationic lipid according to claim 1, characterized in that, Each B0 is an independent connection key. R B The side chain is a hydrogen atom, a hydroxyl group, or an amino acid; the side chain of the amino acid is preferably any of the following structures:

4. The asymmetric cationic lipid according to claim 1, characterized in that, It contains at least one nitrogen atom as a cyclic atom, and optionally also contains one or more of oxygen atoms, sulfur atoms and nitrogen atoms as cyclic atom, and the remaining cyclic atom is a carbon atom; It has a single-ring or double-ring structure, and each ring is independently a four-, five-, six-, or seven-membered ring; preferably, the two rings in the double-ring structure share at least two ring-forming atoms; preferably, Selected from Any of the following, wherein the *-marked N atom is associated with L1, L2, R1, R2, L4, or R M Connected; When substitution is used, it is preferable to use a form in which one hydrogen atom on the ring is replaced by a hydroxyl group; Preferably, Choose from any of the following structures: Preferably, Choose from any of the following structures:

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

6. The asymmetric cationic lipid according to claim 1, characterized in that, The structure of the asymmetric cationic lipid is selected from any one of formulas (2) to (7):

7. The asymmetric cationic lipid according to claim 1, characterized in that, Both M1 and M2 are or all or all or all Among them, M1 contains at least one R M With any R in M2 M They are not the same.

8. The asymmetric cationic lipid according to claim 1, characterized in that, Each R0 is independently a linear, branched, or cyclic C 1-40 Alkyl, C 2-40 alkenyl, C 2-40 alkynyl group, C 1-40 Heteroalkyl or fat-soluble vitamin residues; each R0 is independently in a substituted or unsubstituted form; in the substituted form of said R0, each substituent is independently C 1-6 Alkyl, hydroxyl, C 1-6 Hydroxyalkyl, halogen, aryl or aralkyl, preferably methyl, hydroxy, hydroxymethyl, fluorine atom or benzyl; Preferably, R0 is selected from any of the following structures or alternative forms thereof: Vitamin A residues, vitamin D residues, vitamin E residues, and vitamin K residues; wherein each tp is an independent integer from 0 to 20, and each R p Independently linear C 1-20 Alkyl, C 2-20 alkenyl or C 2-20 alkynyl group; the C 2-20 The alkenyl group contains one or two carbon-carbon double bonds; the C 2-20 The alkynyl group contains one or two carbon-carbon triple bonds; More preferably, R0 is selected from any of the following structures: More preferably, each Z0 in R1 and R2 is independently selected from any one of the following: linking bond, -SS-, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -OC(=O)O-, -NHC(=O)O-, and -OC(=O)NH-; Most preferably, R1 and R2 are each independently selected from any of the following structures:

9. The asymmetric cationic lipid according to claim 1, characterized in that, Z0 in L1 and L2 are each independently selected from any one of the following: linker bond, -C(=O)-, -OC(=O)-, -C(=O)O-, -NHC(=O)NH-, -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, and -NHC(=S)NH-; Preferably, L1 and L2 are each independently -(CH2). t -(CH2) t -; More preferably, L1 and L2 are each independently selected from any one of the following: a connecting bond, -CH2-, -(CH2)2-, and -(CH2)3-.

10. The asymmetric cationic lipid according to claim 1, characterized in that, Z0 in L3 and L4 are each independently selected from any one of the following: linker bond, -C(=O)-, -N(CH3)-, -OC(=O)-, -C(=O)O-, -NHC(=O)-, -C(=O)NH-, -OC(=O)O-, -NHC(=O)O-, -OC(=O)NH-, -NHC(=O)NH-, and -NHC(=S)NH-.

11. The asymmetric cationic lipid according to claim 1, selected from any of the following structures:

12. A lipid composition, characterized in that, Contains the asymmetric cationic lipid according to any one of claims 1-11.

13. The lipid composition according to claim 12, characterized in that, The lipid composition further comprises one or more of phospholipids, steroid lipids, polyethylene glycol-modified lipids, another cationic lipid, and anionic lipids; preferably, the lipid composition further comprises any one of phospholipids, steroid lipids, and polyethylene glycol-modified lipids; more preferably, the lipid composition further comprises any two of phospholipids, steroid lipids, and polyethylene glycol-modified lipids; even more preferably, the lipid composition further comprises phospholipids, steroid lipids, and polyethylene glycol-modified lipids; most preferably, the lipid composition further comprises phospholipids, steroid lipids, polyethylene glycol-modified lipids, and another cationic lipid; or the lipid composition further comprises phospholipids, steroid lipids, polyethylene glycol-modified lipids, and anionic lipids; in any of the foregoing embodiments, the polyethylene glycol-modified lipid is selected from any one of non-targeted polyethylene glycol-modified lipids, targeted polyethylene glycol-modified lipids, and combinations thereof; Preferably, in any of the foregoing embodiments, the phospholipid is selected from 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-dimyristoyl-sn-glycerol-3-phosphate choline, 1,2-dioleoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-distearateoyl-sn-glycerol-3-phosphate choline, 1,2-diundecanoyl-sn-glycerol-3-phosphate choline, 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline, 1,2-dicarboxylic acid ... -Di-O-octadecyl-sn-glycerol-3-phosphate choline, 1-oleoyl-2-cholesterolylhemisuccino-sn-glycerol-3-phosphate choline, 1-hexadecyl-sn-glycerol-3-phosphate choline, 1,2-dilinanoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidanoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diphydanyl-s n-Glyceryl-3-phosphate ethanolamine, 1,2-distearatel-sn-glyceryl-3-phosphate ethanolamine, 1,2-dilinoleyl-sn-glyceryl-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glyceryl-3-phosphate ethanolamine, 1,2-diarachidonicyl-sn-glyceryl-3-phosphate ethanolamine, 1,2-bis(docosahexaenoyl)-sn-glyceryl-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glyceryl-3-phosphate-rac-(1-glycerol) sodium salt, dioleoylphosphatidylserine Dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, and combinations thereof; Preferably, in any of the foregoing embodiments, the steroid lipids are selected from cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, rapeseed sterol, tomatine, ursolic acid, α-tocopherol, and combinations thereof. Preferably, in any of the foregoing embodiments, the non-targeted PEGylated lipid is selected from polyethylene glycol-dispalmitoylphosphatidylcholine, polyethylene glycol-dimyristylglycerol, polyethylene glycol-distearate phosphatidylethanolamine, polyethylene glycol-dioleoylphosphatidylethanolamine, polyethylene glycol-cholesterol, polyethylene glycol-diacylglycerol, polyethylene glycol-dialkoxypropyl, and combinations thereof; more preferably, the non-targeted PEGylated lipid is selected from polyethylene glycol 500-dispalmitoylphosphatidylglycerol. Phosphatidylcholine, polyethylene glycol 2000-dispalmitoylphosphatidylcholine, polyethylene glycol 500-distearylphosphatidylethanolamine, polyethylene glycol 2000-distearylphosphatidylethanolamine, polyethylene glycol 500-dioleoylphosphatidylethanolamine, polyethylene glycol 2000-dioleoylphosphatidylethanolamine, polyethylene glycol 500-dimyristoylglycerol, polyethylene glycol 2000-dimyristoylglycerol, and combinations thereof; or, the non-targeted PEGylated lipid is selected from... any combination thereof, where n1 is an integer from 10 to 250; Preferably, in any of the foregoing embodiments, the targeted polyethylene glycol-modified lipid is a folic acid or N-acetylgalactosamine-modified polyethylene glycol-modified lipid; more preferably, the targeted polyethylene glycol-modified lipid is selected from... any combination thereof, where n2 is an integer from 10 to 250; Preferably, in any of the foregoing embodiments, the other cationic lipid is selected from 1,2-dioleoyl-3-trimethylammonium-propane (methyl sulfate), 1,2-dioleoyloxy-3-trimethylammonium propane chloride, 1-[2-(oleoyloxy)ethyl]-2-oleo-3-(2-hydroxyethyl)imidazoline chloride, 1,2-dioleoyl-3-dimethylamino-propane, 2,3-di(tetradecanoyloxy)propyltrimethylazone chloride, dicedyldimethylammonium chloride, dicedyldimethylammonium bromide, N,N-dioleo-N,N-dimethylammonium chloride, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)prop-1-ammonium, and 3,6-bis{4-[bis(2-hydroxydodecyl)amino]butyl}piperazine-2,5-dione , any one of the following: 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecane-2-ol), methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester, 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine, ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 8-[(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino]octanoate (heptadecane-9-yl) ester and ((2-(2-hydroxyethoxy)ethyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); Preferably, in any of the foregoing cases, the anionic lipid is selected from any one of 1,2-dioleoyl-sn-glycerol-3-phosphate sodium salt, 1,2-dimyristoyl-sn-glycerol-3-phosphate sodium salt, bis(monooleoylglycerol)ammonium phosphate salt, and cardiolipin.

14. The lipid composition according to claim 13, characterized in that, The asymmetric cationic lipids account for 30% to 65% of the total lipids, preferably 35% to 55%; The phospholipids account for 2% to 15% of the total lipids, preferably 5% to 12%; The steroid lipids account for 25% to 50% of the total lipids, preferably 38% to 50%; The PEGylated lipids account for 0.5% to 10% of the total lipids, preferably from 1% to 3%, wherein the targeted PEGylated lipids account for 0-0.5% of the total lipids.

15. A lipid pharmaceutical composition, characterized in that, The lipid composition comprising any one of claims 12-14 further comprises one or more pharmaceutically active ingredients.

16. The lipid pharmaceutical composition according to claim 15, characterized in that, The active pharmaceutical ingredient is a nucleic acid, a small molecule, an oligopeptide, a polypeptide, or a protein, preferably a nucleic acid, more preferably DNA, RNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir, or ribozyme, and most preferably mRNA or siRNA.

17. The lipid pharmaceutical composition according to claim 15, characterized in that, The lipid pharmaceutical composition is used to prepare a drug, which is selected from any one of antitumor agents, antibiotics, antiviral agents, antifungal agents, antiparasitic agents, and vaccines.

18. A lipid pharmaceutical composition formulation, characterized in that, The lipid pharmaceutical composition comprising any one of claims 15-17 further comprises a working solution; wherein the working solution is preferably a pharmaceutically acceptable carrier, more preferably any one of deionized water, ultrapure water, phosphate buffer, and physiological saline, and even more preferably phosphate buffer or physiological saline.

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