An amino acid cationic lipid

Abstract

The present invention provides a novel amino acid cationic lipid, the structure of which is shown in the general formula (1), wherein the definitions of the various symbols are consistent with those described herein. The amino acid cationic lipid is a pharmaceutically acceptable, biodegradable or highly biocompatible lipid, and has the advantages of low toxicity, low immunogenicity and high biocompatibility. The amino acids or amino acid derivatives used in the preparation process are simple and easy to obtain, and can be obtained naturally or by simple synthesis, and have the advantages of simplicity, safety and cost saving. The novel amino acid cationic lipid of the present invention may also contain a degradable group between the amino acid residue and the lipophilic tail chain. The presence of the degradable group enables the lipid nanoparticles LNP prepared therefrom to be degraded in time within the endosome, thereby solving the problem that the LNP prepared from the non-degradable lipids in the prior art will be stored in the endosome and acidify the endosomal environment, thereby hindering the endosomal escape of drug molecules (such as nucleic acids), and solving the problem that the drugs delivered into the cell cannot function. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of drug delivery, and specifically relates to a pharmaceutical carrier cationic lipid, in particular to an amino acid cationic lipid, and a lipid composition comprising the amino acid cationic lipid, a lipid pharmaceutical composition, and preparations and applications thereof. Background Art

[0002] Liposomes are closed vesicles with a bilayer structure. They possess low immunogenicity and high biocompatibility, enabling the delivery of drugs or active molecules into cells. They are widely used to deliver nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, peptide drugs, and protein drugs. For drugs or small molecules with poor stability or rapid degradation, liposome delivery can improve efficacy, reduce toxicity, enhance stability, and improve targeting.

[0003] Lipid nanoparticles (LNPs) contain lipids and drug molecules or active molecules and are widely used for the delivery of small molecules and nucleic acid drugs. They have recently attracted much attention due to their great success as a delivery platform for COVID-19 mRNA vaccines. LNPs contain cationic lipids, phospholipids, sterol lipids, and PEGylated lipids. Cationic lipids interact with drug molecules (such as negatively charged nucleic acids) through electrostatics, while phospholipids prevent lipid oxidation or attach ligands to the surface of lipid nanoparticles. Sterol lipids have strong membrane fusogenicity, promoting the intracellular uptake and cytoplasmic entry of drug molecules. PEGylated lipids are located on the surface of lipid nanoparticles, improving their hydrophilicity, avoiding rapid clearance by the immune system, preventing particle aggregation, and increasing stability. For example, nucleic acid molecules are very fragile and easily affected by factors such as nucleases and temperature, and are degraded and ineffective during storage and transportation. Ionizable cationic lipids are neutral at physiological pH, but positively charged in acidic environments. They will combine with negatively charged nucleic acids and form LNPs with other lipids. After entering the endosome, the cationic lipids can ionize under the acidic conditions of the endosome cavity and carry partial positive charges to promote the escape of nucleic acids from the endosomal cavity and release them into the cytoplasm to exert therapeutic effects, thereby improving the transfection rate of nucleic acid drugs.

[0004] Although cationic lipids have made the latest progress in drug delivery, there is still a need in the art for selectable cationic lipids suitable for conventional therapeutic uses. Document WO2021026358A1 also reports that nitrogenous lipids can be protonated and carry a positive charge or partial positive charge under physiological pH conditions. In the prior art, cationic lipid DOTAP can be used for mRNA transfection in various cell types. Although effective in vitro, the permanent cationic quaternary ammonium group causes the liposomes to be rapidly cleared from the systemic circulation and target organs, and exhibits toxicity. Amino acid ether lipids are disclosed in CN104168887A, and the ether bonds they contain are relatively stable in vivo. Cationic lipids containing only ether bonds can inhibit the degradation of LNPs, and the storage of LNPs can prevent drug molecules (such as nucleic acid molecules) from being released from the endosome cavity into the cytoplasm to exert their effects. Therefore, in the present invention, we utilize the chemical diversity of amino acids to design and synthesize a series of amino acid cationic lipids that are stably present or degradable with amino acid residues as core structures and containing one or more lipophilic tail chains. Summary of the Invention

[0005] The present invention provides a novel amino acid cationic lipid and a preparation method thereof, a lipid composition comprising the amino acid cationic lipid, a lipid pharmaceutical composition containing the lipid composition and a preparation thereof, a liposome or lipid nanoparticle containing the lipid composition, and in particular an LNP-nucleic acid pharmaceutical composition containing the lipid composition and a preparation thereof, which have the advantages of high delivery efficiency, safety, low toxicity, and high biocompatibility, and can improve the therapeutic and / or preventive effects of drugs.

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

[0007] One embodiment of the present invention provides an amino acid cationic lipid:

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

[0009]

[0010] Wherein, AA is the residue of an amino acid or an amino acid derivative;

[0011] Each time B1 and B2 appear, they are independently connected or C 1-30 alkylene;

[0012] L1 and L2 are each independently a connecting bond or a divalent connecting group;

[0013] L5 and L6 are each independently a linking bond or a divalent linking group;

[0014] Each occurrence of L3 is independently a linking bond or a divalent linking group;

[0015] R1 and R2 are each independently -(CH2) t NR e R f , straight chain C 1-30 Alkylene, branched C 1-30 Alkylene or Wherein, t is an integer of 0-12, t1 and t2 are each independently an integer of 0-5, t3 and t4 are each independently 0 or 1, and t1, t2, t3, and t4 are not 0 at the same time; R e 、R f Each independently is C 1-15 Alkyl, C 2-15 Alkenyl and C 2-155 Any of alkynyl groups;

[0016] Each occurrence of R3 is independently a hydrogen atom, -R d 、-OR d 、C 3-6 Carbocyclic group, nitrogen-containing heterocyclic group, -NR d R d 、-SR d 、-C(=O)R d 、-C(=O)OR d 、-OC(=O)R d 、-OC(=O)OR d Or functional groups R that can interact with biologically related substances 01 ; Among them, R d Each occurrence is independently C 1-12 alkyl;

[0017] a, b, c are each independently 1 or 2; when the fragment -L5-B1-L1-R1 and / or -L6-B2-L2-R2 and / or -L3-R3 is derived from the amino terminus of the amino acid, a, b, c are each independently 1 or 2; when the fragment -L5-B1-L1-R1 and / or -L6-B2-L2-R2 and / or -L3-R3 is derived from the carboxyl terminus, hydroxyl terminus or thiol terminus of the amino acid, a, b, c are each independently 1; when a is 2, the two -L5-B1-L1-R1 fragments are the same or different; when b is 2, the two -L6-B2-L2-R2 fragments are the same or different; when c is 2, the two -L3-R3 fragments are the same or different;

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

[0019] The present invention also provides a lipid composition, the embodiment of which is:

[0020] A lipid composition contains an amino acid cationic lipid having a structure represented by formula (1).

[0021] The present invention also provides a lipid pharmaceutical composition, the embodiments of which are as follows:

[0022] A lipid pharmaceutical composition comprises a lipid composition and a drug, wherein the lipid composition comprises an amino acid cationic lipid having a structure represented by formula (1), and the drug is selected from any one of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs.

[0023] The present invention also provides a lipid pharmaceutical composition preparation, the implementation scheme is as follows:

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

[0025] The present invention also provides a liposome or lipid nanoparticle, the embodiments of which are as follows:

[0026] A liposome or lipid nanoparticle contains a lipid composition, wherein the lipid composition contains an amino acid cationic lipid with a structure shown in formula (1).

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The novel amino acid cationic lipids of the present invention are molecules with a hydrophilic portion and a lipophilic portion. The hydrophilic portion can be provided by amino acid residues, while the lipophilic portion can include one or more lipophilic tail chains. The amino acids or amino acid derivatives used in the preparation process are readily available, either naturally derived or synthesized, offering the advantages of simplicity, safety, and cost-effective production.

[0029] The tertiary amine portion of the novel amino acid cationic lipid of the present invention is derived from amino acids or amino acid residues. Compared with quaternary amines that can produce toxicity, tertiary amines can ionize part of the positive charge and combine with drugs (such as negatively charged nucleic acid molecules), thereby increasing the intracellular transport rate of drugs.

[0030] The novel amino acid cationic lipid of the present invention is a pharmaceutically acceptable, biodegradable or biocompatible lipid, and has the advantages of low toxicity, low immunogenicity and high biocompatibility.

[0031] The novel amino acid cationic lipids of the present invention may contain a degradable group between the amino acid residue and the lipophilic tail chain. The presence of the degradable group enables the lipid composition prepared therefrom to be degraded in a timely manner within the endosome, thereby solving the problem in the prior art that lipid compositions prepared from non-degradable lipids will accumulate in the endosome and acidify the endosomal environment, thereby hindering the endosomal escape of drug molecules (such as nucleic acids), thereby solving the problem that drugs delivered into cells cannot function.

[0032] The novel amino acid cationic lipid of the present invention may contain a targeting group. The lipid composition prepared from the cationic lipid can also have a targeting function, thereby further improving the therapeutic and / or diagnostic effects of the drug.

[0033] The novel amino acid cationic lipids of the present invention can achieve chemical diversity by substituting at the N-terminus, C-terminus, hydroxyl terminus or thiol terminus of an amino acid or an amino acid derivative. The amino acid can also be a polypeptide formed by 2-20 identical or different amino acid residues.

[0034] The amino acid cationic lipids provided by the present invention are stimuli-responsive and can mediate biorecognition events. Lipid compositions and lipid pharmaceutical compositions prepared from amino acid cationic lipids have a specific morphology under normal biological conditions. Once in the body and at the site of a lesion, changes in the biological microenvironment of the lesion cause changes in the assembly morphology and bonding pattern of the lipid composition or lipid pharmaceutical composition, allowing the loaded drug molecules to be released, exerting a therapeutic or preventive effect.

[0035] The amino acid cationic lipid of the present invention can also be coupled with a targeting group, and the lipid composition and lipid pharmaceutical composition prepared therefrom can better achieve targeted delivery and controlled release of drugs.

[0036] Implementation Method

[0037] Terminology

[0038] In the present invention, unless otherwise described, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. The disclosures of all patents and other publications cited herein are incorporated herein by reference in their entirety. In the event that any description or interpretation of a term herein conflicts with any document incorporated herein by reference, the description and interpretation of the following terms shall prevail. Unless otherwise indicated, each term has the following meaning.

[0039] In the present invention, when a structure has isomers, unless otherwise specified, any of the isomers may be present. For example, a structure having cis-trans isomers may be either the cis structure or the trans structure; a structure having E / Z isomers may be either the E structure or the Z structure; and a structure having optical activity may be either levorotatory or dextrorotatory.

[0040] In the present invention, the interpretation of numerical intervals includes numerical intervals marked with a dash (such as 0-12), numerical intervals marked with a wavy line such as (0-12), and numerical intervals marked with "to / to" (such as 0 to 12, 1 to 12). In the present invention, unless otherwise specified, integer intervals marked in the form of intervals can represent a group consisting of all integers within the interval range, and the range includes two endpoints. For example, the integer range 0-12 represents a group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. The numerical ranges in the present invention, including but not limited to numerical ranges represented by integers, non-integers, percentages, and fractions, all include two endpoints unless otherwise specified.

[0041] Numerical values in the present invention generally refer to a range of ±10%, and in some cases this range may be increased to ±15%, but not exceeding ±20%. These values are based on a predetermined value. For example, if the molar percentage of steroid lipids in a solution containing a solvent is approximately 40% of the total lipids, this generally includes a molar percentage of steroid lipids of 30% to 50%.

[0042] In the present invention, unless otherwise specified, "any" includes any one, any two, and any two or more.

[0043] In the present invention, unless otherwise specified, the terms "include", "comprise", "contain" and similar expressions should be interpreted as "including but not limited to" in an open and inclusive sense in this specification and claims.

[0044] In the present invention, when two or more objects are "independently preferred" and have multiple levels of preference, it is not required that all of them are selected from the same level of preference group. One can be preferred in a large range and the other in a small range, or one can be the largest range and the other can be any preferred situation, or they can be selected from the same level of preference.

[0045] In the present invention, "each occurrence is independently any of the options in the definition" not only means that different groups can be independently any of the options in the definition, but also means that different positions in the same group can also be independently any of the options in the definition, for example, "each occurrence is independently a linker, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, -NH-, -O(CR cR c ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-, wherein R c Each occurrence is independently a hydrogen atom or a C 1-12 Alkyl", in the group "-NR c C(=O)NR c -" in the middle, two R c are each independently a hydrogen atom or a C 1-12 Alkyl, that is, two R c Can be the same or different.

[0046] The divalent linking group in the present invention, such as an alkylene group, an alkylene group, an arylene group, an amide bond, etc., is not particularly limited. When connecting to other groups, either of the two connecting ends can be selected. For example, when an amide bond is used as the divalent linking group between Group A and Group B, it can be GroupA-C(=O)NH-GroupB or GroupB-NHC(=O)-GroupA.

[0047] In the structural formula of the present invention, when the terminal group of the linking group is easily confused with the substituent contained in the linking group, To mark the position of the linker to connect other groups, such as in the structural formula In the To mark the two positions of the divalent linking group that connects to other groups, the above two structural formulas represent -CH(CH2CH2CH3)2- and -CH2CH2CH(CH3)2-CH2CH2-, respectively.

[0048] In the present invention, the range of carbon atoms in a group is marked in the subscript position of C in the form of a subscript, indicating the number of carbon atoms in the group, for example, C 1-12 means "having 1 to 12 carbon atoms", C 1-30 means "having 1 to 30 carbon atoms". "Substituted C 1-12 "Alkyl" refers to C 1-12 A compound obtained by replacing the hydrogen atom of an alkyl group. 1-12"Substituted alkyl" refers to a compound having 1 to 12 carbon atoms obtained by replacing the hydrogen atoms of an alkyl group. For example, when a group can be selected from C 1-12 When the alkylene group is present, any alkylene group with the number of carbon atoms in the range indicated by the subscript can be selected, that is, C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 、C 11 、C 12 In the present invention, unless otherwise specified, subscripts marked in interval form represent any integer that can be selected from the range, and the range includes both endpoints.

[0049] The heteroatoms in the present invention are not particularly limited, and include but are not limited to O, S, N, P, Si, F, Cl, Br, I, B, etc.

[0050] In the present invention, the heteroatom used for substitution is referred to as a "substituent atom", and any group used for substitution is referred to as a "substituent".

[0051] As used herein, "substituted" means any of the above groups (e.g., aliphatic, hydrocarbyl, alkyl, or alkylene) wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom, such as, but not limited to, a halogen atom such as F, Cl, Br, and I; an oxo group (=O); a hydroxyl group (-OH); an oxyl group (-OR d , where R d C 1-12 Alkyl); carboxyl (-COOH,); amine group (-NR c R c , two R c Each independently is H, C 1-12 alkyl); C 1-12 In some embodiments, the substituent is C 1-12 In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as a fluoro group. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0052] As used herein, "optional" or "optionally" (e.g., optionally substituted) means that the subsequently described event or circumstances may or may not occur, and that the description includes instances where the event or circumstances occur as well as instances where the event or circumstances do not occur. For example, "optionally substituted hydrocarbyl" means that the hydrocarbyl may or may not be substituted, and that the description includes both substituted and unsubstituted hydrocarbyl groups.

[0053] In the present invention, "carbon chain linking group" refers to a linking group in which all the main chain atoms are carbon atoms, while the side chain portion allows heteroatoms or groups containing heteroatoms to replace the hydrogen atoms of the main chain carbon atoms. When the "main chain atoms" are heteroatoms, they are also called "main chain heteroatoms", such as AS-CH2-B, AO-CH2-B, (atomic spacing is recorded as 4) is considered to contain main chain heteroatoms. Carbon chain linkers can be divided into alkylene groups and carbon chain linkers with heteroatoms in the side groups; the carbon chain linkers with heteroatoms in the side groups include but are not limited to oxo (=O), thio (=S), amino (connected to the main chain carbon through a carbon-nitrogen double bond), oxygen heteroalkyl in the form of ether bond, thio heteroalkyl in the form of thioether bond, nitrogen heteroalkyl in the form of tertiary amino, etc. The main chain of the "carbon chain linker" is composed entirely of carbon atoms, and the side groups of the carbon chain are allowed to contain heteroatoms. That is, it is connected by methylene or substituted methylene. The substituted methylene can be replaced by one monovalent substituent, two monovalent substituents or one divalent substituent (such as divalent oxygen, such as forming a three-membered ring together with the divalent methylene). The substituted methylene group may be a single hydrogen atom substituted (e.g., -CH(CH3)-), two hydrogen atoms substituted separately (e.g., -(CH3)C(OCH3)-), or two hydrogen atoms substituted simultaneously (e.g., carbonyl, thiocarbonyl, -C(=NH)-, -C(=N + H2)-), can also be a cyclic side group (such as The atomic spacing is denoted as 1).

[0054] In the present invention, a compound or a group can be substituted and hybridized at the same time, for example, a nitrophenyl group replaces a hydrogen atom, or -CH2-CH2-CH2- is replaced by -CH2-S-CH(CH3)-.

[0055] In the present invention, "connecting bond" refers to a group that only plays a connecting role and does not contain any atoms. When a group is defined as a connecting bond, it also means that the group may not exist.

[0056] As used herein, a "group" contains at least one atom and refers to a free radical formed by the loss of one or more atoms from a compound. A group formed by the loss of some of these groups is also referred to as a residue. The valence of a group is not particularly limited; for example, groups can be classified as monovalent, divalent, trivalent, tetravalent, etc., and even centrivalent groups. Groups with a valence of 2 or greater are collectively referred to as linkers. Linkers can also contain only one atom, such as oxy and thiol groups.

[0057] In the present invention, "hydrocarbon" refers to a hydrocarbon compound composed of carbon atoms and hydrogen atoms.

[0058] In this disclosure, hydrocarbons are classified into aliphatic hydrocarbons and aromatic hydrocarbons based on their hydrocarbon group. Hydrocarbons that do not contain either a benzene ring or a hydrocarbon-substituted benzene ring are defined as aliphatic hydrocarbons. Hydrocarbons that contain at least one benzene ring or a hydrocarbon-substituted benzene ring are defined as aromatic hydrocarbons. Aromatic hydrocarbons may contain aliphatic hydrocarbon groups, such as toluene, diphenylmethane, and 2,3-dihydroindene.

[0059] In this disclosure, hydrocarbons are classified into saturated hydrocarbons and unsaturated hydrocarbons based on their degree of saturation. All aromatic hydrocarbons are unsaturated hydrocarbons. Saturated aliphatic hydrocarbons are also called alkanes. The degree of unsaturation of unsaturated aliphatic hydrocarbons is not particularly limited. Examples include, but are not limited to, alkenes (containing double bonds), alkynes (containing triple bonds), and dienes (containing conjugated double bonds). When the aliphatic hydrocarbon portion of an aromatic hydrocarbon is a saturated structure, it is also called an aralkane, such as toluene.

[0060] In the present invention, there is no particular limitation on the structure of hydrocarbons, and the hydrocarbons may be in the form of a straight-chain structure without side groups, a branched-chain structure with side groups, a cyclic structure, a dendritic structure, a comb-like structure, a hyperbranched structure, or the like. Where not particularly defined, the straight-chain structure without side groups, the branched-chain structure with side groups, and the cyclic structure are preferably straight-chain hydrocarbons, branched-chain hydrocarbons, and cyclic hydrocarbons, respectively. Among them, hydrocarbons without cyclic structures are generally referred to as open-chain hydrocarbons, including but not limited to straight-chain structures without side groups and branched-chain structures with side groups. Open-chain hydrocarbons belong to aliphatic hydrocarbons. Therefore, straight-chain hydrocarbons may also be referred to as straight-chain aliphatic hydrocarbons. Branched-chain hydrocarbons may also be referred to as branched-chain aliphatic hydrocarbons.

[0061] In the present invention, compounds formed by replacing a carbon atom at any position in a hydrocarbon with a heteroatom are collectively referred to as heterohydrocarbons.

[0062] In the present invention, heteroaliphatic hydrocarbons refer to heteroaliphatic hydrocarbons derived from aliphatic hydrocarbons, including heteroaliphatic cyclic hydrocarbons and heteroaliphatic open chain hydrocarbons, etc. Saturated heteroaliphatic hydrocarbons are heteroalkanes.

[0063] In the present invention, a "hydrocarbyl group" refers to a hydrocarbon residue formed by losing at least one hydrogen atom. Depending on the number of hydrogen atoms lost, a hydrocarbon group can be classified as a monovalent hydrocarbon group (losing one hydrogen atom), a divalent hydrocarbon group (losing two hydrogen atoms, also known as a hydrocarbylene group), a trivalent hydrocarbon group (losing three hydrogen atoms), and so on. When n hydrogen atoms are lost, the valence of the resulting hydrocarbon group is n. Unless otherwise specified, a hydrocarbon group in the present invention specifically refers to a monovalent hydrocarbon group. Unless otherwise expressly stated in this specification, a hydrocarbon group is optionally substituted.

[0064] In the present invention, the source of the hydrocarbon group is not particularly limited. For example, the hydrocarbon group may be derived from aliphatic hydrocarbons or aromatic hydrocarbons, saturated hydrocarbons or unsaturated hydrocarbons, straight-chain hydrocarbons, branched-chain hydrocarbons or cyclic hydrocarbons, hydrocarbons or heterohydrocarbons, etc. From the perspective of saturation, the hydrocarbon group may be derived from, for example, alkanes, alkenes, alkynes, diolefins, etc.; for cyclic hydrocarbons, the hydrocarbon group may be derived from, for example, alicyclic hydrocarbons or aromatic hydrocarbons, monocyclic hydrocarbons or polycyclic hydrocarbons; and for heterocyclic hydrocarbons, the hydrocarbon group may be derived from, for example, alicyclic heterocyclic hydrocarbons or aromatic heterocyclic hydrocarbons.

[0065] As used herein, "aliphatic hydrocarbon group" refers to the residue formed by the loss of at least one hydrogen atom from an aliphatic hydrocarbon. Unless otherwise specified, an aliphatic hydrocarbon group herein refers specifically to a monovalent aliphatic hydrocarbon group. Aliphatic hydrocarbon groups include saturated aliphatic hydrocarbon groups and unsaturated aliphatic hydrocarbon groups. Unless otherwise specified in this specification, an aliphatic hydrocarbon group is optionally substituted.

[0066] In the present invention, "alkyl" refers to a hydrocarbon group formed from an alkane. Unless otherwise specified, it refers to a hydrocarbon group formed by losing a hydrogen atom at any position. It may be linear or branched, and may be substituted or unsubstituted. Specifically, "propyl" refers to either n-propyl or isopropyl, and "propylene" refers to either 1,3-propylene, 1,2-propylene, or isopropylene. Unless otherwise specified in this specification, an alkyl group is optionally substituted.

[0067] In the present invention, "unsaturated hydrocarbon group" refers to a hydrocarbon group formed by losing a hydrogen atom from an unsaturated hydrocarbon. Hydrocarbon groups formed by losing a hydrogen atom from an unsaturated carbon atom can be categorized as alkenyl, alkynyl, and dienyl groups, such as propenyl and propynyl. Hydrocarbon groups formed by losing a hydrogen atom from a saturated carbon atom from an unsaturated hydrocarbon can be categorized as olefinic, alkyne, and dienyl groups, depending on the type of unsaturated bond, such as allyl and propargyl.

[0068] In the present invention, "alkenyl" or "alkenyl group" means a substituted or unsubstituted straight or branched chain alkenyl group comprising two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon double bond. The symbol "C 2-15 "Alkenyl" means a substituted or unsubstituted straight or branched chain alkenyl group comprising 2 to 15 carbon atoms and at least one carbon-carbon double bond, i.e., an alkenyl group may comprise one, two, three, four or more carbon-carbon double bonds. Unless otherwise specifically stated, the alkenyl groups described herein refer to both unsubstituted and substituted alkenyl groups. Unless otherwise specifically stated in the specification, an alkenyl group is optionally substituted.

[0069] As used herein, "alkynyl" or "alkynyl group" means an optionally substituted straight or branched chain hydrocarbon comprising two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more carbon atoms) and at least one carbon-carbon triple bond. 2-15"Alkynyl" means a substituted or unsubstituted straight or branched chain alkynyl group comprising 2 to 15 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group may comprise one, two, three, four or more carbon-carbon triple bonds. Unless otherwise specifically stated, the alkynyl groups described herein refer to both unsubstituted and substituted alkynyl groups. Unless otherwise specifically stated in the specification, an alkynyl group is optionally substituted.

[0070] As used herein, "alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the remainder of the molecule to a free radical group, consisting only of carbon and hydrogen, and which may be saturated or unsaturated. For example, an alkylene group (C 1-24 alkylene), alkylene having one to twelve carbon atoms (C 1-12 alkylene groups), specifically exemplified by methylene, ethylene, propylene, n-butylene, vinylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. Unless otherwise specifically stated in the specification, an alkylene group is optionally substituted.

[0071] In the present invention, "alkylene" refers to a divalent alkyl group, including open-chain alkylene and divalent cycloalkyl. Open-chain alkylene refers to a divalent alkyl group without a ring structure, and divalent cycloalkyl refers to a divalent alkyl group with a ring structure. Unless otherwise specified in this specification, an alkylene group is optionally substituted.

[0072] In the present invention, "molecular weight" represents the mass of a compound molecule, and "average molecular weight" represents the mass of the general compound components in a macroscopic substance. Unless otherwise specified, "average molecular weight" generally refers to the "number average molecular weight" M. n . For the number average molecular weight, it can be the molecular weight of a polydisperse block or substance, or the molecular weight of a monodisperse block or substance. Unless otherwise specified, the unit of measurement for "molecular weight" and "average molecular weight" is Dalton, Da. The "degree of polymerization" can also be used to characterize the molecular weight of the polyethylene glycol chain, specifically referring to the number of repeating units (ethylene oxide units, EO units) in a compound molecule. Accordingly, the "average degree of polymerization", "number average degree of polymerization" or "number of EO units" are used to characterize the average value or number average of the number of repeating units.

[0073] In the context of the present invention, "about" generally refers to ±0.5%.

[0074] In the present invention, the terms "stable" and "degradable" are relative concepts. Detailed examples of stable and degradable groups are given in paragraphs

[0134] to

[0145] of CN113402405A.

[0075] In the present invention, "hydroxy protecting group" includes all groups that can be used as a protecting group for a common hydroxyl group. The hydroxy protecting group is preferably an alkanoyl group (such as acetyl, tert-butyryl), an aralkanoyl group (such as benzoyl), a benzyl group, a trityl group, a trimethylsilyl group, a tert-butyldimethylsilyl group, an allyl group, an acetal group or a ketal group. The removal of the acetyl group is generally carried out under alkaline conditions, and the most commonly used methods are aminolysis with NH3 / MeOH and methanolysis catalyzed by methanol anion; the benzyl group can be easily removed by palladium-catalyzed hydrogenolysis in a neutral solution at room temperature, and can also be reduced and cleaved by metal sodium in ethanol or liquid ammonia; the trityl group is generally removed by catalytic hydrogenolysis; the trimethylsilyl group is usually removed using a reagent containing fluoride ions (such as tetrabutylammonium fluoride / anhydrous THF, etc.); tert-butyldimethylsilyl ether is relatively stable and can withstand the ester hydrolysis conditions of alcoholic potassium hydroxide and mild reducing conditions (such as Zn / CH3OH, etc.), and can be removed by fluoride ions (such as Bu4N + F - ) can be removed in tetrahydrofuran solution or with aqueous acetic acid at room temperature.

[0076] In the present invention, "carboxyl protecting group" refers to a protecting group that can be converted into a carboxyl group by the deprotection reaction of hydrolysis or carboxyl protecting group. Carboxyl protecting group is preferably an alkyl group (such as a methyl group, an ethyl group, a tert-butyl group) or an aralkyl group (such as a benzyl group), more preferably a tert-butyl group (tBu), a methyl group (Me) or an ethyl group (Et). In the present invention, "protected carboxyl" refers to a group formed after a carboxyl group is protected by a suitable carboxyl protecting group, preferably a methoxycarbonyl group, an ethoxycarbonyl group, a tert-butyloxycarbonyl group or a benzyloxycarbonyl group. The carboxyl protecting group can be removed by hydrolysis under the catalysis of an acid or base, and can be eliminated by thermal decomposition reaction occasionally. For example, the tert-butyl group can be removed under mild acidic conditions, and the benzyl group can be removed by hydrogenolysis. The reagent for removing the carboxyl protecting group is selected from TFA, H2O, LiOH, NaOH, KOH, MeOH, EtOH and a combination thereof, preferably a combination of TFA and H2O, a combination of LiOH and MeOH or a combination of LiOH and EtOH. The protected carboxyl group is deprotected to produce the corresponding free acid, and the deprotection is carried out in the presence of a base. The base and the free acid formed by the deprotection form a pharmaceutically acceptable salt.

[0077] In the present invention, "amino-protecting group" includes all groups that can be used as conventional amino-protecting groups, such as aryl C 1-6 Alkyl, C 1-6 Alkoxy C 1-6 Alkyl, C 1-6 Alkoxycarbonyl, aryloxycarbonyl, C 1-6Alkylsulfonyl, arylsulfonyl, or silyl, etc. The amino protecting group is preferably Boc tert-butyloxycarbonyl, Moz p-methoxybenzyloxycarbonyl, or Fmoc 9-fluorenylmethyleneoxycarbonyl. The reagent for removing the amino protecting group is selected from TFA, H2O, LiOH, MeOH, EtOH, and combinations thereof, preferably a combination of TFA and H2O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. The reagent for removing the Boc protecting group is TFA or HCl / EA; preferably TFA. The deprotecting agent used in the Fmoc protecting group removal reaction is a 20% piperidine solution in N,N-dimethylformamide (DMF).

[0078] As used herein, "cationic" refers to the ability of a corresponding structure to permanently or non-permanently acquire a positive charge in response to certain conditions (e.g., pH). Thus, cations include both permanent cations and cationizable cations. A permanent cation refers to a compound, group, or atom that is positively charged at any pH or hydrogen ion activity of its environment. Typically, the positive charge is generated by the presence of a quaternary nitrogen atom. When a compound carries multiple such positive charges, it can be referred to as a permanent cation. A cationizable refers to a compound, group, or atom that is positively charged at relatively low pH and uncharged at relatively high pH of its environment. Furthermore, in a non-aqueous environment where pH cannot be determined, a cationizable compound, group, or atom is positively charged at high hydrogen ion concentrations and uncharged at low hydrogen ion concentrations or activity. Whether it is charged or uncharged at a given pH or hydrogen ion concentration depends on the properties of the cationizable or polycationizable compound, particularly the pKa of the corresponding cationizable group or atom. In a dilute aqueous environment, the fraction of cationizable compounds, groups, or atoms that are positively charged can be estimated using the so-called Henderson-Hasselbalch equation, which is well known to those skilled in the art. For example, in some embodiments, if a compound or moiety is cationizable, it is preferred that it be positively charged at a pH of about 1 to 9, preferably 4 to 9, 5 to 8, or even 6 to 8, more preferably at a pH of 9 or less, 8 or less, or 7 or less, and most preferably at a physiological pH (e.g., about 7.3 to 7.4), i.e., under physiological conditions, particularly the physiological salt conditions of cells in vivo. In other embodiments, it is preferred that the cationizable compound or moiety be predominantly neutral at physiological pH (e.g., about 7.0-7.4), but become positively charged at lower pH values. In some embodiments, the preferred range for the pKa of the cationizable compound or moiety is from about 5 to about 7.

[0079] In the present invention, "cationic component / compound" typically refers to a charged molecule that is positively charged (cationic) at a pH typically between about 1 and 9. In some embodiments, the cationic component / compound is preferably charged at a pH of 9 or less (e.g., 5 to 9), 8 or less (e.g., 5 to 8), 7 or less (e.g., 5 to 7), and most preferably at a physiological pH (e.g., about 7.3 to 7.4). Thus, a cationic peptide, protein, polysaccharide, lipid, or polymer according to one embodiment of the present invention is positively charged under physiological conditions, particularly under physiological salt conditions of cells in vivo.

[0080] In the present invention, liposome nanoparticles, cationic peptides, proteins, polysaccharides, lipids or polymers are uncharged, have neutral charge or are electrically neutral under physiological conditions, particularly under the physiological conditions of cells in vivo. Cationic peptides or proteins preferably contain a large amount of cationic amino acids, such as Arg, His, Lys or Orn (particularly cationic amino acids more than anionic amino acid residues such as Asp or Glu) that are larger than the quantity of other amino acid residues or contain the assembly mainly formed by cationic amino acid residues. Statement "cation" can also refer to "polycationic" components / cationic components / compounds and can also refer to cationic lipids that can be positively charged. For example, cationic lipids contain one or more amine groups with a positive charge, and preferred cationic lipids are ionizable, so that they can exist in positively charged form or neutral form according to pH. The ionization of cationic lipids affects the surface charge of lipid nanoparticles (LNP) under different pH conditions. This charge state can affect plasma protein absorption, blood clearance and tissue distribution and the ability of forming a non-double-layer structure that is crucial to the intracellular delivery of nucleic acid.

[0081] In the present invention, "PEGylated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion.

[0082] In the present invention, "neutral lipid" refers to any of a number of lipid substances that exist in an uncharged or neutral zwitterionic form at a selected pH, preferably a phospholipid, and can be of synthetic or natural origin.

[0083] In the present invention, "steroid lipid" refers to a steroid or a steroid analog.

[0084] In the present invention, "amino acid residue" includes amino acids in which a hydrogen atom is removed from the amino group and / or a hydroxyl group is removed from the carboxyl group and / or a hydrogen atom is removed from the sulfhydryl group and / or the amino group is protected and / or the carboxyl group is protected and / or the sulfhydryl group is protected. Loosely speaking, an amino acid residue can be referred to as an amino acid. The source of the amino acid in the present invention is not particularly limited unless otherwise specified, and can be a natural source, a non-natural source, or a mixture of the two. The amino acid structure type in the present invention is not particularly limited unless otherwise specified, and can refer to L-type, D-type, or a mixture of the two. In one embodiment of the present invention, the amino acid is a hydrophobic amino acid selected from any one of tryptophan (Trp), phenylalanine (Phe), valine (Val), isoleucine (Ile), leucine (Leu) and tyrosine (Tyr). In another embodiment of the present invention, the amino acid is a hydrophilic amino acid selected from any one of glutamic acid (Glu), aspartic acid (Asp), histidine (His), glutamine (Gln), asparagine (Asn), serine (Ser), threonine (Thr), proline (Pro), glycine (Gly), lysine (Lys) and arginine (Arg), preferably glycine or lysine, more preferably lysine.

[0085] The modified form in the present invention refers to a structural form that can be converted into the target reactive group through any chemical change process such as oxidation, reduction, hydration, dehydration, electronic rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, deprotonation, substitution, deprotection, change of leaving group, etc.

[0086] In the present invention, the "changed form of a reactive group" refers to a reactive group that remains active (still a reactive group) after undergoing at least one chemical change process such as oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, deprotonation, substitution, deprotection, change of leaving group, or an inactive form after being protected.

[0087] The term "minor modification" in the present invention refers to a chemical modification process that can be completed through a simple chemical reaction process. The simple chemical reaction process mainly includes chemical reaction processes such as deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and conversion of leaving groups.

[0088] "Minor changes" correspond to "minor modifications" and refer to structures that can form the target reactive group after undergoing simple chemical reactions such as deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and leaving group conversion. Such conversion of leaving groups can, for example, be converted from an ester to an acyl chloride.

[0089] The term "any suitable" in "any suitable linker" or "any suitable reactive group" as used herein refers to a structure that conforms to the fundamental principles of chemical structure and enables the preparation method of the present invention to be successfully implemented. Chemical structures described in this manner can be considered to have a clear and defined scope.

[0090] When at least two structural types are listed, "any combination" of the listed structural types refers to a combination of any two or more structures of the aforementioned related structural types; and there is no limitation on the number of structural units. The number of any structural unit can be zero, one, or more than one. When the number of structural units of the same type is more than one, they can be structural units of the same or different chemical structures, and the total number of structural units is at least two. For example, any combination of alkylene, divalent cycloalkyl, divalent cycloalkenyl, divalent cycloalkynyl, divalent cycloalkadienyl, arylene, carbon-carbon double bond, carbon-carbon triple bond, conjugated carbon-carbon double bond, divalent aliphatic heterocyclic linking group, divalent aromatic heterocyclic linking group, and carbon chain linking group containing heteroatoms in the side group, for example, -Ph-CH2-Ph-(arylene-alkylene-arylene), -CH2-Ph-CH2CH2-(alkylene-arylene-alkylene, wherein the number of alkylene groups is 2 and has different chemical structures), or the structure in which the benzene ring in the above examples is replaced by a hexyl ring, a diazahexyl ring, or 1-(2-pyridyl)hexahydro-1H-1,4-diazepine. For another example, cycloalkenylalkyl group = cycloalkenyl + alkylene group = cycloalkenyl group as a substituent of the alkyl group, and cycloalkadienylalkyl group = cycloalkadienyl group as a substituent of the alkyl group.

[0091] In the present invention, the "N / P ratio" refers to the molar ratio of nitrogen atoms in the cationic lipid to phosphate in the nucleic acid.

[0092] In the present invention, "nucleic acid" refers to DNA or RNA or a modified form thereof, which contains purine or pyrimidine bases (adenine "A", cytosine "C", guanine "G", thymine "T") present in DNA or purine or pyrimidine bases (adenine "A", cytosine "C", guanine "G", uracil "U") present in RNA.

[0093] In the present invention, "RNA" refers to ribonucleic acid that may be naturally occurring or non-naturally occurring. For example, RNA may include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides or linkers. RNA may include a cap structure, a chain-terminating nucleoside, a stem-loop, a polyadenylate sequence and / or a polyadenylation signal. RNA may have a nucleotide sequence encoding a polypeptide of interest. For example, RNA may be messenger RNA (mRNA). Translation of an mRNA encoding a specific polypeptide, for example, translation of an mRNA in vivo within a mammalian cell, may produce the encoded polypeptide. RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single-stranded guide RNA (sgRNA), self-amplifying RNA (saran), circular RNA (circRNA), cas9 mRNA and mixtures thereof.

[0094] In the present invention, antisense oligonucleotides or small interfering RNA (siRNA) can inhibit the expression of target genes and target proteins in vitro or in vivo.

[0095] In the present invention, FLuc mRNA can express luciferase protein, which emits bioluminescence in the presence of luciferin substrate, so FLuc is often used in mammalian cell culture to measure gene expression and cell activity.

[0096] In the present invention, "inhibiting the expression of a target gene" refers to the ability of nucleic acid to silence, reduce or inhibit the expression of a target gene. To test the degree of gene silencing, a test sample (e.g., a cell sample in a culture medium expressing a target gene) is contacted with a nucleic acid that inhibits the expression of a target gene. The expression of the target gene in a test sample or test animal is compared with the expression of the target gene in a control sample (e.g., a cell sample in a culture medium expressing a target gene) that is not contacted with or not administered with nucleic acid. The expression of the target gene in the control sample can be assigned a value of 100%. In a specific embodiment, when the target gene expression level in the test sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% relative to the target gene expression level in the control sample or control mammal, the expression of the target gene is inhibited.

[0097] In the present invention, methods for determining the expression level of the target gene include, but are not limited to, dot blot, northern blot, in situ hybridization, ELISA, immunoprecipitation, enzyme action, and phenotypic assay.

[0098] In the present invention, "transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo, or in vivo.

[0099] In the present invention, "antigen" typically refers to a substance that can be recognized by the immune system, preferably recognized by the adaptive immune system, and can trigger an antigen-specific immune response, for example, by forming antibodies and / or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen can be or can comprise a peptide or protein that can be presented to a T cell by MHC. In the sense of the present invention, an antigen can be a translation product of a provided nucleic acid molecule (preferably an mRNA as defined herein). In this context, fragments, variants, and derivatives of peptides and proteins comprising at least one epitope are also understood to be antigens.

[0100] In the present invention, "delivery" refers to providing an entity to a target, for example, delivering a drug and / or therapeutic agent and / or preventive agent to a subject, which is a tissue and / or cell of a human and / or other animal.

[0101] As used herein, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with a therapeutic agent and, within the scope of reasonable medical judgment, suitable for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reaction, or other problems or complications commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is an exemplary carrier. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like. The composition may also contain a small amount of a wetting agent, emulsifier, or pH buffer, as needed. Oral formulations may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Specifically, for example, excipients include, but are not limited to, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), demulcents, emulsifiers, fillers (diluents), film formers or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration. More specifically, excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dicalcium phosphate, calcium stearate, cross-linked sodium carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, citric acid, cross-linked polyvinyl pyrrolidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, phenylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, and xylitol.

[0102] The pharmaceutical composition of the present invention can act systemically and / or locally. For this purpose, they can be administered by suitable routes, for example, by injection (such as intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermal administration; or by oral, buccal, nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation administration. For these routes of administration, the pharmaceutical composition of the present invention can be administered in suitable dosage forms. The dosage forms include but are not limited to tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

[0103] In the present invention, a vaccine is a preventive or therapeutic material that provides at least one antigen or antigenic function that can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0104] As used herein, "treatment" refers to the care and treatment of a patient for the purpose of combating a disease, disorder, or condition, and is intended to include delaying the progression of the disease, disorder, or condition, alleviating or relieving symptoms and complications, and / or curing or eliminating the disease, disorder, or condition. The patient to be treated is preferably a mammal, particularly a human.

[0105] 1. Amino acid cationic lipids

[0106] One embodiment of the present invention:

[0107] An amino acid cationic lipid, characterized in that the structure is as shown in the general formula (1):

[0108]

[0109] Wherein, AA is the residue of an amino acid or an amino acid derivative;

[0110] Each time B1 and B2 appear, they are independently connected or C 1-30 alkylene;

[0111] L1 and L2 are each independently a connecting bond or a divalent connecting group;

[0112] L5 and L6 are each independently a linking bond or a divalent linking group;

[0113] Each occurrence of L3 is independently a linking bond or a divalent linking group;

[0114] R1 and R2 are each independently -(CH2) t NR e R f , straight chain C 1-30 Alkylene, branched C 1-30 Alkylene or Wherein, t is an integer of 0-12, t1 and t2 are each independently an integer of 0-5, t3 and t4 are each independently 0 or 1, and t1, t2, t3, and t4 are not 0 at the same time; R e 、R f Each independently is C 1-15 Alkyl, C 2-15 Alkenyl and C 2-15 Any of alkynyl groups;

[0115] Each occurrence of R3 is independently a hydrogen atom, -R d 、-OR d 、C 3-6 Carbocyclic group, nitrogen-containing heterocyclic group, -NR d R d 、-SR d 、-C(=O)R d 、-C(=O)OR d 、-OC(=O)R d 、-OC(=O)OR d Or functional groups R that can interact with biologically related substances 01 ; Among them, R d Each occurrence is independently C 1-12 alkyl;

[0116] a, b, c are each independently 1 or 2; when the fragment -L5-B1-L1-R1 and / or -L6-B2-L2-R2 and / or -L3-R3 is derived from the amino terminus of the amino acid, a, b, c are each independently 1 or 2; when the fragment -L5-B1-L1-R1 and / or -L6-B2-L2-R2 and / or -L3-R3 is derived from the carboxyl terminus, hydroxyl terminus or thiol terminus of the amino acid, a, b, c are each independently 1; when a is 2, the two -L5-B1-L1-R1 fragments are the same or different; when b is 2, the two -L6-B2-L2-R2 fragments are the same or different; when c is 2, the two -L3-R3 fragments are the same or different;

[0117] or a salt, tautomer, stereoisomer or solvate thereof.

[0118] 1.1. Amino acid or amino acid derivative residue AA

[0119] In the present invention, AA is the residue of an amino acid or an amino acid derivative.

[0120] In a specific embodiment of the present invention, the aforementioned amino acid or amino acid derivative is preferably arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, histidine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine and an amino acid derivative of any of the aforementioned amino acids.

[0121] In a specific embodiment of the present invention, the amino acid or amino acid derivative residue AA is preferably any one of the following structures:

[0122] Among them, R a Each occurrence is independently any one of a linker, H, methyl, ethyl, propyl, and isopropyl; or AA is any one of the aforementioned structures, wherein 1-2 carbonyl groups are independently terminated by oxygen atoms or secondary amine atoms: or

[0123] In a specific embodiment of the present invention, the amino acid or amino acid derivative residue AA is preferably any one of the following structures:

[0124]

[0125]

[0126] 1.2.B1, B2

[0127] In the present invention, each occurrence of B1 and B2 is independently a connecting bond or C 1-30 Alkylene.

[0128] In a specific embodiment of the present invention, B1 and B2 are each independently a connecting bond or C 1-20 Alkylene; specifically any one of the following:

[0129] Case (1): B1 and B2 are each independently C 1-20 Alkylene, specifically B1 and B2 are each independently any one of methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene and eicosylene; more preferably B1 and B2 are each independently C 2-10 alkylene;

[0130] Case (2): One of B1 and B2 is a connecting bond, and the other is C 1-20 alkylene;

[0131] Case (3): B1 and B2 are both connecting bonds.

[0132] 1.3.L1, L2, L3, L5, L6

[0133] 1.3.1.L1, L2

[0134] In the present invention, L1 and L2 are each independently a linking bond or a divalent linking group.

[0135] In a specific embodiment of the present invention, L1 and L2 are each independently a linker, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, -NH-, -O(CR c R c ) s O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-, wherein R c Each occurrence is independently a hydrogen atom or a C 1-12 Alkyl, s is 1, 2, 3 or 4; more preferably L1, L2 is one of the following:

[0136] Case (1): L1 and L2 are each independently selected from -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, -O(CH2) s Any one of O-, -S-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH-, and -NHC(=O)S-;

[0137] Case (2): One of L1 and L2 is a connecting bond, and the other is -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -O-, or -O(CH2) sAny one of O-, -S-, -C(=O)S-, -SC(=O)-, -NHC(=O)-, -C(=O)NH-, -NHC(=O)NH-, -OC(=O)NH-, -NHC(=O)O-, -SC(=O)NH-, and -NHC(=O)S-;

[0138] Case (3): L1 and L2 are both connecting bonds.

[0139] In a specific embodiment of the present invention, preferably L1 and L2 are each independently any one of a linker, -C(=O)-, -O-, -NH-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NHC(=O)- and -C(=O)NH-.

[0140] 1.3.2.L3

[0141] In the present invention, each occurrence of L3 is independently a linking bond or a divalent linking group.

[0142] In a specific embodiment of the present invention, L3 is preferably a divalent linking group selected from any one, any two or a combination of any two or more of L7, L8 and Z divalent linking groups; more preferably, it is any one of -L7-, -L7-Z-, -Z-L7-, -Z-L7-Z-, -L7-Z-L8-, -Z-L7-Z-L8-, -L7-Z-L8-Z-, -Z-L7-Z-L8-Z- and -L7-Z-L8-Z-L7-Z-; wherein, L7 and L8 are carbon chain linking groups, each independently being -(CR a R b ) t -(CR a R b ) o -(CR a R b ) p -, t, o, p are each independently an integer of 0-12, and t, o, p are not 0 at the same time, R a and R b Each occurrence is independently a hydrogen atom or a C 1-12 Alkyl; each occurrence of Z is independently -C(=O)-, -NH-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c-、-NR c C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-, wherein R c Each occurrence is independently H or C 1-12 alkyl.

[0143] In a specific embodiment of the present invention, preferably the aforementioned L3 is -(CH2) t -、-(CH2) t Z-, -Z(CH2) t -、-(CH2) t Z(CH2) t -、-Z(CH2) t Z-, -(CH2) t Z(CH2) t Z-, -Z(CH2) t Z(CH2) t - and -Z(CH2) t Z(CH2) t Any one of Z-, wherein t is an integer from 1 to 12; preferably -(CH2) t -、-(CH2) t O-, -(CH2) t C(=O)-、-(CH2) t NH-, -(CH2) t C(=O)O-、-(CH2) t OC(=O)-、-(CH2) t C(=O)NH-、-(CH2) t NHC(=O)-、-(CH2) t OC(=O)O-、-(CH2) t NHC(=O)O-、-(CH2) t OC(=O)NH-、-(CH2) t NHC(=O)NH-、-O(CH2) t -, -C(=O)(CH2) t -、-C(=O)O(CH2) t -, -OC(=O)(CH2) t -、-C(=O)NH(CH2) t -, -NHC(=O)(CH2) t -、-OC(=O)O(CH2) t -, -NHC(=O)O(CH2) t -、-OC(=O)NH(CH2)t -、-NHC(=O)NH(CH2) t -、-(CH2) t O(CH2) t -、-(CH2) t C(=O)(CH2) t -、-(CH2) t C(=O)O(CH2) t -、-(CH2) t OC(=O)(CH2) t -、-(CH2) t C(=O)NH(CH2) t -、-O(CH2) t O-、-(CH2) t NHC(=O)(CH2) t -、-(CH2) t OC(=O)O(CH2) t -、-(CH2) t NHC(=O)O(CH2) t -、-(CH2) t OC(=O)NH(CH2) t -、-(CH2) t NHC(=O)NH(CH2) t -、-C(=O)(CH2) t C(=O)-、-C(=O)O(CH2) t C(=O)O-、-OC(=O)(CH2) t OC(=O)-、-C(=O)O(CH2) t OC(=O)-、-OC(=O)(CH2) t C(=O)O-、-OC(=O)O(CH2) t OC(=O)O-、-C(=O)NH(CH2) t C(=O)NH-、-NHC(=O)(CH2) t NHC(=O)-、-NHC(=O)(CH2) t C(=O)NH-、-C(=O)NH(CH2) t NHC(=O)-、-NHC(=O)O(CH2) t NHC(=O)O-、-OC(=O)NH(CH2) t OC(=O)NH-、-C(=O)(CH2) t O-、-NHC(=O)O(CH2) tOC(=O)NH-, -OC(=O)NH(CH2) t NHC(=O)O-, -C(=O)(CH2) t C(=O)O-, -NHC(=O)NH(CH2) t NHC(=O)NH-, -C(=O)(CH2) t OC(=O)-、-C(=O)(CH2) t OC(=O)O-, -C(=O)(CH2) t NHC(=O)O-, -C(=O)(CH2) t OC(=O)NH-, -C(=O)(CH2) t NHC(=O)NH-, -C(=O)(CH2) t C(=O)O(CH2) t -, -C(=O)(CH2) t OC(=O)(CH2) t -, -C(=O)(CH2) t OC(=O)O(CH2) t -, -C(=O)(CH2) t NHC(=O)O(CH2) t -, -C(=O)(CH2) t OC(=O)NH(CH2) t -, -C(=O)(CH2) t NHC(=O)NH(CH2) t - and -C(=O)(CH2) t C(=O)(CH2) t Any of NHC(=O)O-; most preferably -(CH2) t -、-(CH2) t NH-, -(CH2) t O-, -(CH2) t C(=O)O-、-(CH2) t OC(=O)-、-O(CH2) t -Any one.

[0144] 1.3.3.L5, L6

[0145] In the present invention, L5 and L6, when appearing each time, are independently a linking bond or a divalent linking group.

[0146] In a specific embodiment of the present invention, L5 and L6 are each independently any one of a linker, -O-, -NH-, -C(=O)-, -OC(=O)- and -C(=O)O-; more preferably, one of L5 and L6 is a linker, and the other is -O-, -NH-, -C(=O)-, -OC(=O)-, -C(=O)O-; more preferably, L5 and L6 are both linkers, or both are -O-, or both are -NH-, or both are -C(=O)O-.

[0147] 1.4.R1, R2, R3

[0148] 1.4.1.R1, R2

[0149] In the present invention, R1 and R2 are each independently a linear alkyl group, a branched alkyl group or

[0150] In a specific embodiment of the present invention, the linear alkyl group is C 1-25 Straight chain alkyl, more preferably C 1-17 The linear alkyl group is specifically any one of pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl and heptadecyl.

[0151] In one embodiment of the present invention, the branched alkyl group is represented by Among them, R e 、R f Each independently is C 1-15 Alkyl; more preferably, each independently selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl; more preferably, the branched alkyl group is selected from any one of the following structures:

[0152]

[0153] In a specific embodiment of the present invention, Select any one of the following structures:

[0154] 1.4.2.R3

[0155] In the present invention, each occurrence of R3 is independently a hydrogen atom, -R d 、-OR d 、C 3-6 Carbocyclic group, nitrogen-containing heterocyclic group, -NR d R d 、-SR d 、-C(=O)R d 、-C(=O)OR d 、-OC(=O)R d、-OC(=O)OR d Or functional groups R that can interact with biologically related substances 01 ; Among them, R d Each occurrence is independently C 1-12 alkyl.

[0156] In one embodiment of the present invention, preferably each occurrence of R3 is independently a hydrogen atom, an alkyl group, an alkoxy group, -C(=O)OR d 、-OC(=O)R d 、-OC(=O)OR d , epoxy, hydroxy, protected hydroxy, sulfhydryl, protected sulfhydryl, carboxyl, protected carboxyl, amino, protected amino, aldehyde, protected aldehyde, active ester, carbonate, carbamate, isocyanate, isothiocyanate, succinimide, maleimide, protected maleimide, dimethylamino, alkenyl, alkenoate, azido, cyano, dithiopyridyl, α-haloacetyl alkynyl, alkynyl, folic acid, rhodamine, biotinyl, monosaccharide, polysaccharide, Any one of the following, where R d Each occurrence is independently C 1-12 alkyl.

[0157] In one embodiment of the present invention, each occurrence of R3 is independently a functional group R 01 , the R 01 is a functional group with therapeutic targeting; preferably, it is a residue of any one of folic acid and N-acetylgalactosamine or a residue of a functional derivative of any one of them; further preferably, it is any one of the following structures:

[0158]

[0159] 1.5. -L3-R3, -L5-B1-L1-R1, and -L6-B2-L2-R2 fragments

[0160] 1.5.1.-L3-R3 fragment

[0161] In a specific embodiment of the present invention, each occurrence of -L3-R3 consisting of L3 described in Section 1.3.2 and R3 described in Section 1.4.2 is independently selected from any one of the following structures:

[0162]

[0163]

[0164] 1.5.2.-L5-B1-L1-R1 and -L6-B2-L2-R2 fragments

[0165] In a specific embodiment of the present invention, -L5-B1-L1-R1 and -L6-B2-L2-R2 are each independently selected from any one of the following structures:

[0166]

[0167] 1.6. Examples of general structural formulas

[0168] In a specific embodiment of the present invention, according to L1 and L2 described in 1.3.1, the structure of the amino acid cationic lipid of the present invention is selected from any one of the following general formulas:

[0169]

[0170] Wherein, s is 1, 2, 3 or 4; the definitions of AA, B1, B2, L3, L5, B6, R1, R2, R3, a, b, and c are the same as those described in general formula (1) and are not repeated here.

[0171] In a specific embodiment of the present invention, the structure of the amino acid cationic lipid preferably satisfies any one of the following general formulas:

[0172]

[0173] wherein the definitions of AA, B1, B2, L1, L2, L3, L5, B6, R1, R2, R3, and c are consistent with those in the general formula (1) and are not repeated here, and B1, B2, L1, L2, L5, and L6 are not connecting bonds; more preferably, each occurrence of L1, L2, L5, and L6 is independently any one of -O-, -NH-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH-, and -NHC(=O)O-.

[0174] In a specific embodiment of the present invention, the structure of the amino acid cationic lipid preferably satisfies any one of the following general formulas:

[0175]

[0176] wherein the definitions of B1, B2, L1, L2, L3, R1, R2, and R3 are consistent with those described in general formula (1) and are not repeated here, and B1, B2, L1, L2, and L3 are not connecting bonds; more preferably, each occurrence of L1 and L2 is independently any one of -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH-, and -NHC(=O)O-; more preferably, each occurrence of L1 and L2 is independently any one of -C(=O)-, -C(=O)O-, -OC(=O)-, and -OC(=O)O-.

[0177] 1.7. Specific structure examples

[0178] In one embodiment of the present invention, the structure of the amino acid cationic lipid is preferably selected from any one of the following structures:

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187] 2. Preparation of Amino Acid Cationic Lipids

[0188] In the present invention, any of the aforementioned amino acid cationic lipids can be prepared by methods including but not limited to the following:

[0189] Method 1:

[0190] Step 1: react one molecule of A-1 (AA1) with one or two identical or different molecules of A-2 (R3'-F1) to generate a small molecule intermediate A-3 containing a divalent linker L3. Wherein, the small molecule A-1 is an amino acid or an amino acid derivative, and A-1 contains one, two, or three identical or different amino acid terminal groups; the small molecule A-2 contains a reactive group F1, which can react with the amino acid terminal group of AA1 to form a divalent linker L3, and F1 is preferably -OH, -COOH, -NH2, -Br, -CHO, -OMs, -COCl, etc., and R3' is R3 or contains R 01 "AA" is an amino acid derivative residue containing two identical or different amino acid terminal groups;

[0191] Step 2: Small molecule A-4 (R2-F2) and small molecule A-5 (F3-B2-F N ) reacts to generate a divalent linker L2 with a reactive group F at one end. N 、Small molecule intermediate A-6 (R2-L2-B2-F N ); wherein, small molecule A-4 contains a reactive group F2, and small molecule A-5 contains a heterofunctional group F3 and F N , F2 can react with F3 to generate a divalent linker L2, F N is a reactive group capable of reacting with a carboxyl group, a hydroxyl group, or an amino group, preferably -OH, -COOH, -NH2, -F, -Cl, -Br, -COCl, wait;

[0192] Step 3: One or two identical or different small molecule intermediates A-6 (R2-L2-B2-F N ) and small molecule intermediate A-3 The reaction is carried out to obtain an amino acid derivative A-7 containing a divalent linker L6. Among them, AA' is an amino acid derivative residue containing one amino acid terminal group;

[0193] Step 4: Amino acid derivative A-7 With one or two molecules of small molecule intermediate A-8 (R1-L1-B1-F C , synthesized according to the method of step 2 or purchased) to react to generate amino acid cationic lipid A-9' containing a divalent linker L5 Among them, the small molecule A-8 contains a group F that can react with the end group of an amino acid (such as a carboxyl group, a hydroxyl group, a thiol group, or an amino group). C , preferably -OH, -COOH, -NH2, -Br,

[0194] When R3' is equal to R3, the resulting structure A-9' corresponds to the structure shown in general formula (1);

[0195] When R3' is not equal to R3, A-9' is subjected to terminal micro-modification to obtain A-9, which corresponds to the structure shown in general formula (1); the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group;

[0196] The definitions of AA, L1, L2, L3, L5, L6, B1, B2, R3, R1, R2, a, b, and c are the same as those described in general formula (1) and are not repeated here.

[0197] The aforementioned small molecule raw materials A-1, A-2, A-3, A-4, A-5, A-8, etc. can be purchased or synthesized independently. For example, the small molecule A-3 in Example 1 is It can be obtained by lysine protected with amino Boc and ethyl bromide The raw material was synthesized independently, and then Boc was removed to obtain the small molecule A-3 in Example 6. It can be purchased, so step 1 can be omitted; in Example 8, A-3 is You can use and It is obtained by reacting the raw materials and then deprotecting them.

[0198] Step 1

[0199]

[0200] Step 2

[0201]

[0202] Step 3

[0203]

[0204] Step 4

[0205]

[0206] In this method, when A-3 in step 3 When the two amino acid end groups of AA are the same, A-3 can also react with two or four molecules of A-6 (R2-L2-B2-F N ) to obtain A-10'; in this case, L5 and L6 are the same, B1 and B2 are the same, L1 and L2 are the same, R1 and R2 are the same, a and b are the same, and the definitions of L1, L2, L3, L5, L6, B1, B2, R3, R1, R2, a, b, and c are the same as those in the general formula (1) and are not repeated here; F N, AA", R3' are consistent with the above, and will not be repeated here;

[0207] When R3' is equal to R3, the resulting structure A-10' corresponds to the structure shown in general formula (1);

[0208] When R3' is not equal to R3, A-10' is subjected to terminal micro-modification to obtain A-10 corresponding to the structure shown in general formula (1); the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group.

[0209] The aforementioned preparation steps and reactions with multiple molecules can be carried out in a single step or in separate steps.

[0210]

[0211] 2.2.Method 2:

[0212] Step 1: Combine one molecule of B-1 (AA1) with one or two molecules of the same or different B-2 (R2-L2-B2-F N , synthesize according to the method of step 2 of 2.1. Method 1 or purchase) to generate intermediate B-3 containing a divalent linker L6 Among them, small molecule B-1 is an amino acid or amino acid derivative containing one, two or three identical or different amino acid end groups; small molecule B-2 contains a divalent linker L2 and a reactive group F N , F N Can react with the amino acid end group in B-1 to form a divalent linking group L6, preferably -OH, -COOH, -NH2, -F, -Cl, -Br, -COCl, etc.; AA" is an amino acid derivative residue containing two identical or different amino acid terminal groups;

[0213] Step 2: Monomolecular intermediate B-3 With two molecules of the same or different B-4 (R1-L1-B1-F C , synthesize according to the method of step 2 of 2.1. Method 1 or purchase) to generate intermediate B-5 containing a divalent linking group L5 Among them, the small molecule B-4 contains a divalent linker L1 and a reactive group F C , F C Can react with the amino acid end group in B-3 to form a divalent linker L5, F C Preferably -OH, -COOH, -NH2, -F, -Cl, -Br; AA' is an amino acid derivative residue containing one amino acid terminal group;

[0214] Step 3: One molecule of intermediate B-5 reacts with one or two molecules of the same or different B-6 (R3'-F4) to generate an amino acid cationic lipid B-7' containing a divalent linker L3. Among them, the small molecule B-6 contains a reactive group F4, which can react with the amino acid end group of AA' to form a divalent linker L3, and F4 is preferably -OH, -COOH, -NH2, -F, -Cl, -Br, AA is an amino acid or amino acid derivative residue;

[0215] When R3' is equal to R3, the resulting structure B-7' corresponds to the structure shown in general formula (1);

[0216] When R3' is not equal to R3, B-7' is subjected to terminal micro-modification to obtain B-7, which corresponds to the structure shown in general formula (1); the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group;

[0217] The definitions of L1, L2, L3, L5, L6, B1, B2, R3, R1, R2, a, b, and c are the same as those described in general formula (1) and will not be repeated here.

[0218] The aforementioned small molecule raw materials B-1, B-2, B-4, and B-6 can be purchased or synthesized independently.

[0219] Step 1

[0220]

[0221] Step 2

[0222]

[0223] Step 3

[0224]

[0225] 2.3. Method 3:

[0226] Step 1: a molecule of C-1( The amino acid derivative C-3 containing the divalent linking groups L3 and L5 is reacted with one or two identical or different C-2 molecules (R1-L1-B1-Fc, synthesized or purchased according to the method of 2.1. Method 1, step 1) to generate an amino acid derivative C-3 containing the divalent linking groups L3 and L5. Among them, small molecule C-1 is an amino acid or amino acid derivative containing two identical or different amino acid end groups; small molecule C-2 contains a reactive group F c, which can react with the amino acid terminal group of C-1 to form a divalent linking group L5, L5 is the same, preferably -OH, -COOH, -NH2, -Br, etc.; AA' is an amino acid derivative residue containing one amino acid terminal group;

[0227] Step 2: Amino acid derivative C-3 With the small molecule intermediate C-4(R2-L2-B2-F N , synthesized according to the method of step 2 of 2.1. Method 1 or purchased) should generate an amino acid cationic lipid C-5' containing a divalent linker L6 Among them, the small molecule C-4 contains a group F that can react with carboxyl, hydroxyl or amino groups. N , preferably -OH, -COOH, -NH2, -Br, -COCl, etc.; AA is an amino acid or amino acid derivative residue;

[0228] When R3' is equal to R3, the resulting structure C-5' corresponds to the structure shown in general formula (1);

[0229] When R3' is not equal to R3, C-5' is subjected to terminal micro-modification to obtain C-5, which corresponds to the structure shown in general formula (1); the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group;

[0230] Wherein, the definitions of L1, L2, L3, L5, L6, B1, B2, R3, R1, R2, a, b, and c are consistent with those described in formula (1) and are not repeated here;

[0231] The aforementioned small molecule raw materials C-1, C-2, and C-4 can be purchased or synthesized independently.

[0232] Step 1

[0233]

[0234] Step 2

[0235]

[0236] In the aforementioned preparation methods 1-3:

[0237] The amino acid terminal groups are protected or unprotected, including but not limited to amino, carboxyl, hydroxyl, sulfhydryl, protected amino, protected carboxyl, protected hydroxyl, protected sulfhydryl; the R 01 The slightly changed form refers to the structural form that can form the target reactive group after simple chemical reaction processes such as deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and change of leaving group.

[0238] a, b, and c are each independently 1 or 2, depending on the type of amino acid end group connected to -L6-B2-L2-R2, -L5-B1-L1-R1, and -L3-R3 and the reaction feed amount. When a is 2, each fragment can be the same or different, depending on the reaction between the amino acid derivative and each fragment. If the reaction with two different fragments is sequential, the reaction is different, and if the reaction with two identical fragments is single-step or step-by-step, the reaction is the same. For example, in method 1, b is 1 or 2, depending on the reaction with F N The end group type of the reacted AA" is R2-L2-B2-F N Feeding situation; when with F N When the terminal group of the reacted AA" is a carboxyl group or a hydroxyl group, b is 1; when it is an amino group, b is 1 or 2; and when b is 2, the two -L6-B2-L2-R2 fragments can be the same or different, that is, when F N When reacting with the amino terminal group of AA", A-3 can react successively with different A-6 to obtain A-7 with b being 2 and two different -L6-B2-L2-R2 fragments. Alternatively, one molecule of A-3 can react with two identical molecules of A-6 in a single step or in multiple steps to obtain A-7 with b being 2 and two identical -L6-B2-L2-R2 fragments.

[0239] R2 in the reaction raw material R2-F2 can be an etherified aliphatic hydrocarbon derivative residue Wherein, t is independently an integer from 0 to 12 each time it occurs; R e 、R f Each independently is C 1-15 Alkyl, C 1-15 Alkenyl and C 2-15 More specifically, R1-F1 can be It can be purchased or synthesized independently. When synthesizing independently, aldehyde-alcohol addition can be used, for example, a molecule With two molecules R e -OH is added to obtain At this time R e and R f Same; R1-F1 can also be It can be purchased or synthesized independently. It is obtained by reacting with a relevant alkylating agent, wherein the alkylating agent is preferably a halide, such as It can be obtained by reacting one molecule of TBS-protected glycerol with two molecules of bromohexane followed by deprotection.

[0240] 2.4. Description of relevant raw materials and / or steps in the preparation process

[0241] 2.4.1. Condensation agents, oxidizing agents, and reducing agents

[0242] In the present invention, the condensing agent used in the reaction is not limited, but preferably includes N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU). DCC is most preferred. The amount of the condensing agent used is generally 1 to 20 times the molar equivalent of the carboxylic acid, preferably 5 to 10 times. A suitable catalyst (such as 4-dimethylaminopyridine) may be added to this reaction.

[0243] In the present invention, the oxidant used in the reaction is not particularly limited, as long as it is a compound or a combination of multiple compounds that can increase the valence of the substrate, preferably phenyliodonium di(trifluoroacetate), 1,4-benzoquinone, benzyltrimethylammonium tribromide, pyridinium dichromate, potassium dichromate, ozone, oxygen, hypofluoric acid, sodium hypochlorite, cobalt acetate, cobalt acetate, manganese acetate, palladium acetate, copper acetate, monoperoxyphthalic acid, iodine, N-iodosuccinimide, iodobenzoylbenzene, 2-iodobenzoic acid, dimethyldioxycyclopropane, dimethyl sulfoxide-oxalyl chloride, dimethyl sulfoxide-acetic anhydride, DDQ, dichlorotris(triphenylphosphine)ruthenium, manganese dioxide, diacetoxyiodobenzene, periodic acid, sodium periodate, sodium periodate-osmium tetroxide, potassium permanganate, sodium perborate, peroxybenzoic acid, dibenzoyl peroxide, Nickel peroxide, hydrogen peroxide, cumene hydroperoxide, tert-butyl peroxide, peracetic acid, m-chloroperbenzoic acid, N-chlorosuccinimide, pyridinium chlorochromate, palladium chloride-copper chloride, urea hydrogen peroxide complex, triphenylmethyl tetrafluoroborate, tributyltin oxide, cobalt trifluoride, vanadium trifluoride, chromium trioxide, manganese triacetate, TEMPO, ceric ammonium nitrate, bromine, N-pyridine oxide, silver oxide, O-ethyl peroxycarbonic acid, manganese acetylacetonate, vanadium acetylacetonate, aluminum isopropoxide, potassium persulfate, dichloroiodobenzene, etc., or a combination thereof, more preferably oxygen, sodium hypochlorite, hydrogen peroxide, dichloroiodobenzene, potassium persulfate, etc., or a combination thereof, the amount of the oxidant being 1 to 50 times, preferably 1 to 20 times, and more preferably 5 to 10 times, the molar equivalent of the hydroxyl group in the intermediate compound.

[0244] In the present invention, the reducing agent used in the reaction is not particularly limited, as long as it can reduce the Schiff base generated by ammonia and aldehyde or ketone to an amino group; preferably, one or a combination of sodium borohydride, sodium cyanoborohydride, lithium aluminum hydride, borane, diborane, diisobutylaluminum hydride, diisopinocamphor borane, lithium borohydride, zinc borohydride, borane-pyridine, borane-methyl sulfide, borane-tetrahydrofuran, etc.; more preferably, sodium cyanoborohydride. The equivalent of the reducing agent is 1 to 50 times the molar equivalent of the amino group to be modified, preferably 1 to 20 times, and more preferably 5 to 10 times.

[0245] In the present invention, the reaction temperature is 0 to 200°C, preferably 0 to 100°C, more preferably 0 to 25°C, and the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours. The obtained product can be purified by purification methods such as extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, or supercritical extraction.

[0246] In the present invention, the reaction solvent can be a solvent-free solvent or an aprotic solvent. The aprotic solvent includes toluene, benzene, xylene, acetonitrile, ethyl acetate, diethyl ether, methyl tert-butyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, preferably tetrahydrofuran, dichloromethane, dimethyl sulfoxide, and dimethylformamide.

[0247] In the present invention, the base used in the reaction is an inorganic base or an organic base, preferably an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole, or diisopropylethylamine); preferably triethylamine and pyridine. The amount of the base used is 1 to 50 times the molar equivalent of the carboxylic acid, preferably 1 to 10 times, and more preferably 2 to 3 times.

[0248] 2.4.2. Protection and Deprotection of Related Groups During the Reaction

[0249] The present invention also involves the "protection" and "deprotection" processes of related groups during the reaction. To prevent the functional groups from affecting the reaction, they are typically protected. Furthermore, when there are two or more functional groups, the remaining functional groups are protected in order to selectively allow only the target functional group to react. Protecting groups not only stably protect the target functional group but also need to be easily removable as needed. Therefore, in organic synthesis, it is important to deprotect only the protecting groups bonded to the designated functional groups under appropriate conditions.

[0250] In the present invention, the definitions of "carboxyl protecting group" and "amino protecting group" are consistent with those in the "Term Description" section and will not be repeated here.

[0251] In the present invention, the hydroxyl group protected by the hydroxyl-protecting group is not particularly limited, and may be, for example, an alcoholic hydroxyl group, a phenolic hydroxyl group, or the like. The amino group protected by the amino-protecting group is not particularly limited, and may be, for example, a primary amine, a secondary amine, a hydrazine, an amide, or the like. The amino group in the present invention is not particularly limited, and includes, but is not limited to, a primary amino group, a secondary amino group, a tertiary amino group, and a quaternary ammonium ion.

[0252] In the present invention, the deprotection of the protected hydroxyl group is related to the type of hydroxyl protecting group. The type of the hydroxyl protecting group is not particularly limited. Taking benzyl, silyl ether, and tert-butyl as examples for protecting the terminal hydroxyl group, the corresponding deprotection methods are:

[0253] A: Deprotection of benzyl protecting group

[0254] Benzyl deprotection can be achieved by utilizing the hydrogenation reaction of a hydrogenation reducing agent and a hydrogen donor. The water content in this reaction system should be less than 1% for the reaction to proceed smoothly.

[0255] The hydrogenation reduction catalyst is not limited, and is preferably palladium and nickel, but the carrier is not limited, but preferably aluminum oxide or carbon, more preferably carbon. The amount of palladium is 1 to 100wt% of the protected hydroxy compound, preferably 1 to 20wt% of the protected hydroxy compound.

[0256] The reaction solvent is not particularly limited, as long as both the raw materials and the product can be dissolved. However, methanol, ethanol, ethyl acetate, tetrahydrofuran, and acetic acid are preferred, with methanol being more preferred. The hydrogen donor is not particularly limited, but hydrogen, cyclohexene, 2-propanol, ammonium formate, and the like are preferred. The reaction temperature is preferably 25 to 40°C. The reaction time is not particularly limited and is inversely correlated with the amount of catalyst used, but is preferably 1 to 5 hours.

[0257] B: Deprotection of silyl ether protecting groups

[0258] Compounds used for this type of hydroxyl protection include trimethylsilyl ether, triethylsilyl ether, dimethyl tert-butylsilyl ether, tert-butyldiphenylsilyl ether, etc. Deprotection of this type of silyl ether is performed using a fluorine-containing compound, preferably tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid, potassium fluoride, and more preferably tetrabutylammonium fluoride and potassium fluoride. The amount of the fluorine-containing reagent used is 5 to 20 times the molar equivalent of the protected hydroxyl group, preferably 8 to 15 times the molar equivalent of the initiator. If the amount of the fluorine-containing reagent is less than 5 times the molar equivalent of the protected hydroxyl group, incomplete deprotection will result. When the amount of the deprotection reagent is greater than 20 times the molar equivalent of the protected hydroxyl group, the excess reagent or compound will cause problems in purification and may be mixed into subsequent steps, causing side reactions. The reaction solvent is not particularly limited, as long as it can dissolve the reactants and products. Aprotic solvents are preferred, and tetrahydrofuran and dichloromethane are more preferred. The reaction temperature is preferably 0 to 30°C. When the temperature is below 0°C, the reaction rate is slow and the protecting group cannot be completely removed.

[0259] C: Deprotection of tert-butyl protecting group

[0260] Deprotection of the tert-butyl group is carried out under acidic conditions, preferably at a solution pH of 0 to 4. The acid is not particularly limited, but is preferably acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, or nitric acid, with hydrochloric acid being more preferred. The reaction solvent is not particularly limited, as long as it can dissolve the reactants and products, and is preferably water. The reaction temperature is preferably 0 to 30°C.

[0261] 2.4.3. Alkylation reaction

[0262] The alkylation reaction of the present invention is preferably based on the alkylation of hydroxyl, sulfhydryl or amino groups, corresponding to the formation of ether bonds, thioether bonds, secondary amino groups or tertiary amino groups, respectively. Examples are as follows:

[0263] 2.4.3.1. Alkylation of the Substrate Alcohol with Sulfonates and Halides

[0264] In the presence of a base, an amine intermediate is obtained by nucleophilic substitution of substrate alcohol with sulfonic ester derivatives and halide. Wherein, the molar equivalent of sulfonic ester and halide is 1 to 50 times, preferably 1 to 5 times, of substrate alcohol. When the molar equivalent of the molar equivalent of sulfonic ester and halide is less than 1 times of molar equivalent of substrate alcohol, the reaction substitution is incomplete and is difficult to purify. And when the molar equivalent of sulfonic ester and halide is greater than 50 times of substrate alcohol, excessive reagent brings trouble to purification and may be mixed into subsequent steps, thereby causing next step side reaction to increase, increasing purification difficulty.

[0265] The resulting product is a mixture of an ether intermediate and excess sulfonate and halide, which can be purified by methods such as anion exchange resin, osmosis, and ultrafiltration. The anion exchange resin is not particularly limited, as long as the target product can undergo ion exchange and adsorption on the resin. Ion exchange resins containing tertiary amines or quaternary ammonium salts, such as dextran, agarose, polypropionate, polystyrene, and polystyrene, as backbones are preferred. The solvent for osmosis and ultrafiltration is not limited, and can generally be water or an organic solvent. The organic solvent is not particularly limited, as long as the product can be dissolved therein. Dichloromethane, chloroform, and the like are preferred.

[0266] The reaction solvent is not limited, but is preferably an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, more preferably dimethylformamide, dichloromethane, dimethyl sulfoxide or tetrahydrofuran.

[0267] The base includes an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine) or an inorganic base (such as sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium carbonate or potassium hydroxide), preferably an organic base, more preferably triethylamine or pyridine. The molar amount of the base is 1 to 50 times the molar equivalent of the sulfonic acid ester or halide, preferably 1 to 10 times, more preferably 3 to 5 times.

[0268] 2.4.3.2. Alkylation of substrate amine with sulfonate or halide

[0269] A. Alkylation of substrate amine with sulfonate and halide

[0270] In the presence of a base, an amine intermediate is obtained by nucleophilic substitution of a substrate amine with a sulfonate derivative and a halide. The molar equivalent of the sulfonate and halide is 1 to 50 times, preferably 1 to 5 times, that of the substrate amine. When the molar equivalent of the sulfonate and halide is less than 1 molar equivalent of the substrate amine, the substitution reaction is incomplete and purification is difficult. When the molar equivalent of the sulfonate and halide is greater than 50 times that of the substrate amine, the excess reagents cause trouble for purification and may be mixed into subsequent steps, thereby increasing side reactions in the next step and increasing the difficulty of purification.

[0271] The resulting product is a mixture of an amine intermediate and excess sulfonate and halide, which can be purified by column chromatography, anion exchange resin, osmosis, ultrafiltration, and the like. The anion exchange resin is not particularly limited, as long as the target product can undergo ion exchange and adsorption on the resin. Ion exchange resins containing tertiary amines or quaternary ammonium salts, such as dextran, agarose, polypropionate, polystyrene, and polystyrene, as backbones are preferred. The solvent for osmosis and ultrafiltration is not limited, and can generally be water or an organic solvent. The organic solvent is not particularly limited, as long as the product can be dissolved therein. Dichloromethane, chloroform, and the like are preferred.

[0272] The reaction solvent is not limited, but is preferably an aprotic solvent such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, more preferably dimethylformamide, dichloromethane, dimethyl sulfoxide or tetrahydrofuran.

[0273] The base includes an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine) or an inorganic base (such as sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium carbonate or potassium hydroxide), preferably an organic base, more preferably triethylamine or pyridine. The molar amount of the base is 1 to 50 times the molar equivalent of the sulfonic acid ester or halide, preferably 1 to 10 times, more preferably 3 to 5 times.

[0274] 2.4.3.3. Alkylation reaction of substrate amine with aldehyde derivatives

[0275] After the substrate amine reacts with the aldehyde derivative to obtain the imine intermediate, the intermediate is obtained under the action of a reducing agent. The molar equivalent of the aldehyde derivative is 1 to 20 times that of the substrate amine, preferably 1 to 2 times, and more preferably 1 to 1.5 times. When the molar equivalent of the aldehyde derivative is greater than 20 times that of the substrate amine, the excess reagent brings trouble to the purification and may be mixed into the subsequent steps, increasing the difficulty of purification. When the molar equivalent of the aldehyde derivative is less than 1 times that of the substrate amine, the reaction is incomplete, increasing the difficulty of purification. The product after the reaction can be purified by means of cation exchange resin, osmosis, ultrafiltration, etc. There is no particular limitation on the cation exchange resin, as long as it can exchange with the quaternary ammonium cation to achieve a separation effect. The solvent for osmosis and ultrafiltration is not limited, and can generally be water or an organic solvent. The organic solvent is not particularly limited, as long as the product can be dissolved in it, preferably dichloromethane, chloroform, etc.

[0276] The reaction solvent is not limited, and is preferably an organic solvent such as methanol, ethanol, water, toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide; more preferably water and methanol.

[0277] The reducing agent is not particularly limited, as long as it can reduce the imine to an amine. Preferred are sodium borohydride, lithium aluminum hydride, sodium cyanoborohydride, Zn / AcOH, and the like, with sodium cyanoborohydride being more preferred. The amount of reducing agent used is generally 0.5 to 50 times, more preferably 1 to 10 times, the amount of the aldehyde derivative.

[0278] 2.4.3.4. Linear functionalization of the terminal end

[0279] The method of terminal linear functionalization is not particularly limited and is related to the type of the final functional group or its protected form. It mainly includes the functionalization of the terminal hydroxyl group and the conversion of the reactive group to the target functional group or its protected form.

[0280] The preparation method for functionalizing the terminal hydroxyl group is to obtain functional groups of types A to J or their protected forms by functionalization starting from the terminal hydroxyl group of A. The specific preparation method is described in paragraphs

[0960] to

[1205] of document CN104530417A. The general reaction formula is as follows:

[0281]

[0282] Wherein, q and q1 are each independently 0 or 1; Z1 and Z2 are each independently a divalent linking group; R 01 It is a functional group that can react with biologically related substances.

[0283] The conversion of reactive groups to target functional groups or their protected forms can be achieved by any of the following methods:

[0284] Method 1: Direct modification: Direct modification of reactive groups to obtain the target functional group or its protected form. Examples include the conversion of carboxyl groups to acyl halides, hydrazides, esters, thioesters, and dithioesters, as well as the conversion of hydroxyl groups, sulfhydryl groups, alkynyl groups, amino groups, and carboxyl groups to their corresponding protected structures. Another example is the modification of hydroxyl groups and amino groups with acid anhydrides.

[0285] Method 2: A coupling reaction between two reactive groups. Using a heterofunctionalizing reagent containing one reactive group and the target functional group or its protected form as a starting material, the target functional group or its protected form is introduced via a reaction between one of the reactive groups and the reactive group at the A terminus. The reaction method and manner between the two reactive groups are not particularly limited. The reaction conditions are related to the type of divalent linker formed by the reaction, and existing publicly available techniques can be employed. Examples include alkylation, alkenyl addition, alkynyl addition, Schiff base reaction combined with reduction, and condensation reactions. Alkylation reactions are preferably based on the alkylation of sulfhydryl or amino groups, corresponding to the formation of thioether bonds, secondary amino groups, or tertiary amino groups, respectively. Condensation reactions include, but are not limited to, those forming ester groups, thioester groups, amide groups, imine bonds, hydrazone bonds, and carbamate groups. Another example is the click reaction, where heterofunctionalized reagents containing groups such as azide, alkynyl, alkenyl, trithioester, sulfhydryl, dienyl, furyl, 1,2,4,5-tetrazine, and cyanate are used as raw materials to introduce the target functional group or its protected form. The reaction between the two reactive groups is accompanied by the formation of a new bond. Typical examples of the newly formed divalent linking groups include amide bonds, urethane bonds, ester groups, secondary amine bonds, thioether bonds, and triazole groups.

[0286] Method 3: Obtain the target functional group or its protected form through a combination of direct modification and coupling reaction.

[0287] In the present invention, the raw materials used in each preparation method can be purchased or synthesized by oneself.

[0288] The intermediates and final products prepared in the present invention can be purified by purification methods including, but not limited to, extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, thin film dialysis, or supercritical extraction. The structure and molecular weight of the final product can be characterized by methods including, but not limited to, nuclear magnetic resonance, electrophoresis, UV-visible spectrophotometry, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism, and mass spectrometry.

[0289] Lipid composition

[0290] In the present invention, a lipid composition comprises any of the amino acid cationic lipids described above, such as the one represented by general formula (1).

[0291] In a specific embodiment of the present invention, the lipid composition preferably contains, in addition to the cationic lipid having the structure shown in general formula (1), one or more of a phospholipid, a steroid lipid, and a pegylated lipid, selected from any one of the following:

[0292] Case (1): also contains phospholipids;

[0293] Case (2): also contains steroid lipids;

[0294] Case (3): also contains PEGylated lipids;

[0295] Case (4): also contains phospholipids and steroid lipids;

[0296] Case (5): also contains phospholipids and PEGylated lipids;

[0297] Case (6): also contains steroid lipids and PEGylated lipids;

[0298] Case (7): also contains phospholipids, steroid lipids and PEGylated lipids;

[0299] More preferably, the three lipids of neutral lipid, steroid lipid and PEGylated lipid are contained simultaneously.

[0300] In a specific embodiment of the present invention, the phospholipids in the lipid composition are preferably 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diondecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG) , palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine (LPE) and combinations thereof.

[0301] In a specific embodiment of the present invention, the steroid lipid in the lipid composition is preferably any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and a combination thereof.

[0302] In a specific embodiment of the present invention, the PEGylated lipid in the lipid composition is preferably polyethylene glycol-1,2 dimyristin (PEG-DMG), polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE), PEG-cholesterol, polyethylene glycol-diacylglycerol (PEG-DAG), polyethylene glycol-dialkoxypropyl (PEG-DAA), specifically including any one of polyethylene glycol 500-dipalmitoylphosphatidylcholine, polyethylene glycol 2000-dipalmitoylphosphatidylcholine, polyethylene glycol 500-stearoylphosphatidylethanolamine, polyethylene glycol 2000-distearoylphosphatidylethanolamine, polyethylene glycol 500-1,2-oleoylphosphatidylethanolamine, polyethylene glycol 2000-1,2-oleoylphosphatidylethanolamine and polyethylene glycol 2000-2,3-dimyristoylglycerol (PEG-DMG) and a combination thereof.

[0303] In a specific embodiment of the present invention, the PEGylated lipid in the lipid composition is preferably any one of the following structures and a combination thereof:

[0304]

[0305]

[0306] Here, n1 is an integer of 25 to 300. More preferably, n1 is any one of 44, 45, 46, 47, and 48.

[0307] In a specific embodiment of the present invention, it is preferred that any of the aforementioned lipid compositions contain 20-80% of amino acid cationic lipids represented by formula (1), 5-15% of phospholipids, 25-55% of steroid lipids and 0.5-10% of pegylated lipids, wherein the percentages are the molar percentages of each lipid in the total lipids in the solution containing the solvent.

[0308] In a specific embodiment of the present invention, any of the aforementioned lipid compositions is preferred, wherein the molar percentage of cationic lipids in the total lipids in the solution containing the solvent is 30-65%; more preferably, about 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, or 55%.

[0309] In a specific embodiment of the present invention, preferably, in any of the aforementioned lipid compositions, the molar percentage of phospholipids in the total lipids in the solution containing the solvent is about 7.5-13%; more preferably, about 8%, 9%, 10%, 11%, or 12%.

[0310] In a specific embodiment of the present invention, preferably in any of the aforementioned lipid compositions, the molar percentage of steroid lipids in the total lipids in the solution containing the solvent is 35-50%, more preferably about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%.

[0311] In a specific embodiment of the present invention, in any of the aforementioned lipid compositions, the molar percentage of PEGylated lipids in the total lipids in the solution containing the solvent is preferably 0.5-5%; preferably 1-3%; more preferably about 1.5%, 1.6%, 1.7%, 1.8%, or 1.9%.

[0312] 3.2. Preparation of lipid compositions

[0313] In the present invention, the lipid composition can be prepared by the following methods, including but not limited to ethanol injection method, microfluidics method, T-tube mixing method, membrane extrusion method, preferably ethanol injection method and microfluidics method.

[0314] 4. Lipid pharmaceutical compositions and their preparations

[0315] 4.1. Lipid Pharmaceutical Compositions

[0316] One embodiment of the present invention provides a lipid pharmaceutical composition comprising any of the lipid compositions described above and a drug, wherein the lipid composition comprises any of the amino acid cationic lipids described above having a structure as represented by general formula (1), and the drug is selected from any one of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs.

[0317] In a specific embodiment of the present invention, in the lipid pharmaceutical composition, the nucleic acid drug is selected from any one of RNA, DNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir and ribozyme, and the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA and siRNA; preferably, the nucleic acid drug is any one of DNA, mRNA, miRNA and siRNA.

[0318] In one embodiment of the present invention, the lipid pharmaceutical composition is preferably used as a drug selected from any one of the following drugs: antitumor agents, antiviral agents, antifungal agents and vaccines.

[0319] In a specific embodiment of the present invention, the drugs preferably include but are not limited to doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracycline, nitrogen mustard, thiotepa, chlorambucil, razithromycin, melphalan, carmustine, lomustine, busulfan, dibromomannitol, mitomycin C, cis-dichlorodiamine platinum (II), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine, dibucaine, chlorpromazine, propranolol, dimerol, labetalol, clonidine, hydralazine, imipramine, amitriptyline, doxepin , phenytoin, diphenhydramine, chlorpheniramine, promethazine, gentamicin, ciprofloxacin, cefoxitin, miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, netifine, amphotericin B, antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma drugs, vitamins, sedatives and imaging agents, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, colchicine, daunorubicin, dihydroxyanthraquinone, mithramycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, puromycin, maytansine.

[0320] In a specific embodiment of the present invention, the N / P ratio of the lipid composition to the nucleic acid is preferably (0.1-100):1, more preferably (0.2-30):1, and most preferably (0.5-20):1;

[0321] 4.2. Lipid Pharmaceutical Composition Formulations

[0322] In a specific embodiment of the present invention, the drug in the lipid pharmaceutical composition is a nucleic acid drug, and the lipid pharmaceutical composition preparation working solution is deionized water, ultrapure water, phosphate buffer or normal saline, more preferably phosphate buffer or normal saline, most preferably normal saline; preferably lipid composition: working solution = (0.05-20) g: 100 mL, more preferably (0.1-10) g: 100 mL, most preferably (0.2-5) g: 100 mL.

[0323] In a specific embodiment of the present invention, a lipid pharmaceutical composition preparation contains the aforementioned lipid pharmaceutical composition and a pharmaceutically acceptable diluent or excipient, wherein the diluent or excipient is preferably any one of deionized water, ultrapure water, phosphate buffer and normal saline, more preferably phosphate buffer or normal saline, and most preferably normal saline.

[0324] In the present invention, the preparation of the lipid pharmaceutical composition preparation comprises the following steps:

[0325] (1) equilibrating the lipid composition in the diluent or excipient;

[0326] (2) adding the nucleic acid drug to the mixture of the equilibrated lipid composition and the diluent or excipient for compounding;

[0327] Among them, preferably, the equilibration time is 0.1 to 12 hours, preferably 0.2 to 6 hours, more preferably 0.5 to 3 hours; preferably, the recombination time is 0.1 to 12 hours, preferably 0.2 to 5 hours, more preferably 0.5 to 2 hours.

[0328] 5. Liposomes or lipid nanoparticles and their preparation

[0329] 5.1. Liposomes or lipid nanoparticles

[0330] In a specific embodiment of the present invention, a liposome or lipid nanoparticle contains any of the lipid pharmaceutical compositions described above.

[0331] In a specific embodiment of the present invention, the aforementioned lipid nanoparticles are preferably LNP-pharmaceutical compositions, LPP-pharmaceutical compositions or PNP-pharmaceutical compositions; preferably LNP-pharmaceutical compositions; more preferably LNP-nucleic acid pharmaceutical compositions; more preferably LNP-mRNA pharmaceutical compositions.

[0332] 5.2. Preparation of liposomes or lipid nanoparticles

[0333] In a specific embodiment of the present invention, liposomes can be prepared by the following methods, including but not limited to thin film dispersion method, ultrasonic dispersion method, reverse phase evaporation method, freeze drying method, freeze-thawing method, emulsion method and injection method, preferably thin film dispersion method, ultrasonic dispersion method and / or reverse phase evaporation method.

[0334] In a specific embodiment of the present invention, lipid nanoparticles can be prepared by the following methods, including but not limited to microemulsion, double emulsion, high shear homogenization ultrasound, thin film hydration extrusion, and microfluidics.

[0335] In a specific embodiment of the present invention, the liposomes are prepared by a thin film dispersion method, which comprises the following steps:

[0336] (1) Weighing cationic lipids, steroid lipids, neutral lipids, and PEGylated lipids, dissolving them fully in an organic solvent, shaking well, and removing the organic solvent by rotary evaporation under reduced pressure to form an oil film, which was then pumped dry with a vacuum pump to remove the organic solvent;

[0337] (2) adding phosphate buffer containing a cryoprotectant and sonicating in a water bath to form a translucent emulsion;

[0338] (3) adding the emulsion to a high-pressure homogenizer for over-compression, and then adding the over-compressed emulsion to a liposome extruder for membrane extrusion to form liposomes;

[0339] (4) optionally, drying the liposomes in a freeze dryer to form liposome powder;

[0340] Wherein, preferably, the organic solvent is dichloromethane, chloroform and / or methanol, more preferably chloroform and methanol; preferably, the rotation speed of the vacuum rotary evaporation is 30-300 rpm, more preferably 50-200 rpm, most preferably 100-170 rpm; preferably, the temperature of the vacuum rotary evaporation is 10-200° C., more preferably 20-100° C., most preferably 40-80° C.;

[0341] Preferably, the vacuum pump drying time is 1 to 72 hours, more preferably 5 to 48 hours, and most preferably 15 to 36 hours;

[0342] Preferably, the mass concentration of the cryoprotectant dissolved in phosphate buffer is 0.1-80%, preferably 1-50%, more preferably 5-20%;

[0343] Preferably, the frequency of the water bath ultrasound is 10 to 300 kHz, more preferably 30 to 200 kHz, and most preferably 60 to 150 kHz;

[0344] Preferably, the water bath ultrasound time is 0.1 to 5 h, more preferably 0.2 to 2 h, and most preferably 0.25 to 1 h;

[0345] Preferably, the pressure of the high-pressure homogenizer is 50 to 240 MPa, more preferably 80 to 200 MPa, and most preferably 100 to 150 MPa;

[0346] Preferably, the number of overpressure cycles of the high-pressure homogenizer is any integer between 1 and 50, more preferably any integer between 3 and 20, and most preferably any integer between 5 and 10;

[0347] Preferably, the pressure of the liposome extruder is 50 to 300 MPa, more preferably 80 to 250 MPa, and most preferably 120 to 200 MPa;

[0348] Preferably, the number of membrane passes of the liposome extruder is any integer between 1 and 50, more preferably any integer between 3 and 30, and most preferably any integer between 5 and 20;

[0349] Preferably, the drying time of the freeze dryer is 1 to 120 hours, more preferably 5 to 72 hours, and most preferably 10 to 36 hours.

[0350] In a specific embodiment of the present invention, in the method for preparing liposomes, the ratio of liposomes to phosphate buffer dissolved with a cryoprotectant can be 1 mg: (0.1-100) mL, preferably 1 mg: (0.3-50) mL, and more preferably 1 mg: (0.5-5) mL.

[0351] In a specific embodiment of the present invention, the lipid nanoparticles are preferably prepared using a microfluidic method, the steps of which are as follows:

[0352] (1) dissolving each lipid component in an organic solvent to obtain a lipid composition dissolved in an organic phase; the organic phase is preferably ethanol;

[0353] (2) adding the nucleic acid drug to a buffer solution to obtain an aqueous phase solution; the aqueous phase is preferably a citrate buffer or a sodium acetate buffer solution;

[0354] (3) The organic phase solution and the aqueous phase solution are mixed by a microfluidic device to form a lipid nanoparticle composition, and then purified by ultrafiltration to remove the organic solvent and free nucleic acid molecules.

[0355] The following is a further description of the preparation methods of amino acid cationic lipids, lipid compositions, lipid pharmaceutical composition preparations, and the biological activity testing of lipid pharmaceutical compositions in conjunction with some specific examples. The specific examples are provided to further illustrate the present invention and do not limit the scope of protection of the present invention. Among them, in the examples of preparing cationic lipids, the structure of the final product was characterized by nuclear magnetic resonance and the molecular weight was confirmed by mass spectrometry.

[0356] Example 1.1: Cationic lipid (E1-1)

[0357]

[0358] The preparation process is as follows:

[0359] Step a: Under a nitrogen atmosphere, dicyclohexylcarbodiimide (DCC, 3.63 g, 17.6 mmol) was added to a round-bottom flask containing 2-hexyldecanoic acid (S1-1, 2.05 g, 8.0 mmol), 6-bromohexanol (S1-2, 1.74 g, 9.6 mmol) and 4-(dimethylamino)pyridine (DMAP, 0.24 g, 2.0 mmol) in dichloromethane (50 mL) under nitrogen atmosphere. The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated, and the resulting residue was purified by column chromatography to give 6-bromohexyl-2-hexyldecanoate (S1-3, 2.77 g).

[0360] Step b: S1-3 (2.30 g, 5.5 mmol) was dissolved in 30 mL of DMF, and N,N-diethyllysine (S1-4, 0.51 g, 2.5 mmol, S1-4 is a lysine protected by amino Boc) was added. With ethyl bromide The mixture was stirred overnight at room temperature. After the reaction, the reaction mixture was concentrated under reduced pressure and poured into 30 mL of ethyl acetate. After washing with 10% citric acid (20 mL) and brine (20 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford S1-5 (1.67 g).

[0361] Step c: Under nitrogen, DCC (0.68 g, 3.3 mmol) was added to a round-bottom flask containing compound S1-5 (1.32 g, 1.5 mmol), 2-hexylnonanol (S1-6, 0.41 g, 1.8 mmol), and DMAP (45.00 mg, 0.4 mmol) dissolved in dichloromethane (20 mL) in a nitrogen atmosphere. The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated, and the resulting residue was purified by column chromatography to afford cationic lipid E1-1 (1.33 g). 1 H NMR(400MHz, CDCl3)δ:4.93-4.81(m,1H),4.09(t,4H),3.46(t,1H),3.18-2.99(m,6H) ,2.49(t,4H),2.33-2.25(m,2H),2.01-1.82(m,4H),1.68-1.26(m,96H),0.88(t,18H). MS(ESI):m / z=1090.03([M+H] + ).

[0362]

[0363] Example 1.2: Cationic lipid (E1-2)

[0364]

[0365] The raw material S1-6 in Example 1.1 was replaced with 2-octyl nonanol Cationic lipid E1-2 was prepared following the same reaction steps. The structure of E1-2 was also verified by NMR and mass spectrometry.

[0366] Example 2: Cationic lipid (E2-2)

[0367]

[0368] The preparation process is as follows:

[0369] Step a: S1-3 (2.77 g, 6.6 mmol) was dissolved in 50 mL of DMF, and TBS-N-hydroxyethyl-N-methyllysine (S2-1, 0.96 g, 3.0 mmol, S2-1 is N-methyllysine protected by amino Boc) was added. 2-bromoethanol with TBS-protected hydroxyl group The mixture was reacted with 4% ethyl acetate (1.09 g, 7.9 mmol) and KCO (1.09 g, 7.9 mmol) and stirred at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of ethyl acetate. After washing with 10% citric acid (20 mL) and brine (20 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford S2-2 (2.36 g).

[0370] Step b: To a round-bottom flask containing compound S2-2 (1.99 g, 2.0 mmol), undecanol (S2-3, 0.41 g, 2.4 mmol), and DMAP (61.00 mg, 0.5 mmol) in dichloromethane (30 mL) was added DCC (0.91 g, 4.4 mmol) under nitrogen atmosphere. The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound E2-1 (1.91 g).

[0371] Step c: Compound E2-1 (1.72 g, 1.5 mmol) was dissolved in THF (20 mL) in a nitrogen-protected flask. Tetrabutylammonium fluoride solution (TBAF, 20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by column chromatography to yield cationic lipid E2-2 (1.36 g, 87.3%). 1 H NMR(400MHz, CDCl3)δ:4.08(t,6H),3.54(t,2H),3.46(t,1H),3.18-2.99(m,2H),2.56(s,3H),2. 52(t,2H),2.49(t,4H),2.32-2.25(m,2H),2.01-1.82(m,4H),1.70-1.23(m,84H),0.88(t,15H). MS(ESI):m / z=1035.96([M+H] + ).

[0372]

[0373] Example 3: Cationic lipid (E3-2)

[0374]

[0375] The preparation process is as follows:

[0376] Step a: S1-3 (2.77 g, 6.6 mmol) was dissolved in 50 mL of DMF, and TBS-N-hydroxybutyl-N-methyllysine (S3-1, 1.04 g, 3.0 mmol, S3-1 is N-methyllysine protected by amino Boc) was added. 4-bromobutanol with TBS-protected hydroxyl group The mixture was reacted with 4% ethyl acetate (1.09 g, 7.9 mmol) and KCO (1.09 g, 7.9 mmol) and stirred at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of ethyl acetate. After washing with 10% citric acid (20 mL) and brine (20 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford S3-2 (2.37 g).

[0377] Step b: Under nitrogen, DCC (0.91 g, 4.4 mmol) was added to a round-bottom flask containing compound S3-2 (2.05 g, 2.0 mmol), 1-hexylnonyl-7-hydroxyheptanoate (S3-3, 0.86 g, 2.4 mmol, prepared by reacting 7-hydroxyheptanoic acid with pentadecane-7-ol), and DMAP (61.00 mg, 0.5 mmol) in dichloromethane (30 mL). The mixture was allowed to react at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound E3-1 (2.26 g).

[0378] Step c: Compound E3-1 (2.04 g, 1.5 mmol) was dissolved in THF (20 mL) in a nitrogen-protected flask. TBAF solution (20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by column chromatography to yield cationic lipid E3-2 (1.66 g, 88.9%). 1H NMR(400MHz, CDCl3)δ:4.93-4.81(m,1H),4.08(t,6H),3.68-3.60(t,2H),3.46(t,1H),3.18-2.99(m,2H),2.5 6(s,3H),2.52(t,2H),2.49(t,4H),2.33-2.25(m,4H),2.01-1.82(m,4H),1.69-1.22(m,102H),0.88(t,18H). MS(ESI):m / z=1048.13([M+H] + ).

[0379]

[0380] Example 4: Cationic lipid (E4-2)

[0381]

[0382] The preparation process is as follows:

[0383] Step a: Under a nitrogen atmosphere, glycerol containing a TBS-protected hydroxyl group (S4-1, 2.06 g, 10.0 mmol), K2CO3 (4.14 g, 30.0 mmol), and tetradecane bromide (S4-2, 3.05 g, 11.0 mmol) were dissolved in 60 mL of DMF. The mixture was stirred at 110°C for 16 hours. After the reaction was confirmed to be complete by thin-layer chromatography, the reaction solution was poured into water (60 mL) for precipitation, filtered, and further separated and purified by column chromatography to obtain compound S4-3 (5.35 g, 89.3%).

[0384] Step b: Dissolve the above product S4-3 (4.79 g, 8.0 mmol) in THF (50 mL) in a nitrogen-protected flask. Add TBAF (50 mL, 1 M) and react overnight to remove the TBS protection. After completion of the reaction, concentrate, extract, and combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate. Purify by column chromatography to obtain 1,2-di-O-tetradecyl-5H-glyceride (S4-4, 3.41 g, 87.9%).

[0385] Step c: Dissolve S4-4 (3.03 g, 6.3 mmol) and S4-5 (0.80 g, 2.5 mmol) in anhydrous dichloromethane. Stir thoroughly and combine the two solutions. Add DMAP (30.50 mg, 0.3 mmol), EDCI (1.20 g, 6.3 mmol), and DIPEA (1.13 g, 12.5 mmol) sequentially to the mixture. Stir at room temperature for 9 h. After completion of the reaction, the reaction solution was diluted with dichloromethane (50 mL), washed twice with saturated sodium carbonate solution (20 mL x 2), twice with 20 mL of aqueous solution, and once with saturated brine. The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated to obtain the crude product. The crude product was separated and purified by column chromatography. The target eluate was collected and concentrated to obtain the product E4-1 (1.66 g).

[0386] Step d: Compound E4-1 (1.25 g, 1.0 mmol) was dissolved in THF (20 mL) in a nitrogen-protected flask. TBAF solution (20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by column chromatography to yield cationic lipid E4-2 (1.00 g, 88.1%). 1 H NMR(400MHz, CDCl3)δ:4.51(t,1H),4.13-4.01(m,4H),3.59-3.33(m,16H),2.56(s,3H),2.52 (m,2H),2.34(t,2H),2.13-1.92(m,2H),1.55-1.40(m,8H),1.25-1.17(m,88H),0.88(t,12H). MS(ESI):m / z=1139.04([M+H] + ).

[0387]

[0388] Example 5: Cationic lipid (E5-2)

[0389]

[0390] The preparation process is as follows:

[0391] Step a: Under nitrogen, to a round-bottom flask containing 1,16-heptadecanediene-9-carboxylic acid (S5-1, 2.56 g, 9.0 mmol), S1-2 (1.95 g, 10.8 mmol), and DMAP (0.27 g, 2.3 mmol) in dichloromethane (50 mL) was added DCC (4.08 g, 19.8 mmol). The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound S5-2 (3.35 g).

[0392] Step b: S5-2 (2.96 g, 6.6 mmol) was dissolved in 50 mL of DMF, and S5-3 (1.39 g, 3.0 mmol, S5-3 is an amino Boc-protected N-methyl lysine) was added. 3-Bromo-1,2-propanediol with two hydroxyl groups protected by TBS The mixture was reacted with 4% ethyl acetate (1.09 g, 7.9 mmol) and KCO (1.09 g, 7.9 mmol) and stirred at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of ethyl acetate. After washing with 10% citric acid (20 mL) and brine (20 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford S5-4 (2.48 g).

[0393] Step c: Under nitrogen, to a round-bottom flask containing compound S5-4 (2.15 g, 1.8 mmol), S2-3 (0.82 g, 2.2 mmol), and DMAP (54.90 mg, 0.45 mmol) in dichloromethane (30 mL) was added DCC (0.82 g, 4.0 mmol). The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford the target compound E5-1 (2.00 g).

[0394] Step d: Compound E5-1 (1.76 g, 1.3 mmol) was dissolved in THF (20 mL) in a nitrogen-protected flask. TBAF solution (20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by column chromatography to yield cationic lipid E5-2 (1.28 g, 87.5%). 1H NMR(400MHz, CDCl3)δ:4.09(t,6H),3.67-3.54(m,2H),3.46-3.38(m,2H),3.18-2.99(m,2H),2.56(s,3H) ,2.49(t,4H),2.40-2.37(m,2H),2.33-2.25(m,2H),2.01-1.82(m,4H),1.71-1.22(m,92H),0.88(t,15H). MS(ESI):m / z=1122.03([M+H] + ).

[0395]

[0396] Example 6: Cationic lipid (E6-1)

[0397]

[0398] The preparation process is as follows:

[0399] Step a: Dissolve the above compound S1-3 (1.84 g, 4.4 mmol) in 30 mL of DMF, add N,N-dimethyllysine (S6-1, 0.35 g, 3.0 mmol) and K2CO3 (0.73 g, 5.3 mmol), and stir at room temperature overnight. After the reaction is complete, the reaction mixture is concentrated under reduced pressure and poured into 30 mL of ethyl acetate. After washing with 10% citric acid (20 mL) and brine (20 mL), the organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product is purified by column chromatography to afford compound S6-2 (1.38 g).

[0400] Step b: Under nitrogen, DCC (0.45 g, 2.2 mmol) was added to a round-bottom flask containing compound S6-2 (0.85 g, 1.0 mmol), 1-pentadecanol (S6-3, 0.27 g, 1.2 mmol), and DMAP (30.50 mg, 0.3 mmol) in dichloromethane (10 mL) in a nitrogen atmosphere. The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford cationic lipid E6-1 (0.93 g). 1H NMR(400MHz, CDCl3)δ:4.07(t,6H),3.27(t,1H),2.64-2.53(m,2H),2.45-2.37(m,2H),2.33-2.31(m,2H),2. 29-2.26(m,6H),2.25-2.19(m,2H),2.07-1.95(m,2H),1.66-1.51(m,14H),1.29-1.23(m,88H),0.88(t,15H). MS(ESI):m / z=1118.07([M+H] + ).

[0401]

[0402] Example 7: Cationic lipid (E7-1)

[0403]

[0404] The preparation process is as follows:

[0405] Step a: Under nitrogen, DCC (3.63 g, 17.6 mmol) was added to a round-bottom flask containing TBS-7-hydroxyheptanoic acid (S7-1, 2.08 g, 8.0 mmol), 1-nonanol (S7-2, 1.38 g, 9.6 mmol), and DMAP (0.24 g, 2.0 mmol) dissolved in dichloromethane (50 mL) in a nitrogen atmosphere. The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated, and the resulting residue was purified by column chromatography to afford TBS-7-hydroxyheptanoic acid nonyl ester (S7-3, 2.56 g).

[0406] Step b: Compound S7-3 (1.94 g, 5.0 mmol) was dissolved in THF (20 mL). Under nitrogen, TBAF solution (20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography afforded 7-hydroxynonyl heptanoate (S7-4, 1.20 g, 88.4%).

[0407] Step c: Under nitrogen, DCC (0.91 g, 4.4 mmol) was added to a round-bottom flask containing compound S6-2 (1.70 g, 2.0 mmol), S7-4 (0.65 g, 2.4 mmol), and DMAP (61.00 mg, 0.5 mmol) in dichloromethane (20 mL). The mixture was reacted at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford cationic lipid E7-1 (1.93 g). 1H NMR(400MHz, CDCl3)δ:4.09(t,8H),3.46(t,1H),3.18-2.99(m,2H),2.49(t,4H),2. 32-2.25(m,4H),2.23(s,6H),2.01-1.82(m,4H),1.71-1.22(m,96H),0.88(t,15H). MS(ESI):m / z=1162.06([M+H] + ).

[0408]

[0409] Example 8: Cationic lipid (E8-2)

[0410]

[0411] The preparation process is as follows:

[0412] Step a: Under nitrogen, DCC (5.44 g, 26.4 mmol) was added to a round-bottom flask containing compound S1-1 (3.07 g, 12.0 mmol), 5-hexen-1-ol (S8-1, 1.44 g, 14.4 mmol), and DMAP (0.37 g, 3.0 mmol) in dichloromethane (50 mL). The mixture was reacted at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford the target compound, 5-hexene-2-hexyldecanoate (S8-2, 3.61 g).

[0413] Step b: To a solution of S8-2 (3.18 g, 9.0 mmol) in dichloromethane (50 mL) was added m-chloroperbenzoic acid (3.11 g, 18.0 mmol) and the reaction was stirred at room temperature for 10 h. After completion of the reaction, the reaction mixture was poured into a solution of NaHCO3 (20 mL) and extracted with dichloromethane (20 mL*3). The combined organic layers were washed with Na2S2O3 (aqueous solution), brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by column chromatography to give 5,6-epoxyhexane-2-hexyldecanoate (S8-3, 2.63 g).

[0414] Step c: Compound S2-1 (3.34 g, 10.5 mmol) was added to a solution of S8-3 (2.48 g, 7.0 mmol) in anhydrous ethanol (30 mL). The mixture was stirred at room temperature for 10 h. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to afford compound S8-4 (2.17 g).

[0415] Step d: Under nitrogen, compound S8-4 (1.88 g, 2.8 mmol) was dissolved in 30 mL of anhydrous THF and cooled to 0°C. Tert-butyldimethylsilyl chloride (TBSCl, 0.85 g, 5.6 mmol) was slowly added, followed by imidazole (0.76 g, 11.2 mmol). The reaction was stirred at room temperature for 18 h. After completion of the reaction, the mixture was quenched with ice water (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic layers were combined and washed with saturated NaHCO₃ solution to remove acidic impurities. The mixture was dried over anhydrous sodium sulfate and concentrated by evaporation under reduced pressure to give the crude product, which was purified by column chromatography to afford compound S8-5 (1.87 g).

[0416] Step e: Dissolve S1-3 (1.00 g, 2.4 mmol) in 30 mL of DMF, add S8-5 (1.57 g, 2.0 mmol) and K2CO3 (0.73 g, 5.3 mmol), and stir at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 30 mL of ethyl acetate. After washing with 10% citric acid (20 mL) and brine (20 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford S8-6 (1.82 g).

[0417] Step f: Under nitrogen, to a round-bottom flask containing compound S8-6 (1.46 g, 1.3 mmol), S2-3 (0.27 g, 1.6 mmol), and DMAP (39.65 mg, 0.33 mmol) in dichloromethane (20 mL) was added DCC (0.59 g, 2.9 mmol). The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford the target compound E8-1 (1.42 g).

[0418] Step g: Compound E8-1 (1.28 g, 1.0 mmol) was dissolved in THF (20 mL) in a nitrogen-protected flask. TBAF solution (20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by column chromatography to yield cationic lipid E8-2 (0.92 g, 87.5%). 1H NMR(400MHz, CDCl3)δ:4.07(t,6H),3.55-3.53(m,3H),3.46(t,1H),3.18-2.99(m,2H),2.56(s,3H) ,2.52(t,2H),2.49(t,2H),2.33-2.25(m,4H),2.01-1.82(m,4H),1.71-1.22(m,82H),0.88(t,15H). MS(ESI):m / z=1051.95([M+H] + ).

[0419]

[0420] Example 9: Cationic lipid (E9-3)

[0421]

[0422] The preparation process is as follows:

[0423] Step a: Under nitrogen, DCC (4.53 g, 22.0 mmol) was added to a round-bottom flask containing 2-octyldecanoic acid (S9-2, 2.84 g, 10.0 mmol), 1,4-butanediol (S9-1, 4.53 g, 12.0 mmol), and DMAP (0.31 g, 2.5 mmol) in dichloromethane (60 mL). The mixture was allowed to react at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to obtain the small molecule intermediate alcohol derivative (S9-3, 2.98 g).

[0424] Step b: N-hydroxyethylglutamic acid (S9-4, 1.22 g, 3.0 mmol, S9-4 is glutamic acid protected by amino Boc 2-bromoethanol with TBS-protected hydroxyl group The mixture was stirred for 48 hours. After the reaction was completed, the reaction solution was diluted with dichloromethane (50 mL), washed twice with saturated sodium carbonate solution (30 mL * 2), twice with 30 mL of aqueous solution, and once with saturated brine. The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated to obtain a crude product. The crude product was separated and purified by column chromatography. The target eluate was collected and concentrated to obtain the product E9-1 (1.72 g).

[0425] Step c: Removal of the Boc protecting group. In a clean, dry round-bottom flask, prepare a trifluoroacetic acid / dichloromethane (1:2, v / v) solution. Slowly add a dichloromethane solution of E9-1 (1.30 g, 1.2 mmol) dropwise under an ice bath. Allow to react at room temperature for 2 hours. After completion of the reaction, concentrate the reaction solution, dilute with purified water, and extract with dichloromethane. The organic phase is dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated and recrystallized to yield compound E9-2 (1.07 g, 90.5%).

[0426] Step d: Compound E9-2 (0.79 g, 0.8 mmol) was dissolved in THF (20 mL) in a nitrogen-protected flask. TBAF solution (20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by column chromatography to yield cationic lipid E9-3 (0.61 g, 88.4%). 1 H NMR(400MHz, CDCl3)δ:4.51(t,1H),4.09(t,8H),3.54(t,2H),2.74(t,2H) ,2.34-2.26(m,4H),2.13-1.92(m,2H),1.71-1.22(m,64H),0.88(t,12H). MS(ESI):m / z=868.72([M+H] + ).

[0427]

[0428] Example 10: Cationic lipid (E10-3)

[0429]

[0430] The preparation process is as follows:

[0431] Step a: Under nitrogen, DCC (5.44 g, 26.4 mmol) was added to a round-bottom flask containing compound S1-1 (3.07 g, 12.0 mmol), S9-1 (1.30 g, 14.4 mmol), and DMAP (0.37 g, 3.0 mmol) in dichloromethane (50 mL). The mixture was allowed to react at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to yield the small molecule intermediate alcohol derivative (S10-1, 3.30 g).

[0432] Step b: S10-1 (2.96 g, 9.0 mmol), S10-2 (1.62 g, 3.6 mmol, S10-2 is glutamic acid protected by amino Boc 2-(2-bromoethoxy)ethanol with TBS-protected hydroxyl group The mixture was stirred for 48 hours, and the reaction mixture was diluted with dichloromethane (50 mL), washed twice with saturated sodium carbonate solution (30 mL * 2), twice with 30 mL of aqueous solution, and once with saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was concentrated to obtain a crude product. The crude product was separated and purified by column chromatography, and the target eluate was collected and concentrated to obtain the product E10-1 (1.98 g).

[0433] Step c: Removal of the Boc protecting group. In a clean, dry round-bottom flask, prepare a trifluoroacetic acid / dichloromethane (1:2, v / v) solution. Slowly add a dichloromethane solution of E10-1 (1.61 g, 1.5 mmol) dropwise under an ice bath. Allow to react at room temperature for 2 hours. After completion of the reaction, concentrate the reaction solution, dilute with purified water, and extract with dichloromethane. The organic phase is dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated and recrystallized to yield compound E10-2 (1.31 g, 90.1%).

[0434] Step d: Compound E10-2 (0.97 g, 1.0 mmol) was dissolved in THF (20 mL) in a nitrogen-protected flask. TBAF solution (20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by column chromatography to yield cationic lipid E10-3 (0.76 g, 89.0%).1 H NMR (400MHz,CDCl3)δ: 1 H NMR(400MHz, CDCl3)δ:4.51(t,1H),4.07(t,8H),3.70(t,2H),3.63(t,4H),2.74 (t,2H),2.33-2.25(m,4H),2.13-1.92(m,2H),1.68-1.22(m,56H),0.88(t,12H). MS(ESI):m / z=856.68([M+H] + ).

[0435]

[0436] Example 11: Cationic lipid (E11-2)

[0437]

[0438] The preparation process is as follows:

[0439] Step a: TBS-6-hydroxyhexanoic acid (S11-1, 3.69 g, 15.0 mmol), EDCI (3.46 g, 18.0 mmol), and N-hydroxysuccinimide (NHS, 1.88 g, 16.5 mmol) were dissolved in dichloromethane (50 mL) and stirred at room temperature overnight. After completion of the reaction, the reaction solution was backwashed twice with 0.1 mol / L HCl (20 mL x 2) and once with saturated sodium chloride (20 mL). The dichloromethane phase was collected, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain compound S11-2 (5.07 g, 98.6%).

[0440] Step b: The above compound S11-2 (4.12 g, 12.0 mmol) was dissolved in dichloromethane (50 mL), and 10-nonadecanamine (S11-3, 3.75 g, 13.2 mmol) and triethylamine (TEA, 2.5 mL, 18.0 mmol) were added. The mixture was reacted at room temperature overnight to precipitate a large amount of white solid. The solid was filtered, slurried with methanol (20 mL), filtered, and rinsed twice with methanol. The solid was collected and dried to obtain TBS-6-hydroxy-N-(10-nonadecanylamino)hexanamide (S11-4, 5.15 g, 83.8%).

[0441] Step c: Compound S11-4 (4.61 g, 9.0 mmol) was dissolved in THF (50 mL) in a nitrogen-protected flask. TBAF solution (50 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography afforded compound S11-5 (3.15 g, 87.9%).

[0442] Step d: S11-5 (2.48 g, 6.3 mmol), TBS-N-hydroxybutyl-N-methylglutamic acid (S11-6, 0.87 g, 2.5 mmol, S11-6 is glutamic acid protected by amino Boc 4-bromobutanol with TBS-protected hydroxyl group The mixture was stirred for 48 hours, and the reaction mixture was diluted with dichloromethane (30 mL), washed twice with saturated sodium carbonate solution (20 mL * 2), twice with 20 mL of aqueous solution, and once with saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, and the solvent was concentrated to obtain a crude product. The crude product was separated and purified by column chromatography, and the target eluate was collected and concentrated to obtain the product E11-1 (1.43 g).

[0443] Step e: Compound E11-1 (1.11 g, 1.0 mmol) was dissolved in THF (20 mL) in a nitrogen-protected flask. TBAF solution (20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by column chromatography to yield cationic lipid E11-2 (0.88 g, 88.3%). 1 H NMR(400MHz, CDCl3)δ:4.51(t,1H),4.07(t,4H),3.96-3.82(m,2H),3.68-3.60(t,2H),2.56(s,3 H),2.52(t,2H),2.34(t,2H),2.17(t,4H),2.13-1.92(m,2H),1.69-1.21(m,80H),0.88(t,12H). MS(ESI):m / z=992.89([M+H] + ).

[0444]

[0445] Example 12: Cationic lipid (E12-2)

[0446]

[0447] The preparation process is as follows:

[0448] Step a: Under nitrogen, to a round-bottom flask containing 9-heptadecanol (S12-1, 2.46 g, 9.6 mmol), S11-1 (1.97 g, 8.0 mmol), and DMAP (0.24 g, 2.0 mmol) dissolved in dichloromethane (50 mL) was added DCC (3.63 g, 17.6 mmol) and reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated, and the resulting residue was purified by column chromatography to afford 1-octylnonyl 6-hydroxyhexanoate (S12-2, 3.22 g).

[0449] Step b: Dissolve compound S12-2 (2.91 g, 6.0 mmol) in THF (30 mL) in a nitrogen-protected flask. Add TBAF solution (30 mL, 1 M) and allow to react overnight to remove the TBS protection. After completion of the reaction, concentrate, extract, and combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate. Purify by column chromatography to afford compound S12-3 (1.96 g, 88.2%).

[0450] Step c: Dissolve S12-3 (1.86 g, 5.0 mmol) and S11-6 (0.70 g, 2.0 mmol) in anhydrous dichloromethane. Stir thoroughly and combine the two solutions. Add DMAP (24.40 mg, 0.2 mmol), EDCI (0.96 g, 5.0 mmol), and DIPEA (0.90 g, 10.0 mmol) sequentially to the mixture. Stir and react at room temperature for 48 h. After completion of the reaction, the reaction solution was diluted with dichloromethane (30 mL) and washed twice with saturated sodium carbonate solution (20 mL x 2), twice with 20 mL of aqueous solution, and once with saturated brine. The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated to obtain the crude product. The crude product was separated and purified by column chromatography. The target eluate was collected and concentrated to obtain compound E12-1 (1.18 g).

[0451] Step d: Compound E12-1 (0.84 g, 0.8 mmol) was dissolved in THF (10 mL) in a nitrogen-protected flask. TBAF solution (10 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by column chromatography to yield cationic lipid E12-2 (0.66 g, 88.1%).1 H NMR(400MHz, CDCl3)δ:4.93-4.81(m,2H),4.51(t,1H),4.07(t,4H),3.68-3.60(t,2H),2.56(s,3 H),2.52(t,2H),2.34(t,2H),2.32(t,4H),2.13-1.92(m,2H),1.70-1.21(m,72H),0.88(t,12H). MS(ESI):m / z=938.79([M+H] + ).

[0452]

[0453] Example 13: Cationic lipid (E13-1)

[0454]

[0455] The preparation process is as follows:

[0456] Step a: 2-Heptylnonanol (S13-1, 3.63 g, 15.0 mmol) was dissolved in dry THF (50 mL). Under nitrogen, NaH (60%, 0.60 g, 15.0 mmol) was slowly added with stirring in an ice bath. The mixture was allowed to react in an ice bath for 1 hour. TBS-6-bromohexanol (S13-2, 5.31 g, 18.0 mmol, the hydroxyl group of S13-2 was protected with TBS) was then added and allowed to react overnight at room temperature. After completion of the reaction, the reaction mixture was placed in an ice bath and quenched by the slow addition of 5 mL of water. The reaction solution was poured into 20 mL of water and extracted twice with ethyl acetate (50 mL x 2). The organic phases were combined and backwashed once with saturated aqueous sodium chloride (50 mL). The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a crude oil. The crude oil was purified by column chromatography, concentrated, and pumped dry to obtain the small molecule intermediate S13-3 (3.57 g).

[0457] Step b: Compound S13-3 (3.20 g, 7.0 mmol) was dissolved in THF (50 mL) in a nitrogen-protected flask. TBAF solution (50 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography afforded compound S13-4 (2.12 g, 88.3%).

[0458] Step c: S13-4 (1.72 g, 5.0 mmol) and S13-5 (0.35 g, 2.0 mmol) were dissolved in anhydrous dichloromethane, stirred, and the two solutions were combined. DMAP (24.40 mg, 0.2 mmol), EDCI (0.96 g, 5.0 mmol), and DIPEA (0.90 g, 10.0 mmol) were added sequentially to the mixture, and the reaction was stirred at room temperature for 48 h. After the reaction, the reaction solution was diluted with dichloromethane (30 mL), then washed twice with saturated sodium carbonate solution (20 mL x 2), twice with 20 mL of aqueous solution, and once with saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was concentrated to obtain the crude product. The crude product was separated and purified by column chromatography. The target eluate was collected and concentrated to obtain cationic lipid E13-1 (0.87 g). 1 H NMR(400MHz, CDCl3)δ:4.51(t,1H),4.08(t,4H),3.37(t,4H),3.28-3.24(m,4H) ,2.56(s,6H),2.34(t,2H),2.13-1.92(m,2H),1.72-1.21(m,66H),0.88(t,12H). MS(ESI):m / z=824.76([M+H] + ).

[0459]

[0460] Example 14: Cationic lipid (E14-1)

[0461]

[0462] The preparation process is as follows:

[0463] Step a: Glutamic acid (S14-1, 1.48 g, 6.0 mmol, the amino group of S14-1 was Boc-protected) and 3-dimethylamino-1-propanol (S14-2, 1.55 g, 15.0 mmol) were dissolved in anhydrous dichloromethane. After stirring, the two solutions were combined. DMAP (73.20 mg, 0.6 mmol), EDCI (2.88 g, 15.0 mmol), and DIPEA (2.70 g, 30.0 mmol) were added sequentially to the mixture. The mixture was stirred at room temperature for 48 h. After completion of the reaction, the reaction solution was diluted with dichloromethane (50 mL) and washed twice with saturated sodium carbonate solution (20 mL x 2), twice with 20 mL of aqueous solution, and once with saturated brine. The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated to obtain the crude product. The crude product was separated and purified by column chromatography. The target eluate was collected and concentrated to obtain the product S14-3 (1.31 g).

[0464] Step b: Removal of the Boc protecting group. In a clean, dry round-bottom flask, prepare a solution of trifluoroacetic acid / dichloromethane (1:2, v / v). Slowly add a dichloromethane solution of S14-3 (1.04 g, 2.5 mmol) dropwise under an ice bath. Allow to react at room temperature for 2 hours. After completion of the reaction, concentrate the reaction solution, dilute with purified water, and extract with dichloromethane. The organic phase is dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated and recrystallized to afford compound S14-4 (0.73 g, 92.1%).

[0465] Step c: S14-4 (0.57 g, 1.8 mmol) was dissolved in 30 mL of DMF, and S1-3 (1.66 g, 4.0 mmol) and K2CO3 (0.66 g, 4.8 mmol) were added. The mixture was stirred at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 30 mL of ethyl acetate. After washing with 10% citric acid (20 mL) and brine (20 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to obtain cationic lipid E14-1 (1.47 g). 1 H NMR(400MHz, CDCl3)δ:4.52(t,1H),4.09(t,8H),2.87-2.80(m,4H),2.49(t,4H),2. 34-2.31(m,4H),2.23(s,12H),2.13-1.98(m,4H),1.69-1.21(m,66H),0.88(t,12H). MS(ESI):m / z=994.87([M+H] + ).

[0466]

[0467] Example 15: Cationic lipid (E15-1)

[0468]

[0469] The preparation process is as follows:

[0470] Step a: Under nitrogen, DCC (3.63 g, 17.6 mmol) was added to a round-bottom flask containing compound S12-1 (2.46 g, 9.6 mmol), S7-1 (2.08 g, 8.0 mmol), and DMAP (0.24 g, 2.0 mmol) in dichloromethane (50 mL) in a nitrogen atmosphere. The mixture was reacted at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford TBS-1-octylnonyl7-hydroxyheptanoate (S15-1, 3.26 g).

[0471] Step b: Compound S15-1 (2.99 g, 6.0 mmol) was dissolved in THF (50 mL) in a nitrogen-protected flask. TBAF solution (50 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography afforded compound S15-2 (2.05 g, 88.6%).

[0472] Step c: Under nitrogen, DCC (2.72 g, 13.2 mmol) was added to a round-bottom flask containing compound S15-2 (0.97 g, 2.5 mmol), N-methylglutamic acid (S15-3, 2.11 g, 6.0 mmol, the secondary amino and carboxyl groups of S15-3 were protected with Boc and benzyl, respectively), and DMAP (0.18 g, 1.5 mmol) in dichloromethane (50 mL). The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound S15-4 (3.48 g).

[0473] Step d: Compound S15-4 (2.87 g, 4.0 mmol) was dissolved in methanol (50 mL), and nickel chloride hexahydrate (NiCl2·6H2O, 2.86 g, 12.0 mmol) was added. After stirring, sodium borohydride (NaBH4, 1.37 g, 36.0 mmol) was slowly added. The reaction mixture was stirred at room temperature. After TLC showed that the reaction was complete, 20 mL of methanol was slowly added to the reaction solution to quench the reaction. After stirring for 30 minutes, the reaction solution was filtered through celite and washed with methanol (50 mL). 20 mL of water was added to the reaction solution, mixed well, and extracted three times with ethyl acetate (20 mL*3). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. The mixture was purified by column chromatography, concentrated, and pumped to dryness to obtain compound S15-5 (2.24 g, 89.3%).

[0474] Step e: Under nitrogen, DCC (1.36 g, 6.6 mmol) was added to a round-bottom flask containing compound S2-3 (0.62 g, 3.6 mmol), S15-5 (1.88 g, 3.0 mmol), and DMAP (91.50 mg, 0.8 mmol) in dichloromethane (30 mL) in a nitrogen atmosphere. The mixture was reacted at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound S15-6 (1.96 g).

[0475] Step f: Removal of the Boc protecting group. In a clean, dry round-bottom flask, prepare a solution of trifluoroacetic acid / dichloromethane (1:2, v / v). Slowly add a dichloromethane solution of S15-6 (1.51 g, 2.0 mmol) dropwise under an ice bath. Allow to react at room temperature for 2 hours. After completion of the reaction, concentrate the reaction solution, dilute with purified water, and extract with dichloromethane. The organic phase is dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated and recrystallized to afford compound S15-7 (1.23 g, 90.3%).

[0476] Step g: Compound S15-7 (1.02 g, 1.5 mmol) was dissolved in dry THF (20 mL). NaH (60%, 0.06 g, 1.5 mmol) was slowly added under ice-cooling and the mixture was allowed to react for 1 hour. After the reaction, 4-bromo-1-butanol (S15-8, 0.28 g, 1.8 mmol) was added and stirred under ice-cooling for 1 hour. The reaction mixture was then slowly returned to room temperature and allowed to react overnight. After the reaction, the mixture was placed under ice-cooling and quenched by the slow addition of 2 mL of water. After stirring for 30 minutes, water (10 mL) was added and stirred. The mixture was extracted twice with dichloromethane (20 mL x 2). The organic phases were combined and backwashed once with saturated sodium chloride (20 mL). The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude E15-1. The product was purified by column chromatography, concentrated, and pumped dry to obtain cationic lipid E15-1 (0.58 g). 1 H NMR(400MHz, CDCl3)δ:4.93-4.82(m,1H),4.52(t,1H),4.09(t,4H),3.68-3.60(t,2H),2.57 (s,3H),2.52(t,2H),2.34-2.30(m,4H),2.13-1.92(m,2H),1.70-1.21(m,58H),0.88(t,9H). MS(ESI):m / z=754.66([M+H] + ).

[0477]

[0478] Example 16.1: Cationic lipid (E16-1)

[0479]

[0480] The preparation process is as follows:

[0481] Compound S15-7 (3.41 g, 5.0 mmol) was dissolved in dry THF (50 mL). NaH (60%, 0.20 g, 5.0 mmol) was slowly added under ice-cooling and allowed to react for 1 hour. After the reaction, 2-bromoethanol (S16-1, 0.75 g, 6.0 mmol) was added and stirred under ice-cooling for 1 hour. The reaction mixture was then slowly returned to room temperature and allowed to react overnight. After the reaction, the reaction mixture was placed under ice-cooling and quenched by the slow addition of 5 mL of water. After stirring for 30 minutes, water (20 mL) was added and stirred. The mixture was extracted twice with dichloromethane (50 mL x 2). The organic phases were combined and backwashed once with saturated sodium chloride (50 mL). The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to yield crude E16-1. The product was purified by column chromatography, concentrated, and pumped dry to yield cationic lipid E16-1 (1.93 g). 1 H NMR(400MHz, CDCl3)δ:4.91-4.81(m,1H),4.51(t,1H),4.08(t,4H),3.54(t,2H),2.57(s, 3H),2.52(t,2H),2.34-2.32(m,4H),2.13-1.92(m,2H),1.68-1.21(m,54H),0.88(t,9H). MS(ESI):m / z=726.62([M+H] + ).

[0482]

[0483] Example 16.2: Cationic lipid (E16-2)

[0484]

[0485] The preparation process is as follows:

[0486] Step a: Under nitrogen, to a round-bottom flask containing glutamic acid (S16-2, 2.02 g, 6.0 mmol, one carboxyl group and primary amino group of S16-2 protected by benzyl and Boc, respectively), S15-2 (2.78 g, 7.2 mmol), and DMAP (0.18 g, 1.5 mmol) dissolved in dichloromethane (40 mL) was added DCC (2.72 g, 13.2 mmol). The reaction was allowed to proceed at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound S16-3 (3.45 g).

[0487] Step b: Compound S16-3 (2.82 g, 4.0 mmol) was dissolved in methanol (50 mL), and NiCl2·6H2O (2.86 g, 12.0 mmol) was added. After stirring, NaBH4 (1.37 g, 36.0 mmol) was slowly added. The reaction mixture was stirred at room temperature. After TLC showed that the reaction was complete, 20 mL of methanol was slowly added to the reaction solution to quench the reaction. After stirring for 30 minutes, the reaction mixture was filtered through celite and washed with methanol (50 mL). 20 mL of water was added to the reaction solution, mixed well, and extracted three times with ethyl acetate (20 mL*3). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. The mixture was purified by column chromatography, concentrated, and pumped dry to obtain compound S16-4 (2.19 g, 89.3%).

[0488] Step c: Under nitrogen, DCC (1.36 g, 6.6 mmol) was added to a round-bottom flask containing compound S2-3 (0.62 g, 3.6 mmol), S16-4 (1.84 g, 3.0 mmol), and DMAP (91.50 mg, 0.8 mmol) in dichloromethane (30 mL) in a nitrogen atmosphere. The mixture was reacted at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to give compound S16-5 (1.89 g).

[0489] Step d: Removal of the Boc protecting group. In a clean, dry round-bottom flask, prepare a solution of trifluoroacetic acid / dichloromethane (1:2, v / v). Slowly add a dichloromethane solution of S16-5 (1.54 g, 2.0 mmol) dropwise under an ice bath. Allow to react at room temperature for 2 hours. After completion of the reaction, concentrate the reaction solution, dilute with purified water, and extract with dichloromethane. The organic phase is dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated and recrystallized to afford compound S16-6 (1.21 g, 90.2%).

[0490] Step e: S16-6 (1.00 g, 1.5 mmol) was dissolved in 20 mL of DMF, and S16-1 (0.23 g, 1.8 mmol) and KCO (0.29 g, 2.1 mmol) were added. The mixture was stirred at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 20 mL of ethyl acetate. The mixture was washed sequentially with 10% citric acid (10 mL) and brine (10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to yield cationic lipid E16-2 (0.87 g). 1H NMR(400MHz, CDCl3)δ:4.93-4.81(m,1H),4.51(t,1H),4.08(t,4H),3.54(t,2H),2 .74(t,2H),2.34-2.31(m,4H),2.13-1.92(m,2H),1.69-1.21(m,54H),0.88(t,9H). MS(ESI):m / z=712.60([M+H] + ).

[0491]

[0492] Example 17: Cationic lipid (E17-1)

[0493]

[0494] The preparation process is as follows:

[0495] Step a: Under nitrogen, DCC (2.27 g, 11.0 mmol) was added to a round-bottom flask containing compound S15-5 (3.14 g, 5.0 mmol), S7-4 (1.63 g, 6.0 mmol), and DMAP (0.15 g, 1.3 mmol) in dichloromethane (50 mL) in a nitrogen atmosphere. The mixture was reacted at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound S17-1 (3.68 g).

[0496] Step b: Removal of the Boc protecting group. In a clean, dry round-bottom flask, prepare a solution of trifluoroacetic acid / dichloromethane (1:2, v / v). Slowly add a dichloromethane solution of S17-1 (2.65 g, 3.0 mmol) dropwise under an ice bath. Allow to react at room temperature for 2 hours. After completion of the reaction, concentrate the reaction solution, dilute with purified water, and extract with dichloromethane. The organic phase is dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated and recrystallized to yield compound S17-2 (2.12 g, 90.4%).

[0497] Step c: Compound S17-2 (1.56 g, 2.0 mmol) was dissolved in dry THF (30 mL). NaH (60%, 0.08 g, 2.0 mmol) was slowly added under ice-cooling and the mixture was allowed to react for 1 hour. After the reaction, compound S16-1 (0.30 g, 2.4 mmol) was added and stirred under ice-cooling for 1 hour. The reaction mixture was then slowly returned to room temperature and stirred overnight. After the reaction, the mixture was placed under ice-cooling and quenched by the slow addition of 3 mL of water. After 30 minutes, water (15 mL) was added and stirred. The mixture was extracted twice with dichloromethane (30 mL x 2). The organic phases were combined and backwashed once with saturated sodium chloride (30 mL). The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude E17-1. The product was purified by column chromatography, concentrated, and pumped dry to obtain cationic lipid E17-1 (0.87 g). 1 H NMR(400MHz, CDCl3)δ:4.92-4.81(m,1H),4.51(t,1H),4.08(t,6H),3.54(t,2H),2.56(s, 3H),2.52(t,2H),2.34-2.32(m,6H),2.13-1.92(m,2H),1.70-1.21(m,58H),0.88(t,9H). MS(ESI):m / z=826.67([M+H] + ).

[0498]

[0499] Example 18: Cationic lipid (E18-2)

[0500]

[0501] The preparation process is as follows:

[0502] Step a: Under nitrogen atmosphere, add compound S18-1 (2.10 g, 5.0 mmol, S18-1 is prepared by and To a round-bottom flask containing (prepared by deprotection of the carboxyl group) S7-4 (1.63 g, 6.0 mmol) and DMAP (0.15 g, 1.3 mmol) was added DCC (2.27 g, 11.0 mmol), and the mixture was allowed to react at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound S18-2 (2.75 g).

[0503] Step b: Removal of the Boc protecting group. In a clean, dry round-bottom flask, prepare a trifluoroacetic acid / dichloromethane (1:2, v / v) solution. S18-2 (2.02 g, 3.0 mmol) in dichloromethane was slowly added dropwise under an ice bath. The mixture was allowed to react at room temperature for 2 hours. After completion of the reaction, the reaction mixture was concentrated, diluted with purified water, and extracted with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and recrystallized to afford compound S18-3 (1.55 g, 90.2%).

[0504] Step c: Dissolve S18-3 (1.15 g, 2.0 mmol) in 30 mL of DMF, add S1-3 (1.84 g, 4.4 mmol) and KCO (0.73 g, 5.3 mmol), and stir at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 30 mL of ethyl acetate. After washing with 10% citric acid (20 mL) and brine (20 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford E18-1 (2.03 g).

[0505] Step d: Compound E18-1 (1.25 g, 1.0 mmol) was dissolved in THF (20 mL) in a nitrogen-protected flask. TBAF solution (20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by column chromatography to yield cationic lipid E18-2 (1.01 g, 88.6%). 1 H NMR(400MHz, CDCl3)δ:4.51(t,1H),4.09(t,8H),3.70-3.69(t,2H),4.23(t,2H),2.49(m,4H),2. 34-2.32(m,4H),2.30(t,2H),2.13-1.92(m,2H),1.87(m,2H),1.71-1.21(m,86H),0.88(t,15H). MS(ESI):m / z=1136.95([M+H] + ).

[0506]

[0507] Example 19.1: Cationic lipid (E19-1)

[0508]

[0509] The preparation process is as follows:

[0510] N,N-dimethylaspartic acid (S19-1, 0.32 g, 2.0 mmol) and S3-3 (1.79 g, 5.0 mmol) were dissolved in anhydrous dichloromethane, stirred, and the two solutions were combined. DMAP (24.40 mg, 0.2 mmol), EDCI (0.96 g, 5.0 mmol), and DIPEA (0.90 g, 10.0 mmol) were added sequentially to the mixture, and the reaction was stirred at room temperature for 48 h. After completion of the reaction, the reaction solution was diluted with dichloromethane (30 mL), washed twice with saturated sodium carbonate solution (30 mL x 2), twice with 30 mL of aqueous solution, and once with saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was concentrated to obtain the crude product. The crude product was separated and purified by column chromatography, and the target eluate was collected and concentrated to obtain cationic lipid E19-1 (0.88 g). 1 HNMR(400MHz, CDCl3)δ:4.93-4.81(m,2H),4.08(t,4H),3.40(t,1H),2.56 (s,6H),2.52-2.48(m,2H),2.30(t,4H),1.70-1.22(m,64H),0.88(t,12H). MS(ESI):m / z=838.71([M+H] + ).

[0511]

[0512] Example 19.2: Cationic lipid (E19-2)

[0513]

[0514] The raw material S3-3 in Example 19.1 was replaced by raw material The cationic lipid E19-2 was prepared following the same reaction steps. The structure of E19-2 was also verified by NMR and mass spectrometry.

[0515] Example 20: Cationic lipid (E20-1)

[0516]

[0517] The preparation process is as follows:

[0518] Step a: Under nitrogen, to a round-bottom flask containing compound S7-1 (2.08 g, 8.0 mmol), 10-nonadecanol (S20-1, 2.74 g, 9.6 mmol), and DMAP (0.24 g, 2.0 mmol) in dichloromethane (50 mL) was added DCC (3.63 g, 17.6 mmol) and reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound S20-2 (3.47 g).

[0519] Step b: Compound S20-2 (2.64 g, 5.0 mmol) was dissolved in THF (50 mL) in a nitrogen-protected flask. TBAF solution (50 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography afforded compound S20-3 (1.82 g, 87.9%).

[0520] Step c: S20-3 (1.55 g, 3.8 mmol) and S20-4 (0.36 g, 1.5 mmol) were dissolved in anhydrous dichloromethane, stirred, and the two solutions were combined. DMAP (18.30 mg, 0.15 mmol), EDCI (0.72 g, 3.8 mmol), and DIPEA (0.68 g, 7.5 mmol) were added sequentially to the mixture and stirred at room temperature for 48 h. After completion of the reaction, the reaction solution was diluted with dichloromethane (20 mL), then washed twice with saturated sodium carbonate solution (10 mL x 2), twice with 10 mL of aqueous solution, and once with saturated brine. The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated to obtain the crude product. The crude product was separated and purified by column chromatography. The target eluate was collected and concentrated to obtain cationic lipid E20-1 (0.82 g). 1 H NMR (400MHz, CDCl3) δ: 4.93-4.81(m,2H), 4.08(t,4H), 3.40(t,1H), 2.49-2.51(m,6H), 2.33-2.25(m,8H), 1.71-1.22(m,80H), 0.87(t,12H). MS(ESI):m / z=1028.85([M+H] + ).

[0521]

[0522] Example 21: Cationic lipid (E21-2)

[0523]

[0524] The preparation process is as follows:

[0525] Step a: Under nitrogen, to a round-bottom flask containing compound S3-1 (2.08 g, 6.0 mmol), S3-3 (2.56 g, 7.2 mmol), and DMAP (0.18 g, 1.5 mmol) in dichloromethane (50 mL) was added DCC (2.72 g, 13.2 mmol). The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford the target compound S21-1 (3.42 g).

[0526] Step b: To a solution of S21-1 (2.74 g, 4.0 mmol) in anhydrous ethanol (50 mL) was added 1,2-epoxytridecane (S21-2, 3.17 g, 16.0 mmol) and stirred at room temperature for 10 h. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to afford compound E21-1 (2.00 g).

[0527] Step c: Compound E21-1 (1.62 g, 1.5 mmol) was dissolved in THF (20 mL) in a nitrogen-protected flask. TBAF solution (20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography afforded cationic lipid E21-2 (1.21 g, 83.6%). 1 H NMR(400MHz, CDCl3)δ:4.93-4.81(m,1H),3.68-3.60(t,2H),3.55-3.53(m,2H),3.45(t,1H),3.18-2.98(m,2H), 2.56(s,3H),2.52(t,2H),2.36-2.33(m,4H),2.31(t,4H),2.01-1.82(m,4H),1.71-1.22(m,78H),0.89(t,12H). MS(ESI):m / z=967.89([M+H] + ).

[0528]

[0529] Example 22: Cationic lipid (E22-2)

[0530]

[0531] The preparation process is as follows:

[0532] Step a: 5-Hydroxypentanoic acid (S22-1, 2.32 g, 10.0 mmol, the hydroxyl group of S22-1 was protected with TBS), EDCI (2.30 g, 12.0 mmol), and NHS (1.25 g, 11.0 mmol) were dissolved in dichloromethane (50 mL) and stirred at room temperature overnight. After completion of the reaction, the reaction solution was backwashed twice with 0.1 mol / L HCl (20 mL x 2) and once with saturated sodium chloride (50 mL). The dichloromethane phase was collected, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to yield compound S22-2 (3.22 g, 97.8%).

[0533] Step b: Compound S22-2 (2.96 g, 9.0 mmol) was dissolved in dichloromethane (50 mL). 2-Hexyldecylamine (S22-3, 2.39 g, 9.9 mmol) and TEA (2.8 mL, 18.0 mmol) were added and reacted at room temperature overnight to precipitate a large amount of white solid. The solid was filtered, slurried with methanol (20 mL), filtered, and rinsed twice with methanol. The solid was collected and dried to afford compound S22-4 (3.41 g, 83.3%).

[0534] Step c: Compound S22-4 (3.19 g, 7.0 mmol) was dissolved in THF (30 mL) in a nitrogen-protected flask. TBAF solution (30 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography afforded compound S22-5 (2.13 g, 89.2%).

[0535] Step d: S22-5 (1.88 g, 5.5 mmol) and S22-6 (0.67 g, 2.2 mmol, the hydroxyl group of S22-6 was protected with TBS) were dissolved in anhydrous dichloromethane. The two solutions were combined after stirring. DMAP (26.84 mg, 0.22 mmol), EDCI (1.06 g, 5.5 mmol), and DIPEA (0.99 g, 11.0 mmol) were added sequentially to the mixture. The mixture was stirred at room temperature for 48 h. After completion of the reaction, the reaction solution was diluted with dichloromethane (30 mL) and washed twice with saturated sodium carbonate solution (20 mL x 2), twice with 30 mL of aqueous solution, and once with saturated brine. The organic phase was dried over anhydrous sodium sulfate and the solvent was concentrated to obtain the crude product. The crude product was separated and purified by column chromatography. The target eluate was collected and concentrated to obtain the product E22-1 (1.11 g).

[0536] Step e: Compound E22-1 (0.95 g, 1.0 mmol) was dissolved in THF (10 mL) in a nitrogen-protected flask. TBAF solution (10 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by column chromatography to yield cationic lipid E22-2 (0.74 g, 88.3%). 1 H NMR(400MHz, CDCl3)δ:4.09(t,4H),3.54(t,2H),3.40(t,1H),3.19-3.13(m,4H),2.74(t,2H),2.56 (s,3H),2.52-2.48(m,2H),2.19-2.15(t,4H),2.01-1.98(m,2H),1.71-1.22(m,56H),0.87(t,12H). MS(ESI):m / z=838.72([M+H] + ).

[0537]

[0538] Example 23: Cationic lipid (E23-1)

[0539]

[0540] The preparation process is as follows:

[0541] Step a: Under nitrogen, to a round-bottom flask containing 4-benzyl aspartate (S23-1, 1.62 g, 5.0 mmol, the amino group of S23-1 was protected with Boc), 3-diethylamino-1-propanol (S23-2, 0.79 g, 6.0 mmol), and DMAP (0.15 g, 1.3 mmol) in dichloromethane (30 mL) was added DCC (2.27 g, 11.0 mmol). The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound S23-3 (1.82 g).

[0542] Step b: Removal of the Boc protecting group. In a clean, dry round-bottom flask, prepare a solution of trifluoroacetic acid / dichloromethane (1:2, v / v). Slowly add a dichloromethane solution of S23-3 (1.31 g, 3.0 mmol) dropwise under an ice bath. Allow to react at room temperature for 2 hours. After completion of the reaction, concentrate the reaction solution, dilute with purified water, and extract with dichloromethane. The organic phase is dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated and recrystallized to afford compound S23-4 (0.91 g, 90.0%).

[0543] Step c: Dissolve S1-3 (1.84 g, 4.4 mmol) in 30 mL of DMF, add S23-4 (0.67 g, 2.0 mmol) and K2CO3 (0.73 g, 5.3 mmol), and stir at room temperature overnight. After the reaction is complete, the reaction mixture is concentrated under reduced pressure and poured into 30 mL of ethyl acetate. After washing with 10% citric acid (20 mL) and brine (20 mL), the organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product is purified by column chromatography to afford S23-5 (1.64 g).

[0544] Step d: Compound S23-5 (1.22 g, 1.2 mmol) was dissolved in methanol (20 mL), and NiCl2·6H2O (0.86 g, 3.6 mmol) was added. After stirring, NaBH4 (0.41 g, 10.8 mmol) was slowly added. The reaction was stirred at room temperature. After TLC showed that the reaction was complete, 10 mL of methanol was slowly added to the reaction solution to quench the reaction. After stirring for 30 minutes, the reaction solution was filtered through celite and washed with methanol (20 mL). 20 mL of water was added to the reaction solution, mixed well, and extracted three times with ethyl acetate (10 mL*3). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. The mixture was purified by column chromatography, concentrated, and pumped dry to obtain compound S23-6 (0.98 g).

[0545] Step e: Under nitrogen, DCC (0.36 g, 1.8 mmol) was added to a round-bottom flask containing compound S23-6 (0.74 g, 0.8 mmol), S7-4 (0.23 g, 1.0 mmol), and DMAP (24.40 mg, 0.2 mmol) in dichloromethane (10 mL). The mixture was allowed to react at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford cationic lipid E23-1 (0.81 g, 86.5%). 1 H NMR(400MHz, CDCl3)δ:4.09(t,10H),3.40(t,1H),2.87-2.80(t,2H),2.57(q, 4H),2.51-2.49(m,6H),2.33-2.27(m,4H),1.98-1.15(m,94H),0.89(m,15H). MS(ESI):m / z=1178.02([M+H] + ).

[0546]

[0547] Example 24: Cationic lipid (E24-1)

[0548]

[0549] The raw material S23-2 in Example 23 was replaced with isopentadecyl alcohol (S24-1, 1.31 g, 5.4 mmol), the raw material S7-4 was replaced with 1-(3-hydroxypropyl)-4-methylpiperazine (S24-6, 0.37 g, 2.3 mmol) was prepared according to the same reaction steps to obtain cationic lipid E24-1 (1.02 g, 87.2%). 1 H NMR(400MHz, CDCl3)δ:4.09(t,8H),3.40(t,1H),2.85(t,4H),2.50(m,6H),2.35(t,6H) ,2.30(s,3H),2.25(m,2H),1.80-1.70(m,2H),1.67-1.15(m,89H),0.92-0.89(m,18H). MS(ESI):m / z=1175.07([M+H] + ).

[0550]

[0551] Example 25: Cationic lipid (E25-1)

[0552]

[0553] The preparation process is as follows:

[0554] Step a: Under nitrogen, to a round-bottom flask containing N-methyl-N-tert-butyl-aspartic acid-4-benzyloxycarbonyl ester (S25-1, 1.69 g, 5.0 mmol), S15-2 (2.30 g, 6.0 mmol), and DMAP (0.15 g, 1.3 mmol) dissolved in dichloromethane (30 mL) was added DCC (2.27 g, 11.0 mmol). The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound S25-2 (2.94 g).

[0555] Step b: Compound S25-2 (2.46 g, 3.5 mmol) was dissolved in methanol (50 mL), and NiCl2·6H2O (2.50 g, 10.5 mmol) was added. After stirring, NaBH4 (1.20 g, 31.5 mmol) was slowly added. The reaction mixture was stirred at room temperature. After TLC showed that the reaction was complete, 20 mL of methanol was slowly added to the reaction solution to quench the reaction. After stirring for 30 minutes, the reaction solution was filtered through celite and washed with methanol (50 mL). 50 mL of water was added to the reaction solution, mixed well, and extracted three times with ethyl acetate (20 mL*3). The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. The mixture was purified by column chromatography, concentrated, and pumped to dryness to obtain compound S25-3 (1.92 g, 89.5%).

[0556] Step c: Under nitrogen, DCC (1.22 g, 5.9 mmol) was added to a round-bottom flask containing compound S25-3 (1.66 g, 2.7 mmol), S2-3 (0.56 g, 3.2 mmol), and DMAP (82.35 mg, 0.68 mmol) in dichloromethane (20 mL). The mixture was reacted at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound S25-4 (1.73 g).

[0557] Step d: Removal of the Boc protecting group. In a clean, dry round-bottom flask, prepare a solution of trifluoroacetic acid / dichloromethane (1:2, v / v). Slowly add a dichloromethane solution of S25-4 (1.53 g, 2.0 mmol) dropwise under an ice bath. Allow to react at room temperature for 2 hours. After completion of the reaction, concentrate the reaction solution, dilute with purified water, and extract with dichloromethane. The organic phase is dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated and recrystallized to afford compound S25-5 (1.25 g, 92.5%).

[0558] Step e: Compound S25-5 (1.00 g, 1.5 mmol) was dissolved in dry THF (20 mL). NaH (60%, 0.60 g, 15.0 mmol) was slowly added under ice-cooling and the mixture was allowed to react for 1 hour. After the reaction, compound S15-8 (0.27 g, 1.8 mmol) was added and stirred under ice-cooling for 1 hour. The reaction solution was then slowly returned to room temperature and allowed to react overnight. After the reaction, the reaction mixture was placed under ice-cooling and quenched by the slow addition of 2 mL of methanol. After stirring for 30 minutes, water (10 mL) was added and stirred. The mixture was extracted twice with dichloromethane (20 mL x 2). The organic phases were combined and backwashed once with saturated sodium chloride (20 mL). The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude E25-1. The product was purified by column chromatography, concentrated, and pumped dry to obtain cationic lipid E25-1 (0.83 g).1 H NMR(400MHz, CDCl3)δ:4.93-4.81(m,1H),4.07(t,4H),3.68-3.60(t,2H),3.40(t, 1H),2.56(s,3H),2.52-2.49(m,4H),2.32(t,2H),1.65-1.25(m,58H),0.89(t,9H). MS(ESI):m / z=740.63([M+H] + ).

[0559]

[0560] Example 26: Cationic lipid (E26-1)

[0561]

[0562] The preparation process is as follows:

[0563] Compound S25-5 (2.67 g, 4.0 mmol) was dissolved in dry THF (40 mL). NaH (60%, 1.60 g, 40.0 mmol) was slowly added under ice-cooling and allowed to react for 1 hour. After the reaction, 2-(2-bromoethoxy)ethanol (S26-1, 0.81 g, 4.8 mmol) was added and stirred under ice-cooling for 1 hour. The reaction solution was then slowly returned to room temperature and allowed to react overnight. After the reaction, the reaction mixture was placed under ice-cooling and quenched by the slow addition of 4 mL of methanol. After stirring for 30 minutes, water (15 mL) was added and stirred. The mixture was extracted twice with dichloromethane (40 mL x 2). The organic phases were combined and backwashed once with saturated sodium chloride (40 mL). The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude E26-1. The product was purified by column chromatography, concentrated, and pumped to dryness to obtain cationic lipid E26-1 (2.26 g). 1 H NMR(400MHz, CDCl3)δ:4.93-4.81(m,1H),4.07(t,4H),3.70(t,2H),3.63(t,4H),3.40(t, 1H),2.56(s,3H),2.52(t,2H),2.48(t,2H),2.32(t,2H),1.65-1.25(m,54H),0.89(t,9H). MS(ESI):m / z=756.63([M+H] + ).

[0564]

[0565] Example 27: Cationic lipid (E27-2)

[0566]

[0567] The preparation process is as follows:

[0568] Step a: Compound S18-3 (5.15 g, 9.0 mmol) was added to compound S21-2 (5.80 g, 6.0 mmol) in anhydrous ethanol (100 mL) and stirred at room temperature for 10 h. After the reaction, the mixture was concentrated, extracted, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound S27-1 (2.15 g).

[0569] Step b: Dissolve S1-3 (1.00 g, 2.4 mmol) in 20 mL of DMF, add S27-1 (1.54 g, 2.0 mmol) and K2CO3 (0.73 g, 5.3 mmol), and stir at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 20 mL of ethyl acetate. After washing with 10% citric acid (10 mL) and brine (10 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford E27-1 (1.84 g).

[0570] Step c: Compound E27-1 (1.11 g, 1.0 mmol) was dissolved in THF (10 mL) in a nitrogen-protected flask. TBAF solution (10 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by column chromatography to yield cationic lipid E27-2 (0.83 g, 83.6%). 1 H NMR(400MHz, CDCl3)δ:4.51(t,1H),4.07(t,8H),3.70(t,2H),3.55-3.53(m,1H),2.50(t, 2H),2.35-2.25(m,7H),2.13-1.92(m,2H),1.88(q,2H),1.70-1.25(m,74H),0.89(t,12H). MS(ESI):m / z=996.84([M+H] + ).

[0571]

[0572] Example 28: Cationic lipid (E28-2)

[0573]

[0574] The preparation process is as follows:

[0575] Step a: Under nitrogen, DCC (1.81 g, 8.8 mmol) was added to a round-bottom flask containing compound S15-2 (1.54 g, 4.0 mmol), S28-1 (1.73 g, 4.8 mmol; the secondary amino and hydroxyl groups of S28-1 were protected with Boc and TBS, respectively), and DMAP (0.12 g, 1.0 mmol) in dichloromethane (30 mL). The mixture was allowed to react at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound E28-2 (2.37 g).

[0576] Step b: Compound S28-2 (2.18 g, 3.0 mmol) was dissolved in THF (20 mL) in a nitrogen-protected flask. TBAF solution (20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography afforded compound S28-3 (1.56 g, 85.0%).

[0577] Step c: Under nitrogen, to a round-bottom flask containing compound S28-3 (1.23 g, 2.0 mmol), 2-methyldodecanoic acid (S28-4, 0.51 g, 2.4 mmol), and DMAP (61.00 mg, 0.5 mmol) in dichloromethane (20 mL) was added DCC (0.91 g, 4.4 mmol). The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound E28-1 (1.34 g).

[0578] Step d: Removal of the Boc protecting group. In a clean, dry round-bottom flask, prepare a trifluoroacetic acid / dichloromethane (1:2, v / v) solution. Slowly add a dichloromethane solution of E28-1 (1.05 g, 1.3 mmol) dropwise under an ice bath. Allow to react at room temperature for 2 hours. After completion of the reaction, concentrate the reaction solution, dilute with purified water, and extract with dichloromethane. The organic phase is dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated and recrystallized to yield cationic lipid E28-2 (0.85 g, 91.7%). 1 H NMR(400MHz, CDCl3)δ:5.00(t,1H),4.93-4.81(m,1H),4.61-4.51(m,2H),4. 09(t,2H),4.04(m,1H),2.42-2.26(m,3H),1.65-1.15(m,63H),0.89(t,9H). MS(ESI):m / z=710.63([M+H] + ).

[0579]

[0580] Example 29: Cationic lipid (E29-1)

[0581]

[0582] The preparation process is as follows:

[0583] Step a: S1-3 (1.84 g, 4.4 mmol) was dissolved in 30 mL of DMF, and S29-1 (0.38 g, 2.0 mmol, S29-1 was prepared by serine and The mixture was stirred overnight at room temperature. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 30 mL of ethyl acetate. After washing with 10% citric acid (20 mL) and brine (20 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford S29-2 (1.38 g).

[0584] Step b: Under nitrogen, DCC (0.63 g, 3.1 mmol) was added to a round-bottom flask containing compound S29-2 (1.21 g, 1.4 mmol), S29-3 (0.60 g, 1.7 mmol, S29-3 prepared by reacting 1,6-hexanediol with S1-1), and DMAP (42.70 mg, 0.4 mmol) in dichloromethane (20 mL). The mixture was reacted at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The residue was purified by column chromatography to obtain cationic lipid E29-1 (1.36 g). 1 H NMR(400MHz, CDCl3)δ:4.08(t,8H),3.63(t,2H),3.53(t,1H),3.17-3.12(m,2H),2. 87-2.80(t,2H),2.51(t,4H),2.25-2.23(m,9H),1.65-1.23(m,98H),0.88(t,18H). MS(ESI):m / z=1206.09([M+H] + ).

[0585]

[0586] Example 30: Cationic lipid (E30-2)

[0587]

[0588] The preparation process is as follows:

[0589] Step a: Under nitrogen, a round-bottom flask containing isostearic acid (S30-1, 1.70 g, 6.0 mmol), S1-2 (1.30 g, 7.2 mmol), and DMAP (0.18 g, 1.5 mmol) in dichloromethane (30 mL) was added with DCC (2.72 g, 13.2 mmol) and reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated, and the resulting residue was purified by column chromatography to give compound 6-bromohexyl-2-heptyl undecanoate (S30-2, 2.23 g).

[0590] Step b: Dissolve S30-2 (1.96 g, 4.4 mmol) in 30 mL of DMF, add OTBS-threonine (S30-3, 0.47 g, 2.0 mmol) and KCO (0.73 g, 5.3 mmol), and stir at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 30 mL of ethyl acetate. After washing with 10% citric acid (20 mL) and brine (20 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford S30-4 (1.54 g).

[0591] Step c: Under nitrogen, to a round-bottom flask containing compound S30-4 (1.35 g, 1.4 mmol), S7-4 (0.46 g, 1.7 mmol), and DMAP (42.30 mg, 0.4 mmol) in dichloromethane (30 mL) was added DCC (0.63 g, 3.1 mmol). The mixture was reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford the target compound E30-1 (1.41 g).

[0592] Step d: Compound E30-1 (1.22 g, 1.0 mmol) was dissolved in THF (20 mL) in a nitrogen-protected flask. TBAF solution (20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting mixture was purified by column chromatography to yield cationic lipid E30-2 (0.95 g, 85.7%). 1 H NMR(400MHz, CDCl3)δ:4.09(t,8H),4.05-3.90(m,1H),3.61-3.53(m,1H), 2.50(t,4H),2.32(t,2H),2.25(m,2H),1.70-1.15(m,97H),0.88(t,15H). MS(ESI):m / z=1106.98([M+H] + ).

[0593]

[0594] Example 31: Cationic lipid (E31-1)

[0595]

[0596] The preparation process is as follows:

[0597] Step a: Under nitrogen, to a round-bottom flask containing 3-heptyldecanoic acid (S31-1, 1.62 g, 6.0 mmol), S1-2 (1.30 g, 7.2 mmol), and DMAP (0.18 g, 1.5 mmol) dissolved in dichloromethane (40 mL) was added DCC (2.72 g, 13.2 mmol) and reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, the filtrate was concentrated, and the resulting residue was purified by column chromatography to give 6-bromohexyl-3-heptyldecanoate (S31-2, 2.13 g).

[0598] Step b: S31-2 (1.90 g, 4.4 mmol) was dissolved in 30 mL of DMF, 1-methyl-tryptophan (S31-3, 0.44 g, 2.0 mmol) and K2CO3 (0.73 g, 5.3 mmol) were added, and the mixture was stirred at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 30 mL of ethyl acetate. After washing with 10% citric acid (20 mL) and brine (20 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford S31-4 (1.49 g).

[0599] Step c: Under nitrogen, DCC (0.63 g, 3.1 mmol) was added to a round-bottom flask containing compound S31-4 (1.29 g, 1.4 mmol), S6-3 (0.38 g, 1.7 mmol), and DMAP (42.70 mg, 0.4 mmol) in dichloromethane (20 mL). The mixture was allowed to react at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford cationic lipid E31-1 (1.31 g). 1H NMR(400MHz, CDCl3)δ:7.72-7.70(m,1H),7.28(d,1H),7.19(m,1H),7.05(d,1H),6.82(s,1H),4.64(t,1H) ,4.09(t,6H),3.71(s,3H),3.60-3.38(d,2H),2.51(t,4H),2.20(t,4H),1.70-1.15(m,92H),0.88(t,15H). MS(ESI):m / z=1134.01([M+H] + ).

[0600]

[0601] Example 32: Cationic lipid (E32-2)

[0602]

[0603] The preparation process is as follows:

[0604] Step a: Compound S5-2 (1.97 g, 4.4 mmol) was dissolved in 30 mL of DMF, and OTBS-tyrosine (S32-1, 0.59 g, 2.0 mmol) and KCO (0.73 g, 5.3 mmol) were added. The mixture was stirred at room temperature overnight. After completion of the reaction, the reaction mixture was concentrated under reduced pressure and poured into 30 mL of ethyl acetate. The mixture was washed sequentially with 10% citric acid (20 mL) and brine (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography to afford S32-2 (1.63 g).

[0605] Step b: Under nitrogen, to a round-bottom flask containing compound S32-2 (1.44 g, 1.4 mmol), S7-4 (0.46 g, 1.7 mmol), and DMAP (42.70 mg, 0.4 mmol) in dichloromethane (20 mL) was added DCC (0.63 g, 3.1 mmol) and reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford compound E32-1 (1.48 g).

[0606] Step c: Compound E32-1 (1.28 g, 1.0 mmol) was dissolved in THF (20 mL) in a nitrogen-protected flask. TBAF solution (20 mL, 1 M) was added and allowed to react overnight to remove the TBS protection. After completion of the reaction, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography afforded cationic lipid E32-2 (1.01 g, 86.1%). 1H NMR(400MHz, CDCl3)δ:7.28-6.85(m,4H),4.08(t,8H),3.84(t,1H),3.33-2. 90(m,2H),2.51(t,4H),2.33-2.25(m,4H),1.66-1.25(m,94H),0.88(t,15H). MS(ESI):m / z=1169.00([M+H] + ).

[0607]

[0608] Example 33: Cationic lipid (E33-1)

[0609]

[0610] The preparation process is as follows:

[0611] 1,2-Epoxypentane (S33-1, 0.17 g, 2.0 mmol) and S17-2 (1.56 g, 2.0 mmol) were dissolved in 20 mL of acetonitrile, and calcium trifluoromethanesulfonate (Ca(OTf)2, 0.34 g, 1.0 mmol) was added. The reaction mixture was stirred at room temperature. After TLC showed that the reaction was complete, the acetonitrile was evaporated, water (10 mL) was added, and the mixture was extracted with dichloromethane (20 mL*3). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and evaporated. The crude product was separated and purified by column chromatography, and the target eluate was collected and concentrated to obtain cationic lipid E33-1 (1.65 g, 95.1%). 1 H NMR(400MHz, CDCl3)δ:4.93-4.81(m,1H),4.51(t,1H),4.07(t,6H),3.65-3.58(m,1H),2.56(s, 3H),2.34-2.32(m,6H),2.30-2.28(m,2H),2.13-1.92(m,2H),1.70-1.25(m,62H),0.89(t,12H). MS(ESI):m / z=868.72([M+H] + ).

[0612]

[0613] Example 34: Cationic lipid (E34-1)

[0614]

[0615] The preparation process is as follows:

[0616] Step a: S4-4 (2.43 g, 5.0 mmol) and N,N'-succinimidyl carbonate (DSC, S34-1, 1.92 g, 7.5 mmol) were placed in dichloromethane (40 mL) and stirred under an ice bath. TEA (2.10 mL, 15.0 mmol) was added to the stirred solution, and the reaction mixture was stirred at room temperature overnight. The reaction progress was monitored by TLC. After the reaction was completed, the reaction mixture was diluted with dichloromethane, and the organic layer was washed with water (40 mL) and aqueous sodium bicarbonate solution (40 mL) in sequence, concentrated, and further purified by column chromatography, concentrated, and dried under vacuum to obtain compound S34-2 (2.67 g).

[0617] Step b: Dissolve S34-3 (1.33 g, 6.6 mmol) in dichloromethane (40 mL), add S34-2 (1.88 g, 3.0 mmol) and TEA (0.75 mL, 5.4 mmol) sequentially, and stir at room temperature overnight. After completion of the reaction, concentrate the mixture, and purify the crude product by column chromatography, concentrate, and pump to dryness to afford compound S34-4 (2.86 g).

[0618] Step c: Removal of the tBu protecting group. In a clean, dry round-bottom flask, prepare a solution of trifluoroacetic acid / dichloromethane (1:2, v / v). Slowly add a dichloromethane solution of S34-4 (2.45 g, 2.0 mmol) dropwise under an ice bath. Allow to react at room temperature for 2 hours. After completion of the reaction, concentrate the reaction solution, add purified water, and extract with dichloromethane. The extract is dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated and recrystallized to afford compound S34-5 (2.15 g, 92.2%).

[0619] Step d: Under nitrogen, to a round-bottom flask containing S23-2 (0.16 g, 1.2 mmol), S34-4 (1.17 g, 1.0 mmol), and DMAP (30.50 mg, 0.3 mmol) in dichloromethane (20 mL) was added DCC (0.45 g, 2.2 mmol) and reacted at room temperature for 16 h. After completion of the reaction, the precipitate was removed by filtration, and the filtrate was concentrated. The resulting residue was purified by column chromatography to afford cationic lipid E34-1 (1.11 g). 1 H NMR(400MHz, CDCl3)δ:4.34-4.29(m,1H),4.13-4.01(m,6H),3.59-3.33(m,14H),3.17- 3.00(m,6H),2.87-2.80(m,2H),2.01-1.82(m,2H),1.98-1.21(m,108H),0.88(t,12H). MS(ESI):m / z=1281.15([M+H] + ).

[0620]

[0621] Example 35: Cationic lipid (E35-1)

[0622]

[0623] The preparation process is as follows:

[0624] 1,2-Epoxyhexane (S35-1, 0.20 g, 2.0 mmol) and E28-2 (1.12 g, 2.0 mmol) were dissolved in 20 mL of acetonitrile, followed by the addition of Ca(OTf)2 (0.34 g, 1.0 mmol). The reaction mixture was stirred at room temperature. After TLC indicated completion of the reaction, the acetonitrile was evaporated, water (10 mL) was added, and the mixture was extracted with dichloromethane (10 mL x 3). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and evaporated. The crude product was purified by column chromatography, and the target eluate was collected and concentrated to yield cationic lipid E35-1 (1.51 g, 93.2%). 1 H NMR(400MHz, CDCl3)δ:5.00(t,1H),4.93-4.80(m,1H),4.61-4.51(m,2H),4.09(t,2H) ,4.04(t,1H),3.64-3.57(m,1H),2.42-2.26(m,5H),1.65-1.10(m,69H),0.89(t,12H). MS(ESI):m / z=810.71([M+H] + ).

[0625]

[0626] Example 36: Cationic lipid (E36-1)

[0627]

[0628] The preparation process is as follows:

[0629] Under a nitrogen atmosphere, DCC (0.63 g, 3.1 mmol) was added to a round-bottom flask containing compound S1-5 (1.23 g, 1.4 mmol), S14-2 (0.17 g, 1.7 mmol), and DMAP (42.70 mg, 0.4 mmol) dissolved in dichloromethane (20 mL) under nitrogen atmosphere. The mixture was allowed to react at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, the filtrate was concentrated, and the resulting residue was purified by column chromatography to yield cationic lipid E36-1 (1.14 g). 1H NMR(400MHz, CDCl3)δ:4.09(t,6H),3.46(t,1H),3.18-2.99(m,2H),2.87-2.80(t,2H),2.57( q,4H),2.50(t,4H),2.25-2.23(m,8H),2.01-1.82(m,4H),1.71-1.22(m,74H),0.88(t,12H). MS(ESI):m / z=964.89([M+H] + ).

[0630]

[0631] Example 37. Preparation of PEGylated lipid E37-1

[0632] Example 37.1 Preparation of PEGylated lipid E37-1

[0633]

[0634] The preparation process is as follows:

[0635] Step a: Compound S37-1 (20.00 g, 10.0 mmol, mPEG-OH, Mw approximately 2000, n1 ≈ 45, PDI = 1.03) and toluene (200 mL) were azeotropically removed at 140°C. After evaporating 60 mL of solvent, the reaction mixture was cooled to room temperature. TEA (2.02 g, 20.0 mmol) and MsCl (2.05 g, 18.0 mmol) were added, and the reaction was stirred at room temperature overnight. After completion of the reaction, the reaction solution was poured into water (200 mL) and extracted twice with EtOAc (100 mL x 2). The aqueous phase was retained and further extracted twice with dichloromethane (100 mL x 2). The organic phases were combined, dried, filtered, concentrated, dissolved in isopropanol at 50°C, recrystallized in an ice bath, and filtered to yield compound S37-2 (18.00 g, 90%).

[0636] Step b: Compound S37-2 (18.00 g, 9.0 mmol) was added to 80 mL of water and stirred at room temperature to dissolve. Potassium carbonate (12.42 g, 90.0 mmol), compound S37-3 (9.58 g, 45.0 mmol), and tetra-n-butylammonium bromide (0.29 g, 0.9 mmol) were added, and the reaction mixture was stirred at room temperature for 72 hours. After completion of the reaction, the mixture was extracted twice with dichloromethane (100 mL x 2). The organic phases were combined and backwashed once with saturated sodium chloride solution (100 mL). The retained organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to afford crude compound S37-4. The crude product was purified by column chromatography, concentrated, and pumped to dryness to afford the target compound S37-4 (12.00 g).

[0637] Step c: Compound S37-4 (12.00 g, 6.0 mmol) was dissolved in dry THF (120 mL). NaH (60%, 2.40 g, 60.0 mmol) was slowly added under ice-cooling and the mixture was allowed to react for 1 hour. Compound S37-5 (8.28 g, 30.0 mmol) was added and stirred under ice-cooling for 1 hour. The mixture was then slowly returned to room temperature and allowed to react overnight. After the reaction, the mixture was placed under ice-cooling and quenched by the slow addition of 2 mL of methanol. After stirring for 30 minutes, water (300 mL) was added and stirred. The mixture was extracted twice with EtOAc (150 mL x 2). The aqueous phase was retained and then extracted twice with dichloromethane (100 mL x 2). The organic phases were combined and backwashed once with saturated sodium chloride (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude PEGylated lipid E37-1. The residue was purified by column chromatography, concentrated, and pumped to dryness to obtain PEGylated lipid E37-1 (9.00 g). 1 H NMR (400 MHz, CDCl3) δ: 3.85-3.45 (m, 182H), 3.37 (s, 3H), 3.15 (t, 2H), 2.94 (t, 2H), 2.62 (t, 2H), 1.58-1.48 (m, 4H), 1.36-1.19 (m, 44H), 0.86 (t, 6H). MALDI-TOF analysis determined the molecular weight of E37-1 to be 2447 Da, with a PDI of 1.03.

[0638]

[0639] Example 37.2 Preparation of PEGylated lipid E37-2

[0640]

[0641] The preparation process is as follows:

[0642] Compound S37-4 (11.26 g, 5.0 mmol), compound S37-6 (1.95 g, 6.0 mmol), and triethylamine (TEA, 0.76 g, 7.5 mmol) were dissolved in dichloromethane (100 mL) and stirred at room temperature overnight. The reaction solution was concentrated, dissolved in 100 mL of water, and extracted twice with EtOAc (100 mL*2). The aqueous phase was retained, sodium chloride was added, and the mixture was extracted twice with dichloromethane (100 mL*2). The organic phases were combined and backwashed once with saturated NaCl (100 mL). The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography, concentrated, and pumped to dryness to obtain PEGylated lipid E37-2 (10.1 g, 84.8%). 1H NMR (400 MHz, CDCl3) δ: 3.84-3.45 (m, 182H), 3.37 (s, 3H), 3.35 (t, 2H), 3.18 (t, 2H), 2.27 (t, 2H), 1.56-1.40 (m, 4H), 1.36-1.18 (m, 46H), 0.87 (t, 6H). MALDI-TOF analysis determined the molecular weight of E37-2 to be 2461 Da, with a PDI of 1.03.

[0643]

[0644] Example 38: Preparation of LNP-mRNA Pharmaceutical Composition and Testing of Its Physicochemical Properties

[0645] Example 38.1: Preparation of LNP-mRNA Pharmaceutical Composition

[0646] In this embodiment, multiple groups of LNP-mRNA pharmaceutical compositions containing Fluc-mRNA were prepared for comparison. The phospholipids contained in each group were DSPC, and the sterol lipids contained were cholesterol. The difference was in the two components of cationic lipids and PEGylated lipids. Among them, the control group L-0: the cationic lipid was an amino acid cationic lipid of the prior art (abbreviated as CL-1, prepared by referring to the method disclosed in CN104168887A, with a structure of ) and contains PEGylated lipid PEG2k-DMG (DMG for short); experimental group series (L-1 to L-38): the cationic lipid is the amino acid cationic lipid prepared in the examples of the present application, and the PEGylated lipid is PEG-DMG; experimental group (L-37 to L-38): the cationic lipid is the amino acid cationic lipid prepared in the examples of the present application, and the PEGylated lipid is the PEGylated lipid E37-1 or E37-2 prepared in the present application; specifically as shown in Table 1.

[0647] The preparation method of the LNP-mRNA pharmaceutical composition is as follows:

[0648] Step a: dissolving cationic lipid, DSPC, cholesterol, and PEGylated lipid in ethanol at a molar ratio of 48:9:42:1.5 to obtain an ethanol phase solution;

[0649] Step b: adding Fluc-mRNA to 10-50 mM citrate buffer (pH = 4) to obtain an aqueous solution;

[0650] Step c: The ethanol phase solution and the aqueous phase solution were mixed (1:3 v / v) to prepare LNP-mRNA, and the mixture was washed with DPBS ultrafiltration multiple times to remove ethanol and free molecules, and finally filtered through a 0.2 μm sterile filter to obtain the LNP-mRNA pharmaceutical composition.

[0651] Example 38.2: Physicochemical Properties of LNP-mRNA Pharmaceutical Compositions

[0652] Encapsulation Efficiency Determination: The encapsulation efficiency of the LNP-mRNA compositions was determined using the Quant-it Ribogreen RNA Quantification Kit. The results showed that the lipid compositions of the present invention (L-1 to L-38) had high nucleic acid encapsulation efficiencies for nucleic acid drugs, ranging from 84% to 97%, with most encapsulation efficiencies ranging from 90% to 97%. The results are shown in Table 1 below. The results demonstrate that the lipid compositions prepared from the amino acid cationic lipids in each experimental group were able to effectively encapsulate mRNA, exhibiting superior encapsulation efficiencies compared to existing amino acid cationic lipids. The encapsulation efficiencies of the different amino acid cationic lipids also varied.

[0653] Particle Size Determination: In this example, the particle size of the LNP-mRNA was measured by dynamic light scattering (DLS), and the results are shown in Table 1 below. The LNP-mRNAs measured had high size uniformity, with a PDI of less than 0.3. The particle size of the LNP-mRNA prepared from the lipid composition of the present application was in the range of 90-110 nm.

[0654] Table 1: Summary of the formulations of various lipid compositions and the particle size and encapsulation efficiency of the LNP-mRNA prepared therefrom

[0655]

[0656]

[0657] Example 39: Biological Activity Test of LNP-mRNA Pharmaceutical Composition Preparation

[0658] (1) Cytotoxicity (biocompatibility) research

[0659] The cytotoxicity of the LNP-mRNA pharmaceutical composition of the present invention was tested by MTT staining. The LNP-mRNA pharmaceutical composition was dissolved in culture medium to prepare the desired concentration. If necessary, an appropriate amount of cosolvent was added. Hela cells were used as a cell model at a seeding density of 1×10 43ug) are inoculated into 96-well plates. After inoculation, at 37°C, 4%CO2, the cells are incubated in a cell culture incubator for 24 hours, then old culture medium is abandoned by suction, the dosage of 0.1-0.3ugmRNA in every hole is administered (LNP-mRNA pharmaceutical composition is prepared in embodiment 38) culture medium 100 μL, blank control group adds fresh culture medium 100 μL, and each concentration (0.1ug, 0.15ug, 0.20ug, 0.25ug and 0.3ug) of every group is 6 replicates. After LNP-mRNA pharmaceutical composition preparation is incubated for 24 hours with Hela cells together, 20 μL of PBS buffer solution of the MTT of every hole is added to 5mg / mL and after MTT is incubated for 4 hours with cancer cells, the mixed solution of culture medium and MTT buffer solution is abandoned by suction, 150 μL / hole of DMSO is added, for dissolving the purple crystal formazan of living cells, after vibration is fully, absorbance is tested with microplate reader. The results were calculated based on the measured absorbance values. The results showed that compared with the blank control group, the cell viability of the LNP-mRNA pharmaceutical composition preparation prepared by the present invention was greater than 95%, indicating that the LNP-mRNA pharmaceutical composition preparation of the present invention has good biocompatibility.

[0660] (2) Serum stability evaluation

[0661] The LNP-mRNA pharmaceutical composition was added to a culture medium containing 10% fetal bovine serum (FBS), stirred at 37°C, and samples were taken regularly to determine the change in particle size of the LNP-mRNA. The serum stability of the LNP-mRNA pharmaceutical composition preparation was analyzed by testing its particle size change. The experimental results showed that within 7 days, the particle size changes of the control group and the experimental group were less than 10%, especially the particle size changes of the experimental groups L-1, L-2, L-3, L-6, L-12, L-14, L-19, and L-23 were less than 5%, indicating that the LNP-mRNA pharmaceutical composition preparation prepared by the cationic lipid of the present invention has good serum stability.

[0662] (3) Study on cell transfection activity

[0663] In order to investigate the mRNA transfection rate of each group of LNP-mRNA pharmaceutical compositions prepared in Example 38 of the present invention at the cellular level, Luciferase bioluminescence was used for testing. The LNP-mRNA pharmaceutical composition was dissolved in a culture medium and prepared into the required dose. Hela cells were used as a cell model, and 100 μL / well of the cell suspension was inoculated into a 96-well plate with a black-edged transparent bottom at a seeding density of 6000 cells / well. After inoculation, the cells were incubated in a cell culture incubator for 24 hours, and then administered at a dose of 0.2 μg mRNA per well. The blank control group was added with a corresponding dose of free Fluc-mRNA. After 24 hours of transfection, the old culture medium was removed and replaced with a new culture medium containing D-fluorescein sodium (1.5 mg / mL) substrate. After incubation for 5 minutes, bioluminescence was detected using a microplate reader. The stronger the fluorescence, the more Fluc-mRNA that was transported into the cytoplasm and translated into the corresponding fluorescent protein. The experimental results are shown in Table 2, wherein the relative value of fluorescence intensity is the ratio of the fluorescence intensity value of each group to the fluorescence intensity of the blank control group. The results showed that the LNP-mRNA pharmaceutical compositions prepared by the present invention all had excellent in vitro transfection effects, that is, the LNPs in the experimental groups were all effective nucleic acid delivery vectors, and were basically superior to the L-0 group (except L-3, L-14, L-29 and L-36 groups) prepared by amino acid cationic lipids in the prior art. Among them, the relative fluorescence values of the experimental groups L-1, L-6, L-7, L-20, and L-23 were higher, which may be because they contain two ionizable tertiary amine structures and multiple degradable ester bonds. The partial positive charge ionized by the ionizable tertiary amine binds to the negatively charged nucleic acid, and the degradable ester bond promotes the endosomal escape of LNP-mRNA, and the mRNA is released into the cytoplasm to exert therapeutic effects; compared with the experimental groups L-2 and L-4, the relative fluorescence values are similar, but both are higher than the control group L-0 whose cationic lipids contain ether bonds; the relative fluorescence values of the experimental groups L-3 and L-29 are both lower. The amino acid cationic lipids they use have more hydrophobic tail chains, and their encapsulation efficiency will not be very low, which may be due to the obstruction of the endosomal escape of LNP-mRNA.

[0664] Table 2: Cell transfection test results

[0665]

[0666]

[0667] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

[0668] For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, can be equivalent parameters, concentration and conditions, implement the present invention in a wider range. Although the present invention provides special embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any variation, purposes or improvements to the present invention, including departing from the disclosed scope in the application, and the changes made with conventional techniques known in the art.

Claims

1. An amino acid cationic lipid, characterized in that The structure is shown in formula (3-3): in, Each occurrence of B2 is independently any one of butylene, pentylene, hexylene, heptylene, and octylene, and B2 is optionally substituted, and the substituent is OH; Each occurrence of L2 is independently any one of -OC(=O)-, -C(=O)O-, and -OC(=O)O-; R1 and R2 are each independently a linear C 1-30 Alkyl, branched C 1-30 alkyl Wherein, t is an integer of 0-12, t1 and t2 are each independently an integer of 0-5, and t3 and t4 are 1; R e 、R f Each independently is C 1-15 alkyl; -L3-R3 appears independently each time Any of the following; or a salt, tautomer, stereoisomer or solvate thereof.

2. The amino acid cationic lipid according to claim 1, wherein The B2 is any one of pentylene, hexylene, and heptylene.

3. The amino acid cationic lipid according to claim 1, wherein The straight chain C 1-30 Alkyl is C 1-25 Straight-chain alkyl group.

4. The amino acid cationic lipid according to claim 3, wherein The C 1-25 The linear alkyl group is any one of pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl and heptadecyl.

5. The amino acid cationic lipid according to claim 1, wherein The R e 、R f Each is independently selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl.

6. The amino acid cationic lipid according to claim 1, wherein The branched C 1-30 The alkyl group is selected from any one of the following structures: described Select any one of the following structures:

7. The amino acid cationic lipid according to any one of claims 1 to 6, characterized in that Each occurrence of -B2-L2-R2 is independently selected from any one of the following structures:

8. The amino acid cationic lipid according to claim 1, wherein Its structure is selected from any one of the following structures:

9. An amino acid cationic lipid, characterized in that The structure is as follows:

10. A lipid composition, characterized in that Contains the amino acid cationic lipid according to any one of claims 1 to 9.

11. The lipid composition according to claim 10, characterized in that Further containing one or more of phospholipids, steroid lipids and PEGylated lipids; selected from any one of the following situations: Case (1): also contains phospholipids; Case (2): also contains steroid lipids; Case (3): also contains PEGylated lipids; Case (4): also contains phospholipids and steroid lipids; Case (5): also contains phospholipids and PEGylated lipids; Case (6): also contains steroid lipids and PEGylated lipids; Case (7): Also contains phospholipids, steroid lipids and PEGylated lipids.

12. The lipid composition according to claim 10, characterized in that It also contains three types of lipids: phospholipids, steroid lipids and PEGylated lipids.

13. The lipid composition according to claim 12, characterized in that The phospholipids are selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1 ,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3 -Phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn - any one of glycerol-3-phosphate-rac-(1-glycerol) sodium salt, dioleoylphosphatidylserine, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine, and combinations thereof; or the steroid lipid is selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and combinations thereof; Or the PEGylated lipid is selected from polyethylene glycol-1,2 dimyristin, polyethylene glycol-distearoylphosphatidylethanolamine, PEG-cholesterol, polyethylene glycol-diacylglycerol, polyethylene glycol-dialkoxypropyl, specifically including any one of polyethylene glycol 500-dipalmitoylphosphatidylcholine, polyethylene glycol 2000-dipalmitoylphosphatidylcholine, polyethylene glycol 500-stearoylphosphatidylethanolamine, polyethylene glycol 2000-distearoylphosphatidylethanolamine, polyethylene glycol 500-1,2-dioleoylphosphatidylethanolamine, polyethylene glycol 2000-1,2-dioleoylphosphatidylethanolamine and polyethylene glycol 2000-2,3-dimyristoylglycerol and their combinations; Or the structure of the PEGylated lipid is selected from any one of the following structural formulas and combinations thereof: Wherein, n1 is an integer between 25 and 300.

14. The lipid composition according to any one of claims 11 to 13, characterized in that The invention comprises 20-80% amino acid cationic lipids, 5-15% phospholipids, 25-55% steroid lipids and 0.5-10% PEGylated lipids, wherein the percentages are the molar percentages of each lipid in the total lipids in the solution containing the solvent.

15. The lipid composition according to claim 14, characterized in that The amino acid cationic lipid accounts for 30-65% by mole of the total lipids in the solution containing a solvent.

16. The lipid composition according to claim 14, characterized in that The molar percentage of the amino acid cationic lipid in the total lipids in the solution containing a solvent is any one of 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, and 55%.

17. The lipid composition according to claim 14, characterized in that The molar percentage of the phospholipids in the solution containing the solvent is 7.5-13% of the total lipids.

18. The lipid composition according to claim 14, characterized in that The molar percentage of the phospholipid in the total lipids in the solution containing a solvent is any one of 8%, 9%, 10%, 11% and 12%.

19. The lipid composition according to claim 14, characterized in that The molar percentage of the steroid lipid in the solution containing the solvent is 35-50% of the total lipids.

20. The lipid composition according to claim 14, characterized in that The molar percentage of the steroid lipid in the total lipids in the solution containing the solvent is any one of 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, and 50%.

21. The lipid composition according to claim 14, wherein The molar percentage of the PEGylated lipid in the solution containing the solvent is 0.5-5% of the total lipids.

22. The lipid composition according to claim 14, wherein The molar percentage of the PEGylated lipids in the solution containing the solvent is 1-3% of the total lipids.

23. The lipid composition according to claim 14, wherein The molar percentage of the PEGylated lipid in the total lipids in the solution containing a solvent is any one of 1.5%, 1.6%, 1.7%, 1.8%, and 1.9%.

24. A lipid pharmaceutical composition, characterized in that A lipid composition and a drug according to any one of claims 10 to 23, wherein the drug is selected from any one of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs.

25. The lipid pharmaceutical composition according to claim 24, characterized in that The nucleic acid drug is selected from any one of RNA, DNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir and ribozyme, and the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA and siRNA.

26. The lipid pharmaceutical composition according to claim 24, characterized in that The nucleic acid drug is any one of DNA, mRNA, miRNA and siRNA.

27. The lipid pharmaceutical composition according to any one of claims 25-26, characterized in that The pharmaceutical composition is used as a medicine and is selected from any one of the following medicines: anti-tumor agents, anti-viral agents, anti-fungal agents and vaccines.

28. A lipid pharmaceutical composition preparation, characterized in that Contains the lipid pharmaceutical composition according to claim 27 and a pharmaceutically acceptable diluent or excipient.

29. The lipid pharmaceutical composition preparation according to claim 28, characterized in that The diluent or excipient is any one of deionized water, ultrapure water, phosphate buffer and physiological saline.

30. The lipid pharmaceutical composition preparation according to claim 28, characterized in that The diluent or excipient is phosphate buffer or physiological saline.

31. The lipid pharmaceutical composition preparation according to claim 28, characterized in that The diluent or excipient is physiological saline.

32. A liposome or lipid nanoparticle, characterized in that: Containing the lipid composition according to any one of claims 10 to 23.

33. The liposome or lipid nanoparticle according to claim 32, characterized in that The lipid nanoparticles are LNP-pharmaceutical compositions, LPP-pharmaceutical compositions or PNP-pharmaceutical compositions.

34. The liposome or lipid nanoparticle according to claim 32, wherein The lipid nanoparticles are LNP-pharmaceutical compositions.

35. The liposome or lipid nanoparticle according to claim 32, wherein The lipid nanoparticles are LNP-nucleic acid pharmaceutical compositions.

36. The liposome or lipid nanoparticle according to claim 32, wherein The lipid nanoparticles are LNP-mRNA pharmaceutical compositions.

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