Nitrogen-containing cationic lipids and their applications

Abstract

Provided is a novel cationic lipid having a structure as shown in the general formula (1), specifically relating to a nitrogen-containing cationic lipid, and also relating to a liposome comprising the cationic lipid, a liposome pharmaceutical composition comprising the cationic lipid, and its preparation and application, wherein the definitions of the symbols in the formula are as defined herein. The cationic liposome comprising the cationic lipid shown in formula (1) is capable of improving the loading rate and transport rate of drugs, especially nucleic acid drugs. The end of the novel cationic lipid may also contain a fluorescent group or a targeting group, so that the cationic liposome pharmaceutical composition containing the cationic lipid can have both fluorescent or targeting functions. The aforementioned cationic liposome nucleic acid pharmaceutical composition preparation has good gene compounding ability and high gene transfection ability, further improving the gene therapy and / or diagnostic effect of the drug, and providing more optional cationic lipids for the field of drug delivery.

Description

Technical Field

[0001] The present invention belongs to the field of drug delivery, and specifically relates to a pharmaceutical carrier cationic lipid, and in particular to a nitrogen-containing cationic lipid, a liposome containing the cationic lipid, a liposome nucleic acid drug composition containing the cationic lipid, and a preparation and application thereof. Background Art

[0002] Liposomes are widely used to deliver nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, peptide drugs or protein drugs. In particular, with the approval of two transcript messenger RNA (mRNA) vaccines for the prevention of new coronavirus, lipid nanoparticles (LNPs) carrying mRNA have become a popular delivery technology. In addition to negatively charged mRNA, LNPs also contain four components: ionizable cationic lipids, neutral auxiliary lipids, sterol lipids and PEGylated lipids. Among them, cationic lipids interact with negatively charged mRNA through electrostatics. Auxiliary lipids are generally phospholipids that prevent lipid oxidation or connect ligands to the surface of liposomes or reduce the aggregation of lipid particles. Sterol lipids have strong membrane fusion properties and promote mRNA intracellular uptake and cytoplasmic entry. 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. Among the four lipids used to prepare LNPs, the most critical one is the ionizable cationic lipid, which is non-ionized and carries a neutral charge under physiological conditions, and can be ionized and carries a partial positive charge under acidic conditions. For example, when cationic lipids are used as carriers to deliver nucleic acid drugs, at low pH, the cationic lipids and nucleic acids (such as mRNA encoding antigens or fluorescent proteins) are bound to each other through electrostatic interactions and are encapsulated into LNPs. The encapsulated LNPs maintain an overall neutral surface charge outside the cell to reduce nonspecific interactions and thus enter the cell. After entering the cell, the acidic environment inside the cell endosome will cause the surface charge of the LNPs to become positive, thereby promoting the escape of mRNA from the endosome into the cytoplasm, and further translation into corresponding active molecules (such as antigen molecules or fluorescent proteins) in the cytoplasm, ultimately achieving efficient delivery and transfection of mRNA molecules.

[0003] Despite the recent progress in the use of cationic lipids for drug delivery, there is still a need in the art for alternative cationic lipids suitable for conventional therapeutic uses. WO2021026358A1 reports that nitrogen-containing lipids can be protonated to carry a positive charge or partial positive charge at physiological pH. Therefore, this application designs some new cationic lipids containing nitrogen or multi-stage nitrogen branching. Summary of the Invention

[0004] The present invention provides novel cationic lipids, cationic liposomes containing the cationic lipids, and pharmaceutical compositions and preparations thereof containing the cationic liposomes. The cationic liposome pharmaceutical composition preparation can deliver drugs into cells, increase the drug transport rate, and thus improve the therapeutic effect of nucleic acid drugs.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] One embodiment of the present invention:

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

[0008]

[0009] Where X is N or CR a , the R a H or C 1-12 alkyl;

[0010] L1 and L2 are each independently a linking bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -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 2, 3 or 4;

[0011] L3 is a connecting bond or a divalent connecting group;

[0012] B1 and B2 are each independently a connecting bond or C 1-30 alkylene;

[0013] R1 and R2 are each independently C 1-30 Aliphatic hydrocarbon or C 1-30 Aliphatic hydrocarbon derivative residue, and at least one of R1 and R2 is Wherein, t is an integer from 0 to 12, R e 、Rf Each independently is C1-C 15 Alkyl, C2-C 15 Alkenyl and C2-C 15 Any of alkynyl groups;

[0014] R3 is a hydrogen atom, -R d 、-OR d 、-NR d R d 、-SR d 、-(C=O)R d 、-(C=O)OR d 、-O(C=O)R d 、-O(C=O)OR d or Among them, R d Each occurrence is independently C 1-12 Alkyl, NR d R d The two R's d They can be connected to form a ring, G1 is a terminal branched group with a valence of k+1, j is 0 or 1, and F contains a functional group R 01 , when j is 0, G1 does not exist, when j is 1, G1 induces k Fs, k is an integer from 2 to 8;

[0015] The alkyl, alkylene, aliphatic, alkenyl and alkynyl groups are each independently substituted or unsubstituted.

[0016] The present invention also provides another embodiment:

[0017] A cationic liposome comprises a cationic lipid having a structure as shown in formula (1).

[0018] The present invention also provides another embodiment:

[0019] A liposome pharmaceutical composition comprises cationic liposomes and a drug, wherein the cationic liposomes comprise a cationic lipid having a structure as shown in formula (1).

[0020] The present invention also provides another embodiment:

[0021] A liposome pharmaceutical composition preparation contains the aforementioned liposome pharmaceutical composition and a pharmaceutically acceptable diluent or excipient.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The novel cationic lipid compound of the present invention is a cationic lipid containing multiple nitrogens, which enriches the cationic lipid species and provides more options for the selection of lipid delivery materials. Specifically, it can be applied to the delivery of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs, thereby improving the treatment and / or diagnostic effects of these drugs as preventive and / or therapeutic agents. The end of the novel cationic lipid of the present invention can also contain a fluorescent group or a targeting group, so that the cationic liposome pharmaceutical composition containing the cationic lipid can have both fluorescence or targeting functions, further improving the treatment and / or diagnostic effects of the drug, especially being applied to the delivery of nucleic acid drugs, improving the gene therapy and / or gene diagnosis effects of the drug.

[0024] The novel cationic lipid of the present invention can use the amine in the carbamate bond as a nitrogen branch to draw out a hydrophobic fatty tail chain, and the cationic lipid with the amine in the carbamate bond as a nitrogen branch to draw out a hydrophobic fatty tail chain at one end and the hydrophobic tail chain drawn out at the other end by a carbon branch has the best encapsulation effect and transfection effect.

[0025] Implementation Method

[0026] Terminology

[0027] In the present invention, unless otherwise specified, each term has the following meaning.

[0028] 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.

[0029] In the present invention, the interpretation of numerical intervals includes both numerical intervals marked with dashes (such as 1-6) and numerical intervals marked with wavy lines, such as (1 to 6). 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 both endpoints. For example, the integer range 1-6 represents a group consisting of 1, 2, 3, 4, 5, and 6. The numerical ranges in the present invention, including but not limited to numerical ranges represented by integers, non-integers, percentages, and fractions, all include both endpoints unless otherwise specified.

[0030] In the present invention, formula (2-39) to formula (2-48) refer to formula (2-39), formula (2-40), formula (2-41), formula (2-42), formula (2-43), formula (2-44), formula (2-45), formula (2-46), formula (2-47) and formula (2-48).

[0031] 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%.

[0032] 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.

[0033] 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.

[0034] The divalent linking group in the present invention, such as a hydrocarbylene group, an alkylene group, an arylene group, an amide bond, etc., is not particularly limited. When connecting to other groups, any of the two connecting ends can be selected. For example, when an amide bond is used as the divalent linking group between C-CH2CH2- and -CH2-D, it can be C-CH2CH2-C(=O)NH-CH2-D or C-CH2CH2-NHC(=O)-CH2-D.

[0035] 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.

[0036] 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 1-12When 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.

[0037] 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.

[0038] 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".

[0039] As used herein, "substituted" means any group (e.g., aliphatic, hydrocarbyl, alkyl, or alkylene) in which 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.

[0040] 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).

[0041] The secondary amine bond and hydrazine bond in the present invention refer to "-NH-" with both ends capped by alkylene groups, such as -CH2-NH-CH2-; while -C(=O)-NH- is called an amide bond and is not considered to contain a secondary amine bond.

[0042] In the present invention, a compound, a group or an atom can be substituted and hybridized at the same time, for example, nitrophenyl replaces a hydrogen atom, and -CH2-CH2-CH2- is replaced by -CH2-S-CH(CH3)-.

[0043] 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.

[0044] 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, in -Z-L4-Z-, "each occurrence of Z is independently -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=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 -、-NRc 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 "-Z-L4-Z-" group, the two Z groups can be the same or different, in the group "-NR c C(=O)NR c -" in the middle, two R c may be the same or different and are each independently a hydrogen atom or a C 1-12 alkyl.

[0045] 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.

[0046] In the present invention, "hydrocarbon" refers to a hydrocarbon compound composed of carbon atoms and hydrogen atoms.

[0047] 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.

[0048] 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 two 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.

[0049] 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 selected from the group consisting of straight-chain hydrocarbons, branched-chain hydrocarbons, and cyclic hydrocarbons. 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 straight-chain aliphatic hydrocarbons. Branched-chain hydrocarbons may also be branched-chain aliphatic hydrocarbons.

[0050] 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.

[0051] In the present invention, a "hydrocarbyl group" refers to a hydrocarbon residue formed by the loss of at least one hydrogen atom. Depending on the number of hydrogen atoms lost, hydrocarbon groups can be categorized as monovalent hydrocarbon groups (loss of one hydrogen atom), divalent hydrocarbon groups (loss of two hydrogen atoms, also known as alkylene groups), trivalent hydrocarbon groups (loss of three hydrogen atoms), and so on. When n hydrogen atoms are lost, the valence of the resulting hydrocarbon group is n. Unless otherwise specified, hydrocarbon groups in the present invention specifically refer to monovalent hydrocarbon groups.

[0052] The source of the hydrocarbon group in the present invention is not particularly limited, and may be derived from, for example, aliphatic or aromatic hydrocarbons, saturated or unsaturated hydrocarbons, linear, branched, or cyclic hydrocarbons, or hydrocarbons or heterohydrocarbons. From the perspective of saturation, the hydrocarbon group may be derived from, for example, alkanes, alkenes, alkynes, or diolefins; cyclic hydrocarbons may be derived from, for example, alicyclic hydrocarbons or aromatic hydrocarbons, monocyclic hydrocarbons or polycyclic hydrocarbons; and heterocyclic hydrocarbons may be derived from, for example, alicyclic heterocyclic hydrocarbons or aromatic heterocyclic hydrocarbons.

[0053] In the present invention, "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 in the present invention specifically refers to a monovalent aliphatic hydrocarbon group. Aliphatic hydrocarbon groups include saturated aliphatic hydrocarbon groups and unsaturated aliphatic hydrocarbon groups.

[0054] 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, for example, "propyl" refers to either n-propyl or isopropyl, and "propylene" refers to either 1,3-propylene, 1,2-propylene, or isopropylene.

[0055] 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.

[0056] 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., the 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.

[0057] 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.

[0058] In the present invention, the aliphatic hydrocarbon derivative is preferably an ether-derivatized aliphatic hydrocarbon, an aliphatic hydrocarbon derivative containing 1-2 ether bonds, and more preferably an aliphatic hydrocarbon derivative containing 2 ether bonds.

[0059] In the present invention, "molecular weight" represents the mass of a compound molecule. Unless otherwise specified, the unit of measurement of "molecular weight" is Dalton, Da.

[0060] In the context of the present invention, "about" generally refers to ±0.5%.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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 groups. The amino protecting group is preferably Boc tert-butyloxycarbonyl, Moz p-methoxybenzyloxycarbonyl, and Fmoc 9-fluorenylmethyleneoxycarbonyl. The reagent for removing the amino protecting group is selected from TFA, H2O, LiOH, MeOH, EtOH, and combinations thereof, preferably a combination of TFA and H2O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. The reagent for removing the Boc protecting group is TFA or HCl / EA; TFA is preferred. The deprotecting agent used for removing the Fmoc protecting group is a 20% piperidine solution in N,N-dimethylformamide (DMF).

[0065] In the present invention, "carboxyl activation" refers to the activation treatment of the carboxyl group with a carboxyl activator. After the carboxyl group is activated, it can promote the condensation reaction to proceed better, such as: inhibiting the generation of racemic impurities in the condensation reaction, catalytically accelerating the reaction speed, etc. "Carboxyl activating group" is the residue of the carboxyl activator. The carboxyl activator is a combination of one or more of N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb) and N,N-dicyclohexylcarbodiimide (DCC), preferably a combination of NHS / EDCI, NHS / DCC, HONb / DCC, and most preferably a combination of NHS / EDCI.

[0066] 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.

[0067] In the present invention, "cationic lipid" refers to a lipid that contains a positive charge or is ionizable. In addition to the cationic lipid shown in the general structural formula (1) of the present invention, it also includes but is not limited to N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride ( DOTMA), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 3-(didodecylamino)-N1,N1,4-tri-dodecyl-1-piperazineethylamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tri-dodecyl-1,4-piperazinediethylamine (KL22), 14,25-ditridecyl-15,18,21,24-tetradecyl Aza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino)butyric acid heptatriacontane-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA) and 2,2-dilinoleyl- Any one of 4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate) (SM102), and mixtures thereof.

[0068] In the present invention, "PEGylated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion. In addition to the general structural formula (2) of the present invention, the PEGylated lipids also include, but are not limited to, polyethylene glycol-1,2 dimyristoyl glycerol (PEG-DMG), polyethylene glycol-distearoyl phosphatidylethanolamine (PEG-DSPE), PEG-cholesterol, polyethylene glycol-diacylglycerol (PEG-DAG), polyethylene glycol-dialkoxypropyl (PEG-DAA), specifically including polyethylene glycol 500-dipalmitoyl phosphatidylcholine, polyethylene glycol 2000-dipalmitoyl phosphatidylcholine, polyethylene glycol 500-stearoyl phosphatidylethanolamine, polyethylene glycol 2000-distearoyl phosphatidylethanolamine, polyethylene glycol 500-1,2-oleoyl phosphatidylethanolamine, polyethylene glycol 2000-1,2-oleoyl phosphatidylethanolamine and polyethylene glycol 2000-2,3-dimyristoyl glycerol (PEG-DMG).

[0069] 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. Such lipids include but are not limited to 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) Neutral lipids include any of: palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine (LPE), and combinations thereof. Neutral lipids may be synthetic or naturally derived.

[0070] In the present invention, "steroid lipids" are selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and mixtures thereof.

[0071] 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 acids in the present invention is not particularly limited unless otherwise specified, and can be natural, non-natural, 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.

[0072] The term "functional group source" as used herein refers to a source that is reactive or potentially reactive, has photosensitivity or potentially photosensitivity, or has targeting or potentially targeting properties. The term "potential" refers to a source that can be converted into a reactive group through a chemical process selected from, but not limited to, functionalization (such as grafting, substitution, etc.), deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and leaving group modification, and can emit light or produce targeting properties under external stimuli such as light, heat, enzymes, specific binding molecules, and the in vivo microenvironment. The luminescence mentioned is not particularly limited and includes, but is not limited to, visible light, fluorescence, phosphorescence, and the like.

[0073] 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.

[0074] 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.

[0075] "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 refers to chemical reaction processes such as deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and conversion of leaving groups. "Minor change form" corresponds to "minor modification" and refers to a structural form that can form the target reactive group after undergoing simple chemical reaction processes such as deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and conversion of leaving groups. The conversion of leaving groups, for example, is the conversion of an ester form to an acyl chloride form.

[0076] In the present invention, the "N / P ratio" refers to the molar ratio of ionizable nitrogen atoms in the cationic lipid to phosphate in the nucleic acid.

[0077] In the present invention, "nucleic acid" refers to DNA or RNA or modified forms thereof.

[0078] 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, can produce the encoded polypeptide. RNA may be selected from the non-limiting group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single-stranded guide RNA (sgRNA), cas9 mRNA and mixtures thereof.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] As used herein, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with a therapeutic agent and, within the scope of sound medical judgment, suitable for contact with human and / or other animal tissues without excessive toxicity, irritation, allergic reaction, or other problems or complications commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical compositions are administered intravenously, water is an exemplary carrier. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, and the like. The compositions may also contain small amounts of wetting agents, emulsifiers, or pH buffers, as needed. Oral formulations may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Specifically, for example, excipients include, but are not limited to, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), demulcents, emulsifiers, fillers (diluents), film formers or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration. More specifically, excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dicalcium phosphate, calcium stearate, cross-linked sodium carboxymethylcellulose, cross-linked polyvinyl pyrrolidone, citric acid, cross-linked polyvinyl pyrrolidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, phenylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, and xylitol.

[0085] 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.

[0086] 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.

[0087] In the present invention, treatment refers to the management and care of a patient for the purpose of combating a disease, disorder or condition, and is intended to include delaying the progression of the disease, disorder or condition, alleviating or relieving symptoms and complications, and / or curing or eliminating the disease, disorder or condition. The patient to be treated is preferably a mammal, especially a human. Detailed Description of the Invention

[0089] One embodiment of the present invention is as follows:

[0090] 1.1. A cationic lipid, characterized in that the structure is as shown in the general formula (1):

[0091]

[0092] Where X is N or CR a , the R a H or C 1-12 alkyl;

[0093] L1 and L2 are each independently a linking bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -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 cC(=O)S-, wherein R c Each occurrence is independently a hydrogen atom or a C 1-12 Alkyl, s is 2, 3 or 4;

[0094] L3 is a connecting bond or a divalent connecting group;

[0095] B1 and B2 are each independently a connecting bond or C 1-30 alkylene;

[0096] R1 and R2 are each independently C 1-30 Aliphatic hydrocarbon or C 1-30 Aliphatic hydrocarbon derivative residue, and at least one of R1 and R2 is Wherein, t is an integer from 0 to 12, R e 、R f Each independently is C1-C 15 Alkyl, C2-C 15 Alkenyl and C2-C 15 Any of alkynyl groups;

[0097] R3 is a hydrogen atom, -R d 、-OR d 、-NR d R d 、-SR d 、-(C=O)R d 、-(C=O)OR d 、-O(C=O)R d 、-O(C=O)OR d or Among them, R d Each occurrence is independently C 1-12 Alkyl, NR d R d The two R's d They can be connected to form a ring, G1 is a terminal branched group with a valence of k+1, j is 0 or 1, and F contains a functional group R 01 , when j is 0, G1 does not exist, when j is 1, G1 induces k Fs, k is an integer from 2 to 8;

[0098] The alkyl group, alkylene group, aliphatic hydrocarbon group, aliphatic hydrocarbon derivative residue, alkenyl group and alkynyl group are each independently substituted or unsubstituted.

[0099] 1.1.1.X

[0100] In the present invention, each occurrence of X is independently N or CR a , where R a H or C 1-12 alkyl.

[0101] 1.1.2.L1, L2, L3, L4, L5, L7, L8, Z, Z1, Z2

[0102] In the present invention, the structures of L1, L2, L3, L4, L5, L7, L8, Z, Z1, and Z2 are not particularly limited, and each independently includes, but is not limited to, a linear structure, a branched structure, or a cyclic structure.

[0103] In the present invention, the number of non-hydrogen atoms in L1, L2, L3, L4, L5, L7, L8, Z, Z1, and Z2 is not particularly limited, and each independently preferably has 1 to 50 non-hydrogen atoms; more preferably 1 to 20 non-hydrogen atoms; and more preferably 1 to 10 non-hydrogen atoms. The non-hydrogen atoms are carbon atoms or heteroatoms. The heteroatoms include, but are not limited to, O, S, N, P, Si, B, and the like. When the number of non-hydrogen atoms is 1, the non-hydrogen atom may be a carbon atom or a heteroatom. When the number of non-hydrogen atoms is greater than 1, the type of non-hydrogen atoms is not particularly limited; it may be one, two, or more. When the number of non-hydrogen atoms is greater than 1, it may be any combination of carbon atoms and carbon atoms, carbon atoms and heteroatoms, or heteroatoms and heteroatoms.

[0104] In the present invention, two identical or different reactive groups can react to form a divalent linking group. The reaction conditions are related to the type of divalent linking group generated by the reaction, and existing public technologies can be used. For example: amino groups react with active esters, formic acid active esters, sulfonic acid esters, aldehydes, α, β-unsaturated bonds, carboxylic acid groups, epoxides, isocyanates, and isothiocyanates to obtain divalent linking groups such as amide groups, urethane groups, amino groups, imine groups (which can be further reduced to secondary amino groups), amino groups, amide groups, amino alcohols, urea bonds, and thiourea bonds; sulfhydryl groups react with active esters, formic acid active esters, sulfonic acid esters, sulfhydryl groups, maleimides, aldehydes, α, β-unsaturated bonds, carboxylic acid groups, iodoacetamide, and acid anhydrides to obtain divalent linking groups such as thioester groups, thiocarbonates, thioethers, disulfides, thioethers, thiohemiacetals, thioethers, thioesters, thioethers, and imides; unsaturated bonds react with sulfhydryl groups to obtain thioether groups; Carboxyl groups or acyl halides react with sulfhydryl groups and amino groups to produce thioester groups, amide groups and other groups; hydroxyl groups react with carboxyl groups, isocyanates, epoxides, and chloroformyloxy groups to produce divalent linking groups such as ester groups, carbamate groups, ether bonds, and carbonate groups; carbonyl groups or aldehyde groups react with amino groups, hydrazines, and hydrazides to produce divalent linking groups such as imine bonds, hydrazones, and acylhydrazones; click chemistry reactions of reactive groups such as azide, alkynyl, alkenyl, sulfhydryl, azide, diene, maleimide, 1,2,4-triazolin-3,5-dione, dithioesters, hydroxylamine, hydrazides, acrylates, allyloxy groups, isocyanates, and tetrazole can generate various divalent linking groups containing structures including but not limited to triazole, isoxazole, and thioether bonds.

[0105] There is no particular restriction on the stability of L1, L2, L3, L4, L5, L7, L8, Z, Z1, and Z2, wherein any divalent linking group or any divalent linking group composed of adjacent heteroatom groups is independently a stably existing linking group STAG or a degradable linking group DEGG.

[0106] 1.1.2.1.L1, L2

[0107] In the present invention, L1 and L2 are each independently a linking bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -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 2, 3 or 4.

[0108] In a specific embodiment of the present invention, more preferably L1 and L2 are one of the following situations:

[0109] Case (1): One of L1 and L2 is a connecting bond, and the other is -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -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 Any one of C(=O)S-;

[0110] Case (2): L1 and L2 are both connecting bonds;

[0111] Case (3): L1 and L2 are each independently selected from -O(C=O)-, -(C=O)O-, -O(C=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-.

[0112] In a specific embodiment of the present invention, more preferably, L1 and L2 are each independently selected from any one of -O(C=O)-, -(C=O)O- and -O(C=O)O-.

[0113] In a specific embodiment of the present invention, it is more preferred that one of L1 and L2 is -(C=O)O-, and the other is -O(C=O)- or -(C=O)O-.

[0114] In one embodiment of the present invention, it is more preferred that L1 and L2 are both -(C=O)O.

[0115] In a specific embodiment of the present invention, R c Preferably a hydrogen atom; or R c Preferably C 1-12 Alkyl, more preferably C 1-8 The alkyl group is more preferably any one of methyl, ethyl, propyl, butyl, pentyl and hexyl.

[0116] 1.1.2.2.L7, L8

[0117] In the present invention, L7 and L8 are each independently a linking bond or a divalent linking group, wherein the divalent linking group is selected from -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=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 a hydrogen atom or a C 1-12 alkyl.

[0118] In a specific embodiment of the present invention, R c Preferably a hydrogen atom; or R c Preferably C 1-12 Alkyl, more preferably C 1-8 The alkyl group is more preferably any one of methyl, ethyl, propyl, butyl, pentyl and hexyl.

[0119] 1.1.2.3.L3

[0120] In the present invention, L3 is a linking bond or a divalent linking group.

[0121] In a specific embodiment of the present invention, L3 is a divalent linking group, preferably a divalent linking group selected from any one, any two, or a combination of any two or more of the divalent linking groups of L4, L5, and Z; more preferably, any one of -L4-, -Z-L4-Z-, -L4-Z-L5-, -Z-L4-Z-L5-, and -L4-Z-L5-Z-; wherein, L4 and L5 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)-, -O(C=O)-, -(C=O)O-, -O(C=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, C 1-12 The alkyl group may be substituted or unsubstituted, and is preferably any one of a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, and an octyl group.

[0122] In a specific embodiment of the present invention, R in the aforementioned L3 c Preferred is a hydrogen atom.

[0123] In a specific embodiment of the present invention, L3 is more preferably -(CH2) t -、-(CH2) t Z-, -Z(CH2) t -、-(CH2) t Z(CH2) t - and -Z(CH2) t Z-, wherein t is an integer from 1 to 12, and Z is -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=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-. More preferably, L3 is -(CH2) t -、-(CH2) t 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-、-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 -、-(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 -、-O(CH2) t O-、-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-、-NHC(=O)O(CH2) t OC(=O)NH-、-OC(=O)NH(CH2)t NHC(=O)O-, -NHC(=O)NH(CH2) t NHC(=O)NH-, -C(=O)(CH2) t O-, -C(=O)(CH2) t C(=O)O-, -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- and -C(=O)(CH2) t Any one of NHC(=O)NH-.

[0124] 1.1.3.B1, B2

[0125] In the present invention, B1 and B2 are each independently a connecting bond or C 1-30 Alkylene.

[0126] In a specific embodiment of the present invention, B1 and B2 are preferably each independently a connecting bond or C 1-20 Alkylene; more preferably B1, B2 are any of the following:

[0127] 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 5-12 alkylene;

[0128] Case (2): One of B1 and B2 is a connecting bond, and the other is C 1-20 Alkylene.

[0129] 1.1.4.R1, R2

[0130] In the present invention, R1 and R2 are each independently C 1-30 Aliphatic hydrocarbon or C 1-30 Aliphatic hydrocarbon derivative residue, and at least one of R1 and R2 is Wherein, t is an integer from 0 to 12, R e 、R f Each independently is C1-C 15 Alkyl, C2-C15 Alkenyl and C2-C 15 Any of the alkynyl groups.

[0131] In a specific embodiment of the present invention, preferably C 1-30 The aliphatic hydrocarbon group is a straight-chain alkyl group, a branched-chain alkyl group, a straight-chain alkenyl group, a branched-chain alkenyl group, a straight-chain alkynyl group or a branched-chain alkynyl group; 1-30 When the aliphatic hydrocarbon group is a branched alkyl group, a branched alkenyl group or a branched alkynyl group, it is represented by The C 1-30 The residue of aliphatic hydrocarbon derivative is 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 are not 0 at the same time; wherein, R e 、R f Each independently is C1-C 15 Alkyl, C2-C 15 Alkenyl and C2-C 15 Any of the alkynyl groups.

[0132] In a specific embodiment of the present invention, it is preferred that the C 1-30 Aliphatic hydrocarbon or C 1-30 The aliphatic hydrocarbon derivative residue is selected from any one of the following structures:

[0133]

[0134]

[0135] In a specific embodiment of the present invention, the R in e 、R f Each independently is C 1-15 Alkyl is selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl; Preferably any one of the following structures:

[0136]

[0137] 1.1.5.R3

[0138] In the present invention, R3 is a hydrogen atom, -R d 、-OR d 、-NR d R d 、-SR d 、-(C=O)R d 、-(C=O)OR d 、-O(C=O)R d 、-O(C=O)ORd or Among them, R d Each occurrence is independently C 1-12 Alkyl, NR d R d The two R's d They can be connected to form a ring, G1 is a terminal branched group with a valence of k+1, j is 0 or 1, and F contains a functional group R 01 When j is 0, G1 does not exist. When j is 1, G1 induces k Fs, where k is an integer from 2 to 8.

[0139] In one embodiment of the present invention, preferably each occurrence of R3 is independently a hydrogen atom, R d , OR d 、-(C=O)R d -、-(C=O)OR d 、-O(C=O)R d 、-O(C=O)OR d and any one of, more preferably containing a hydrogen atom, an alkyl group, an alkoxy group, an alcoholic hydroxyl group, a protected alcoholic hydroxyl group, a thiol hydroxyl group, a protected thiol hydroxyl group, a carboxyl group, a protected carboxyl group, an amino group, a protected amino group, an aldehyde group, a protected aldehyde group, an ester group, a carbonate group, a carbamate group, a succinimide group, a maleimide group, a protected maleimide group, a dimethylamino group, an alkenyl group, an alkenoate group, an azido group, an alkynyl group, a folic acid group, a rhodamine group and a biotinyl group; further preferably containing H, -(CH2) t OH, -(CH2) t SH, -OCH3, -OCH2CH3, -(CH2) t NH2, -(CH2) t C(=O)OH, -C(=O)(CH2) t C(=O)OH, -C(=O)CH3, -(CH2) t N3, -C(=O)CH2CH3, -C(=O)OCH3, -OC(=O)OCH3, -C(=O)OCH2CH3, -OC(=O)OCH2CH3, -(CH2) t N(CH3)2, -(CH2) t N(CH2CH3)2, -(CH2) t CHO, Any one of the following, where R d Each occurrence is independently C 1-12 alkyl.

[0140] 1.1.6. Specific structural formula examples

[0141] In a specific embodiment of the present invention, when X in the general structural formula (1) is N, the structure of the cationic lipid of the present invention preferably satisfies any one of the following structural formulas:

[0142]

[0143]

[0144] Wherein, in formula (2-39) to formula (2-48), each occurrence of R1 is independently C 1-30 Aliphatic hydrocarbon or C 1-30 Aliphatic hydrocarbon derivative residue, R2 each occurrence is independently The definitions of s, L3, B1, B2, R3, R1 and R2 are the same as those described in the general formula (1) and are not repeated here.

[0145] 1.1.7. Specific structural examples

[0146] In some specific embodiments of the present invention, a cationic lipid having the structure shown below is finally obtained, including but not limited to any one of the following structures:

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153] 2. Preparation of Cationic Lipids

[0154] In the present invention, any of the aforementioned cationic lipids can be prepared by methods including but not limited to the following:

[0155] Method 1:

[0156] Step 1: react small molecule A-1 with small molecule A-2 to generate a polymer containing a divalent linker L1 and a reactive group F at one end. N , a small molecule intermediate A-3 with R1 at one end; wherein, the small molecule A-1 contains a reactive gene F1, and the small molecule A-2 contains a heterofunctional group F2 and F N , F2 is a reactive group that can react with F1 to form a divalent linking group L1, F Nis a reactive group capable of reacting with an amino group or a secondary amino group, preferably -OMs, -OTs, -CHO, -F, -Cl, or -Br;

[0157] Step 2: Two molecules of the small molecule intermediate A-3 are subjected to an alkylation reaction with a primary amino derivative A-4 containing a nitrogen source end group to obtain a cationic lipid A-5, wherein the R3' end contains a reactive group R 01 or containing R 01 The slightly changed form refers to a chemical process that can be converted into R through deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, or changing the leaving group. 01 Groups;

[0158] When R3' is equal to R3, the resulting structure A-5' corresponds to the structure shown in general formula (1);

[0159] When R3' is not equal to R3, A-5' is subjected to terminal micro-modification to obtain A-5 corresponding to the structure represented by 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; wherein R1 and R2 are the same, B1 and B2 are the same, and L1 and L2 are the same;

[0160] The definitions of L1, L2, L3, B1, B2, R3, R1 and R2 are the same as those described in general formula (1) and are not repeated here.

[0161] The aforementioned small molecule raw materials A-1, A-2, A-4, etc. can be purchased or synthesized independently. For example, the small molecule A-1 in Example 1.1 is It can be achieved by It was synthesized independently as raw materials.

[0162] Step 1

[0163]

[0164] Step 2

[0165]

[0166] 2.2.Method 2:

[0167] Step 1: reacting small molecule B-1 with small molecule B-2 to generate a small molecule intermediate B-3 containing a divalent linker L1, a hydroxyl group at one end, and R1 at the other end; wherein small molecule B-1 contains a reactive group F1, and small molecule B-2 contains a heterofunctional group pair F2 and a hydroxyl group (OH), and F2 is a reactive group that can react with F1 to generate a divalent linker L1;

[0168] Step 2: Oxidize the hydroxyl group of the small molecule intermediate B-3 to an aldehyde group to obtain a small molecule intermediate B-4 containing an aldehyde group, wherein B1' is an alkylene group having one less methylene group than B1;

[0169] Step 3: Two molecules of the small molecule intermediate B-4 containing an aldehyde group are subjected to an addition reaction with a primary amino derivative B-5 containing a nitrogen source end group to obtain a cationic lipid B-6', wherein the R3' end contains a reactive group R 01 or containing R 01 The slightly changed form refers to a chemical process that can be converted into R through deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, or changing the leaving group. 01 Groups;

[0170] When R3' is equal to R3, the resulting structure B-6' corresponds to the structure shown in general formula (1);

[0171] When R3' is not equal to R3, B-6' is subjected to terminal micro-modification to obtain B-6 corresponding to the structure represented by 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, wherein R1 and R2 are the same, B1 and B2 are the same, and L1 and L2 are the same;

[0172] The definitions of L1, L2, L3, B1, B2, R3, R1 and R2 are the same as those described in general formula (1) and are not repeated here.

[0173] The aforementioned small molecule raw materials B-1, B-2, B-5, etc. can be purchased or synthesized independently.

[0174] Step 1

[0175]

[0176] Step 2

[0177]

[0178] Step 3

[0179]

[0180] 2.3. Method 3:

[0181] Step 1: react small molecule C-1 with small molecule C-2 to generate a polymer containing a divalent linker L1 and a reactive group F at one end. N , a small molecule intermediate C-3 with R1 at one end; reacting the small molecule C-1' with the small molecule C-2' to generate a small molecule intermediate C-3 containing a divalent linker L2 and a reactive group F at one endNN , a small molecule intermediate C-3' with R2 at one end; wherein the small molecule C-1 contains a reactive group F1; the small molecule C-2 contains a heterofunctional group F2 and F N , F2 is a reactive group that can react with F1 to form a divalent linking group L1, F N is a reactive group capable of reacting with an amino group or a secondary amino group, preferably -OMs, -OTs, -CHO, -F, -Cl, -Br; the small molecule C-1' contains a reactive group F3; the small molecule C-2' contains a heterofunctional group F4 and F NN , F4 is a reactive group that can react with F3 to form a divalent linking group L2; F NN is a reactive group capable of reacting with an amino group or a secondary amino group, preferably -OMs, -OTs, -CHO, -F, -Cl, -Br, -COOH, -COCl or an activated carboxyl group, wherein the activated carboxyl group refers to a carboxyl group activated by a carboxyl activating agent;

[0182] Step 2: A small molecule intermediate C-3 is subjected to an alkylation reaction with a primary amino derivative C-4 containing a nitrogen source terminal group to obtain a secondary amine derivative C-5;

[0183] Step 3: react the secondary amine derivative C-5 with the small molecule intermediate C-3' to generate the cationic lipid C-6', wherein the R3' end contains a reactive group R 01 or containing R 01 The slightly changed form refers to a chemical process that can be converted into R through deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, or changing the leaving group. 01 Groups;

[0184] When R3' is equal to R3, the resulting structure C-6' corresponds to the structure shown in general formula (1);

[0185] When R3' is not equal to R3, C-6' is subjected to terminal micro-modification to obtain a structure represented by general formula (1) corresponding to C-6; the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group;

[0186] The definitions of L1, L2, L3, B1, B2, R3, R1 and R2 are the same as those described in general formula (1) and are not repeated here.

[0187] The aforementioned small molecule raw materials C-1, C-1', C-2, C-2', C-4, etc. can be purchased or synthesized independently. For example, the small molecule C-1 in Example 6, namely S6-1, is It can be obtained through purchase or self-synthesis.

[0188] Step 1

[0189]

[0190] Step 2

[0191]

[0192] Step 3

[0193]

[0194] In the above-mentioned preparation method, R1 in the reaction raw material R1-F1 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 C1-C 15 Alkyl, C2-C 15 Alkenyl and C2-C 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.

[0195] 2.3. Method 4:

[0196] The trifunctionalized small molecule D-1 containing two identical reactive groups F5 and R3' is reacted with two molecules of D-2 to generate cationic lipid D-3', wherein the small molecule D-2 contains a reactive group F6 that can react with F5 to form a divalent linker L1 or L2, and the R3' end contains a reactive group R 01 or containing R 01 The slightly changed form refers to a chemical process that can be converted into R through deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, or changing the leaving group. 01 Groups;

[0197] When R3' is equal to R3, the resulting structure D-3' corresponds to the structure shown in general formula (1);

[0198] When R3' is not equal to R3, D-3' is subjected to terminal micro-modification to obtain D-3 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; wherein R1 and R2 are the same, and L1 and L2 are the same;

[0199] Wherein, the definitions of X, L1, L2, L3, B1, B2, R3, R1 and R2 are consistent with those described in general formula (1) and are not repeated here.

[0200] The aforementioned small molecule raw materials D-1 and D-2 can be purchased or synthesized independently.

[0201]

[0202] 2.5. Description of relevant raw materials and / or steps in the preparation process

[0203] 2.5.1. Protection and Deprotection of Related Groups During the Reaction

[0204] The present invention also involves the "protection" and "deprotection" processes of related groups during the reaction. To prevent the functional groups from affecting the reaction, they are typically protected. Furthermore, when there are two or more functional groups, the remaining functional groups are protected in order to selectively allow only the target functional group to react. Protecting groups not only stably protect the target functional group but also need to be easily removable as needed. Therefore, in organic synthesis, it is important to deprotect only the protecting groups bonded to the intended functional group under appropriate conditions.

[0205] 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.

[0206] 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 1-6 Alkylsulfonyl, arylsulfonyl, or silyl groups. The amino protecting group is preferably Boc tert-butyloxycarbonyl, Moz p-methoxybenzyloxycarbonyl, and Fmoc 9-fluorenylmethyleneoxycarbonyl. The reagent for removing the amino protecting group is selected from TFA, H2O, LiOH, MeOH, EtOH, and combinations thereof, preferably a combination of TFA and H2O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. The reagent for removing the Boc protecting group is TFA or HCl / EA; TFA is preferred. The deprotecting agent used for removing the Fmoc protecting group is a 20% piperidine solution in N,N-dimethylformamide (DMF).

[0207] 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.

[0208] In the present invention, the deprotection of the protected hydroxyl group is related to the type of hydroxyl protecting group. The type of the hydroxyl protecting group is not particularly limited. Taking benzyl, silyl ether, acetal, and tert-butyl as examples for protecting the terminal hydroxyl group, the corresponding deprotection methods are:

[0209] A: Deprotection of benzyl group

[0210] 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.

[0211] The hydrogenation reduction catalyst is not limited, and is preferably palladium and nickel, but the carrier is not limited, but preferably aluminum oxide or carbon, more preferably carbon. The amount of palladium is 1 to 100 wt% of the protected hydroxy compound, preferably 1 to 20% wt% of the protected hydroxy compound.

[0212] 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.

[0213] B: Deprotection of acetals and ketals

[0214] Preferred acetal or ketal compounds for this type of hydroxyl protection include ethyl vinyl ether, tetrahydropyran, acetone, 2,2-dimethoxypropane, and benzaldehyde. Deprotection of these acetals and ketals is achieved under acidic conditions, preferably with a solution pH of 0 to 4. The acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and nitric acid are preferred, with hydrochloric acid being more preferred. The reaction solvent is not particularly limited, as long as it can dissolve the reactants and products, and water is preferred. The reaction temperature is preferably 0 to 30°C.

[0215] C: Deprotection of silyl ethers

[0216] 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.

[0217] D: Deprotection of tert-butyl group

[0218] 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.

[0219] In the terminal functionalization method, preferably q=0, q1=1, and Z1 is 1,2-methylene. When q is not 0, A and R 01 When there are linkers such as amino acids and succinyl groups, existing technologies in the art for generating Z2 or Z1 (including but not limited to alkylation, condensation, click reaction, etc.) can be used, and the preparation can be carried out with reference to the following linear functionalization steps.

[0220] 2.5.2. Alkylation reaction

[0221] 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:

[0222] 2.5.2.1. Alkylation of the Substrate Alcohol with Sulfonates and Halides

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 2.5.2.2. Alkylation of substrate amine with sulfonate or halide

[0228] A. Alkylation of substrate amine with sulfonate and halide

[0229] 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.

[0230] The resulting product is a mixture of an amine 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. Preferred ion exchange resins are those containing tertiary amines or quaternary ammonium salts, such as dextran, agarose, polypropionate, polystyrene, and polystyrene, as backbones. 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.

[0231] 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.

[0232] 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.

[0233] 2.5.2.3. Alkylation reaction of substrate amine with aldehyde derivatives

[0234] After the substrate amine reacts with the aldehyde derivative to obtain the imine intermediate, the intermediate is obtained under the action of a reducing agent. The molar equivalent of the aldehyde derivative is 1 to 20 times that of the substrate amine, preferably 1 to 2 times, and more preferably 1 to 1.5 times. When the molar equivalent of the aldehyde derivative is greater than 20 times that of the substrate amine, the excess reagent brings trouble to the purification and may be mixed into the subsequent steps, increasing the difficulty of purification. When the molar equivalent of the aldehyde derivative is less than 1 times that of the substrate amine, the reaction is incomplete, increasing the difficulty of purification. The product after the reaction can be purified by means of cation exchange resin, osmosis, ultrafiltration, etc. to obtain the intermediate. There are no special restrictions on the cation exchange resin, as long as it can exchange with the quaternary ammonium cation to achieve a separation effect. There are no restrictions on the solvent for osmosis and ultrafiltration, and it can generally be water or an organic solvent. The organic solvent is not particularly limited as long as the product can be dissolved in it, preferably dichloromethane, chloroform, etc.

[0235] 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.

[0236] 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.

[0237] 2.5.3. Trifunctionalized small molecule D-1

[0238] The trifunctionalized small molecule D-1 contains two identical reactive groups F5 and R3', wherein F5 is a reactive group and R3' contains a reactive group R 01 or containing R 01 The slightly changed form refers to a chemical process that can be converted into R through deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, or changing the leaving group. 01 group.

[0239] Specifically, the trifunctionalized small molecule D-1 includes but is not limited to any one of the following structures: etc., also including the case where the related groups in the aforementioned trifunctionalized small molecules are protected, for example It can also be the case where the amino group is protected, that is

[0240] 2.5.4. Linear functionalization of the terminal end

[0241] 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.

[0242] Linear functionalization of the terminal hydroxyl group, i.e., starting from the terminal hydroxyl group of compound A-5', other functional groups or their protected forms -L3-R3 are obtained through functionalization. The specific preparation method is described in paragraphs

[0960] to

[1205] of document CN104530417A.

[0243] In the present invention, the raw materials used in each preparation method can be purchased or synthesized by oneself.

[0244] The intermediates and final products prepared in the present invention can be purified by purification methods including, but not limited to, extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, thin film dialysis, or supercritical extraction. The structure and molecular weight of the final product can be characterized by methods including, but not limited to, nuclear magnetic resonance, electrophoresis, UV-visible spectrophotometry, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism, and the like.

[0245] Cationic liposomes

[0246] In the present invention, a cationic liposome comprises any of the cationic lipids described above, such as the cationic lipid represented by general formula (1).

[0247] In a specific embodiment of the present invention, the cationic liposome preferably contains, in addition to the cationic lipid having the structure shown in general formula (1), one or more of a neutral lipid, a steroid lipid, and a PEGylated lipid; more preferably, it contains all three lipids. The aforementioned neutral lipid is preferably a phospholipid.

[0248] In a specific embodiment of the present invention, the neutral lipids in the cationic liposomes preferably include but are not limited to 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.

[0249] In a specific embodiment of the present invention, the steroid lipid in the cationic liposome is preferably any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol and mixtures thereof.

[0250] In a specific embodiment of the present invention, the pegylated lipid in the cationic liposome 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).

[0251] In a specific embodiment of the present invention, the structure of the PEGylated lipid in the cationic liposome is preferably as shown in general formula (2):

[0252]

[0253] or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,

[0254] Wherein, L7 and L8 are each independently a linking bond or a divalent linking group, wherein the divalent linking group is selected from -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=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 a hydrogen atom or a C 1-12 alkyl;

[0255] L3 is a connecting bond or a divalent connecting group. When it is a divalent connecting group, it is selected from any one, any two or a combination of any two or more of the divalent connecting groups of L4, L5 and Z; more preferably, it is any one of -L4-, -Z-L4-Z-, -L4-Z-L5-, -Z-L4-Z-L5- and -L4-Z-L5-Z-; wherein, L4 and L5 are carbon chain connecting 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)-, -O(C=O)-, -(C=O)O-, -O(C=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;

[0256] B3 and B4 are each independently a connecting bond or C 1-12 alkylene;

[0257] R1 and R2 are each independently C 1-30 Aliphatic hydrocarbon group;

[0258] R is a hydrogen atom, an alkyl group, an alkoxy group, or -(C=O)R d 、-(C=O)OR d 、-O(C=O)R d 、-O(C=O)OR d or Among them, R d C 1-12Alkyl, G1 is a terminal branched group with a valence of k+1, j is 0 or 1, F contains a functional group, when j is 0, G1 does not exist, when j is 1, G1 leads to k F, and k is an integer of 2-8;

[0259] A is -(CR a R b ) s O- or -O(CR a R b ) s -, where s is 2, 3 or 4, R a and R b are each independently a hydrogen atom or a C 1-12 alkyl;

[0260] n1 is an integer between 20 and 250;

[0261] The alkyl group, alkylene group, alkoxy group and aliphatic hydrocarbon group are each independently substituted or unsubstituted.

[0262] In a specific embodiment of the present invention, the structure of the PEGylated lipid in the cationic liposome is shown in formula (2) and is selected from any one of the following structural formulas:

[0263]

[0264]

[0265] In a specific embodiment of the present invention, it is preferred that any of the aforementioned cationic liposomes contain 20-80% of the cationic lipid represented by formula (1), 5-15% of the neutral lipid, 25-55% of the steroid lipid and 0.5-10% of the PEGylated lipid, wherein the percentages are the molar percentages of each lipid in the total lipids in the solution containing the solvent.

[0266] In a specific embodiment of the present invention, in any of the aforementioned cationic liposomes, the molar percentage of cationic lipids in the total lipids in the solution containing the solvent is preferably 30-65%; more preferably, it is about 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, or 55%.

[0267] In a specific embodiment of the present invention, preferably, in any of the aforementioned cationic liposomes, the molar percentage of neutral lipids in the total lipids in the solution containing the solvent is 7.5-13%; more preferably, it is any one of about 8%, 9%, 10%, 11%, and 12%.

[0268] In a specific embodiment of the present invention, preferably in any of the aforementioned cationic liposomes, 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%.

[0269] In a specific embodiment of the present invention, in any of the aforementioned cationic liposomes, 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%.

[0270] 3.2. Preparation of cationic liposomes

[0271] In the present invention, cationic 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 / or injection method, microfluidic method, preferably thin film dispersion method or injection method.

[0272] 4.1. Cationic liposome pharmaceutical compositions

[0273] One embodiment of the present invention provides a cationic liposome pharmaceutical composition comprising any of the cationic liposomes described above and a drug, wherein the cationic liposomes comprise any of the cationic lipids described above having a structure such as that represented by general formula (1), and the drug includes but is not limited to nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs, and protein drugs.

[0274] In a specific embodiment of the present invention, the cationic liposome pharmaceutical composition is prepared by a simple mixing method or a microfluidic method. Specifically, cationic lipids, neutral lipids, steroid lipids and PEGylated lipids are dissolved in an organic phase according to a certain molar percentage to obtain an organic phase solution; the drug (therapeutic agent or prophylactic agent) is added to the aqueous phase according to a certain N / P ratio to obtain an aqueous phase solution; the aforementioned organic phase solution and aqueous phase solution are mixed (microfluidic mixing or simple mixing) according to an appropriate volume ratio; and post-processing and purification are performed to obtain the cationic liposome pharmaceutical composition.

[0275] In a specific embodiment of the present invention, in the cationic liposome pharmaceutical composition, the preferred drug is a nucleic acid drug, and the nucleic acid drug is selected from any one of RNA, DNA, antisense nucleic acid, plasmid, mRNA (messenger RNA), interfering nucleic acid, aptamer, miRNA inhibitor (antagomir), microRNA (miRNA), ribozyme and small interfering RNA (siRNA); preferably any one of RNA, miRNA and siRNA.

[0276] In a specific embodiment of the present invention, the cationic liposome pharmaceutical composition is preferably used as a drug, including but not limited to anti-tumor agents, antiviral agents, antifungal agents and vaccines.

[0277] In a specific embodiment of the present invention, the drug in the cationic liposome pharmaceutical composition is a nucleic acid drug, and the N / P ratio of the cationic lipid to the nucleic acid is (0.5-20):1; more preferably (1-10):1, more preferably 2:1, 4:1, 6:1 or 10:1.

[0278] In a specific embodiment of the present invention, the aqueous phase for dissolving nucleic acid drugs is preferably deionized water, ultrapure water, phosphate buffer or physiological saline, more preferably phosphate buffer or citrate buffer, most preferably citrate buffer; preferably cationic liposomes: working solution = (0.05-20) g: 100 mL, more preferably (0.1-10) g: 100 mL, most preferably (0.2-5) g: 100 mL.

[0279] 5.1 A Cationic Liposome Pharmaceutical Composition Preparation

[0280] In the present invention, a cationic liposome pharmaceutical composition preparation contains any of the aforementioned cationic liposome pharmaceutical compositions and a pharmaceutically acceptable diluent or excipient, wherein the diluent or excipient is preferably any one of deionized water, ultrapure water, phosphate buffer and normal saline, more preferably phosphate buffer or normal saline, and most preferably normal saline.

[0281] The preparation of cationic lipids, cationic liposomes, and cationic liposome nucleic acid pharmaceutical compositions and the biological activity testing of cationic liposome nucleic acid pharmaceutical compositions are further described below in conjunction with some specific examples. The specific examples are provided to further illustrate the present invention and do not limit the scope of protection of the present invention. Among them, in the examples of preparing cationic lipids, the final product is characterized by nuclear magnetic resonance structure or confirmed by MALDI-TOF molecular weight.

[0282] Example 1:

[0283] Example 1.1: Cationic lipid (E1-1)

[0284]

[0285] Corresponding to the general formula (1), in E1-1, R1 and R2 are B1 and B2 are both hexylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 824 Da.

[0286] The preparation process is as follows:

[0287] Step a: Add 100 mL of anhydrous dichloromethane to compound N-hexyloctylamine (S1-1, 5.33 g, 25.0 mmol) and stir to dissolve at room temperature. Potassium carbonate (K2CO3, 5.95 g, 50.0 mmol), 3-methanesulfonyloxypropionic acid (S1-2, 0.84 g, 5.0 mmol), and tetra-n-butylammonium bromide (0.19 g, 0.6 mmol) were added sequentially, and the reaction mixture was stirred at room temperature for 72 hours. After the reaction, 50 mL of water was added and stirred. The pH was adjusted to 5-7, and the mixture was extracted twice with dichloromethane (50 mL x 2). The organic phases were combined and backwashed once with saturated sodium chloride solution (50 mL). The retained organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain crude compound S1-3. Purification by column chromatography, concentration, and pumping to dryness afforded 3-(N-hexyloctylamino)propionic acid (S1-3, 2.20 g).

[0288] Step b: Under argon atmosphere, dicyclohexylcarbodiimide (DCC, 3.17 g, 15.4 mmol) was added to a round-bottom flask containing S1-3 (2.00 g, 7.0 mmol), 6-bromohexanol (S1-4, 1.51 g, 8.4 mmol) and 4-(dimethylamino)pyridine (DMAP, 0.21 g, 1.8 mmol) dissolved in dichloromethane (50 mL) under argon 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 silica gel column chromatography to give the bromoester S1-5 (2.55 g).

[0289] Step c: Under nitrogen protection, compound 4-amino-1-butanol (S1-6, 0.18 g, 2.0 mmol) was dissolved in acetonitrile (50 mL), and S1-5 (2.24 g, 5.0 mmol) and N, N-diisopropylethylamine (DIPEA, 0.36 g, 4.0 mmol) were added sequentially with slow stirring and stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved with dichloromethane, extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E1-1 (1.32 g). The main data of the H NMR spectrum of E1-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.06 (t, 4H), 3.64-3.61 (m, 2H), 3.24 (t, 4H), 3.03 (t, 4H), 3.02-2.81 (m, 14H), 1.80-1.21 (m, 60H), 0.87 (t, 12H). MALDI-TOF analysis determined the molecular weight of E1-1 to be 823.76 Da.

[0290]

[0291] Example 1.2: Cationic lipid (E1-2)

[0292]

[0293] Corresponding to the general formula (1), in E1-2, R1 and R2 are B1 and B2 are both hexylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is ethylene, R3 is hydroxyl, and the total molecular weight is about 796 Da.

[0294] The preparation process is as follows:

[0295] Under nitrogen protection, compound 2-amino-1-ethanol (S1-7, 0.12 g, 2.0 mmol) was dissolved in acetonitrile (50 mL), and S1-5 (2.24 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were added sequentially with slow stirring and stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved with dichloromethane, extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E1-2 (1.27 g). The main data of the H NMR spectrum of E1-2 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.04 (t, 4H), 3.86-3.78 (m, 2H), 3.23 (t, 4H), 3.03 (t, 4H), 3.02-2.81 (m, 14H), 1.81-1.22 (m, 56H), 0.87 (t, 12H). MALDI-TOF analysis determined the molecular weight of E1-2 to be 795.75 Da.

[0296]

[0297] Example 1.3: Cationic lipid (E1-3)

[0298]

[0299] Corresponding to the general formula (1), in E1-3, R1 and R2 are B1 and B2 are both butylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 768 Da.

[0300] The preparation process is as follows:

[0301] Step a: Under argon atmosphere, DCC (4.53 g, 22.0 mmol) was added to a round-bottom flask containing S1-3 (2.85 g, 10.0 mmol), 4-bromo-n-butanol (S1-8, 1.82 g, 12.0 mmol) and DMAP (0.31 g, 2.5 mmol) dissolved in dichloromethane (150 mL) in an argon atmosphere. The mixture was reacted at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, the filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography to give the bromoester S1-9 (3.59 g).

[0302] Step b: Under nitrogen protection, compound S1-6 (0.18 g, 2.0 mmol) was dissolved in acetonitrile (50 mL), and S1-9 (2.10 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were added sequentially with slow stirring and the reaction was stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved with dichloromethane, and extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E1-3 (1.23 g). The main data of the H NMR spectrum of E1-3 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.07 (t, 4H), 3.64-3.61 (m, 2H), 3.24 (t, 4H), 3.03 (t, 4H), 3.02-2.81 (m, 14H), 1.81-1.22 (m, 52H), 0.87 (t, 12H). MALDI-TOF analysis determined the molecular weight of E1-3 to be 767.70 Da.

[0303]

[0304] Example 2: Cationic lipid (E2-1)

[0305]

[0306] Corresponding to the general formula (1), in E2-1, R1 and R2 are B1 and B2 are both hexylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 768 Da.

[0307] The preparation process is as follows:

[0308] Step a: Compound S1-1 (2.57 g, 12.0 mmol) was dissolved in dichloromethane (50 mL). 6-Bromohexyl-N-succinimidyl carbonate (S2-1, 3.22 g, 10.0 mmol) and triethylamine (TEA, 1.10 mL, 15.0 mmol) were then added sequentially. The mixture was stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated to obtain the crude product. The product was purified by column chromatography, concentrated, and pumped to dryness to obtain the bromoester S2-2 (3.31 g).

[0309] Step b: Under nitrogen protection, compound S1-6 (0.18 g, 2.0 mmol) was dissolved in acetonitrile (50 mL), and S2-2 (2.10 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were added sequentially with slow stirring, and the reaction was stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved with dichloromethane, and extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E2-1 (1.25 g). The main data of the H NMR spectrum of E2-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.05 (t, 4H), 3.65-3.61 (m, 2H), 3.22-3.06 (m, 8H), 2.91-2.63 (m, 6H), 1.81-1.22 (m, 60H), 0.88 (t, 12H). MALDI-TOF analysis determined the molecular weight of E2-1 to be 767.73 Da.

[0310]

[0311] Example 3: Cationic lipid (E3-1)

[0312]

[0313] Corresponding to the general formula (1), in E3-1, R1 and R2 are B1 and B2 are both hexylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 824 Da.

[0314] The preparation process is as follows:

[0315] Step a: Dissolve compound S3-1 (2.89 g, 12.0 mmol) in dichloromethane (50 mL), then add S2-1 (3.22 g, 10.0 mmol) and TEA (1.10 mL, 15.0 mmol) sequentially. Stir and react at room temperature overnight. After completion of the reaction, concentrate the reaction solution to obtain the crude product. Purify by column chromatography, concentrate, and pump dry to obtain the bromoester compound S3-2 (3.49 g).

[0316] Step b: Under nitrogen protection, compound S1-6 (0.18 g, 2.0 mmol) was dissolved in acetonitrile (50 mL), and S3-2 (2.24 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were added sequentially with slow stirring, and the mixture was stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved in dichloromethane, and extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E3-1 (1.35 g). The main data of the H NMR spectrum of E3-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.03 (t, 4H), 3.64-3.61 (m, 2H), 3.20-3.06 (m, 8H), 2.91-2.59 (m, 6H), 1.81-1.22 (m, 68H), 0.85 (t, 12H). MALDI-TOF analysis determined the molecular weight of E3-1 to be 823.75 Da.

[0317]

[0318] Example 4: Cationic lipid (E4-1)

[0319]

[0320] Corresponding to the general formula (1), in E4-1, R1 and R2 are B1 and B2 are both heptylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is butylene group, R3 is hydroxyl group, and the total molecular weight is about 852 Da.

[0321] The preparation process is as follows:

[0322] Step a: Compound S3-1 (2.89 g, 12.0 mmol) was dissolved in dichloromethane (50 mL), followed by the addition of 7-bromoheptyl-N-succinimidyl carbonate (S4-1, 3.36 g, 10.0 mmol) and TEA (1.10 mL, 15.0 mmol). The mixture was stirred at room temperature overnight. After completion of the reaction, the reaction solution was concentrated to obtain the crude product. The product was purified by column chromatography, concentrated, and pumped to dryness to afford the bromoester S4-2 (3.61 g).

[0323] Step b: Under nitrogen protection, compound S1-6 (0.18 g, 2.0 mmol) was dissolved in acetonitrile (50 mL), and S4-2 (2.32 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were added sequentially with slow stirring, and the mixture was stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved in dichloromethane, and extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E4-1 (1.40 g). The main data of the H NMR spectrum of E4-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.05 (t, 4H), 3.64-3.61 (m, 2H), 3.24-3.06 (m, 8H), 2.90-2.61 (m, 6H), 1.82-1.20 (m, 72H), 0.86 (t, 12H). MALDI-TOF analysis determined the molecular weight of E4-1 to be 851.83 Da.

[0324]

[0325] Example 5: Cationic lipid (E5-1)

[0326]

[0327] Corresponding to the general formula (1), in E5-1, R1 is R2 is B1 and B2 are hexylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 823 Da.

[0328] The preparation process is as follows:

[0329] Step a: Under nitrogen, DCC (4.53 g, 22.0 mmol) was added to a round-bottom flask containing 2-hexyldecanoic acid (S5-1, 2.56 g, 10.0 mmol), S1-4 (2.16 g, 12.0 mmol), and DMAP (0.31 g, 2.5 mmol) in dichloromethane (100 mL) in a flask. 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 silica gel column chromatography to afford the bromoester S5-2 (3.39 g).

[0330] Step b: Under nitrogen, compound S1-6 (0.36 g, 4.0 mmol) was dissolved in acetonitrile (50 mL). S5-2 (2.10 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were added sequentially with slow stirring. The mixture was stirred at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to afford compound S5-3 (1.40 g).

[0331] Step c: Under nitrogen protection, compound S5-3 (0.86 g, 2.0 mmol) was dissolved in acetonitrile (30 mL), and S5-4 (1.19 g, 2.5 mmol, wherein S5-4 is a product of S1-4 and The reaction was prepared by the following steps (referring to step b) of Example 1.1) and DIPEA (0.18 g, 2.0 mmol) under stirring at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane, extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E5-1 (1.34 g). The main data of the H NMR spectrum of E5-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.06 (t, 2H), 4.01 (t, 2H), 3.66-3.62 (m, 2H), 3.22 (t, 2H), 3.04 (t, 2H), 3.02-2.81 (m, 10H), 2.29-2.22 (m, 1H), 1.92-1.21 (m, 68H), 0.83 (t, 12H). MALDI-TOF analysis determined the molecular weight of E5-1 to be 822.77 Da.

[0332]

[0333] Example 6.1: Cationic lipid (E6-1)

[0334]

[0335] Corresponding to the general formula (1), in E6-1, R1 is R2 is B1 and B2 are both hexylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 767 Da.

[0336] The preparation process is as follows:

[0337] Under nitrogen protection, compound S5-3 (0.86 g, 2.0 mmol) was dissolved in acetonitrile (30 mL), and S2-2 (1.05 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring, and the reaction was stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved in dichloromethane, and extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E6-1 (1.25 g). The main data of the H NMR spectrum of E6-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.06-4.00 (m, 4H), 3.65 (t, 2H), 3.22-3.09 (m, 4H), 2.90-2.75 (m, 6H), 2.33-2.22 (m, 1H), 1.83-1.22 (m, 64H), 0.86 (t, 12H). MALDI-TOF analysis determined the molecular weight of E6-1 to be 766.87 Da.

[0338]

[0339] Example 6.2: Cationic lipid (E6-2)

[0340]

[0341] Corresponding to the general formula (1), in E6-1, R1 is R2 is B1 and B2 are both hexylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 823 Da.

[0342] The preparation process is as follows:

[0343] Following the methods of steps a and b in Example 5, replacing the 2-hexyldecanoic acid in step a with 2-octyldecanoic acid, S6-1 was prepared. S6-1 was then reacted with S3-2 according to the dosages and procedures of Example 6.1 to yield cationic lipid E6-2. The key H NMR spectrum data for E6-2 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.06-4.02 (m, 4H), 3.65 (t, 2H), 3.22-3.10 (m, 4H), 2.92-2.76 (m, 6H), 2.33-2.24 (m, 1H), 1.79-1.22 (m, 72H), 0.85 (t, 12H). MALDI-TOF analysis determined the molecular weight of E6-2 to be 822.62 Da.

[0344]

[0345] Example 7.1: Cationic lipid (E7-1)

[0346]

[0347] Corresponding to the general formula (1), in E7-1, R1 is R2 is B1 and B2 are both hexylene groups, L1 is an ester group (-OC(=O)O-), L2 is an ester group (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 767 Da.

[0348] The preparation process is as follows:

[0349] Step a: Under nitrogen, DCC (4.53 g, 22.0 mmol) was added to a round-bottom flask containing S7-1 (2.08 g, 10.0 mmol), 7-pentadecanol (S7-2, 2.74 g, 12.0 mmol), and DMAP (0.31 g, 2.5 mmol) in dichloromethane (100 mL) in a flask. 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 silica gel column chromatography to afford the bromoester (S7-3, 3.47 g).

[0350] Step b: Under nitrogen, compound S1-6 (0.36 g, 4.0 mmol) was dissolved in acetonitrile (50 mL). S7-3 (2.10 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were added sequentially with slow stirring. The mixture was stirred at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to afford compound S7-4 (1.40 g).

[0351] Step c: Under nitrogen protection, compound S7-4 (0.86 g, 2.0 mmol) was dissolved in acetonitrile (30 mL), and S2-2 (1.05 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring and the reaction was stirred at room temperature for about 20 h. After the reaction, the reaction solution was concentrated and dissolved with dichloromethane, extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E7-1 (1.24 g). The main data of the H NMR spectrum of E7-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.87-4.79 (m, 1H), 4.03-3.99 (t, 2H), 3.64-3.58 (m, 2H), 3.24-3.06 (m, 4H), 2.90-2.59 (m, 6H), 2.30-2.24 (t, 2H), 1.81-1.22 (m, 64H), 0.85 (t, 12H). MALDI-TOF analysis determined the molecular weight of E7-1 to be 766.71 Da.

[0352]

[0353] Example 7.2: Cationic lipid (E7-2)

[0354]

[0355] Corresponding to the general formula (1), in E7-2, R1 is R2 is B1 and B2 are both hexylene groups, L1 is an ester group (-OC(=O)O-), L2 is an ester group (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 823 Da.

[0356] The preparation process is as follows:

[0357] Following the methods of steps a and b in Example 7, 9-heptadecanol was substituted for 7-pentadecanol to prepare S7-5. S7-5 was then reacted with S3-2 according to the dosages and procedures of Example 7.1 to obtain cationic lipid E7-2. The key H NMR spectrum data for E7-2 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.86-4.80 (m, 1H), 4.02-3.98 (t, 2H), 3.64-3.60 (m, 2H), 3.26-3.08 (m, 4H), 2.90-2.62 (m, 6H), 2.31-2.25 (t, 2H), 1.80-1.22 (m, 72H), 0.85 (t, 12H). MALDI-TOF analysis determined the molecular weight of E7-2 to be 822.68 Da.

[0358]

[0359] Example 8: Cationic lipid (E8-1)

[0360]

[0361] Corresponding to the general formula (1), in E8-1, R1 is R2 is B1 and B2 are hexylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 795 Da.

[0362] The preparation process is as follows:

[0363] Under nitrogen protection, compound S5-3 (0.86 g, 2.0 mmol) was dissolved in acetonitrile (30 mL), and S1-5 (1.12 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring, and the reaction was stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved in dichloromethane, and extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E8-1 (1.29 g). The main data of the H NMR spectrum of E8-1 are as follows: 1H NMR (400 MHz, CDCl3) δ: 4.07 (t, 2H), 4.01 (t, 2H), 3.67-3.62 (m, 2H), 3.22 (t, 2H), 3.04 (t, 2H), 2.99-2.85 (m, 10H), 2.30-2.21 (m, 1H), 1.93-1.47 (m, 20H), 1.45-1.16 (m, 44H), 0.84 (t, 12H). MALDI-TOF analysis determined the molecular weight of E8-1 to be 794.83 Da.

[0364]

[0365] Example 9: Cationic lipid (E9-1)

[0366]

[0367] Corresponding to the general formula (1), in E9-1, R1 is R2 is B1 and B2 are hexylene groups, L1 is an ester group (-OC(=O)-), L2 is an ester group (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 795 Da.

[0368] The preparation process is as follows:

[0369] Step a: Under nitrogen protection, compound S1-6 (0.36 g, 4.0 mmol) was dissolved in acetonitrile (50 mL), and S9-1 (2.24 g, 5.0 mmol, wherein S9-1 is a product of S6-1 and The obtained compound was prepared by reaction (referring to Example 6, step b) and DIPEA (0.36 g, 4.0 mmol) with stirring at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound S9-2 (1.48 g).

[0370] Step b: Under nitrogen protection, compound S9-2 (0.91 g, 2.0 mmol) was dissolved in acetonitrile (30 mL), and S2-2 (1.05 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring and the reaction was stirred at room temperature for about 20 h. After the reaction, the reaction solution was concentrated and dissolved with dichloromethane, extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E9-1 (1.30 g). The main data of the H NMR spectrum of E9-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.87-4.78 (m, 1H), 4.03-3.99 (t, 2H), 3.58 (t, 2H), 3.24-3.06 (m, 4H), 2.90-2.59 (m, 6H), 2.30-2.24 (t, 2H), 1.81-1.22 (m, 68H), 0.85 (t, 12H). MALDI-TOF analysis determined the molecular weight of E9-1 to be 794.74 Da.

[0371]

[0372] Example 10: Cationic lipid (E10-1)

[0373]

[0374] Corresponding to the general formula (1), in E10-1, R1 is R2 is B1 and B2 are both hexamethylene groups, L1 is a carbonate group (-OC(=O)O-), L2 is an ester group (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 783 Da.

[0375] The preparation process is as follows:

[0376] Step a: Under nitrogen, 6-bromohexyl-4-nitrophenyl carbonate (S10-1, 3.45 g, 10.0 mmol, wherein S10-1 is prepared by reacting p-nitrophenyl chloroformate and 6-bromohexanol) was dissolved in dichloromethane (300 mL). S6-2 (9.12 g, 40.0 mmol) was added dropwise under stirring at room temperature, followed by slow dropwise addition of pyridine (1.00 mL, 12.5 mmol) over 10 min, and then DMAP (0.24 g, 2.0 mmol) was added all at once. The reaction was stirred at room temperature for 16 h. After completion of the reaction, the mixture was extracted twice with dichloromethane. The organic phases were combined and washed with brine, then dried over anhydrous magnesium sulfate, filtered, and concentrated to give a crude product. The crude product was separated and purified by silica gel column, and the target eluate was collected and concentrated to give S10-2 (1.21 g).

[0377] Step b: Under nitrogen, compound S1-6 (0.18 g, 2.0 mmol) was dissolved in acetonitrile (50 mL). S10-2 (1.06 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring. The mixture was stirred at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to afford compound S10-3 (0.72 g).

[0378] Step c: Under nitrogen protection, compound S10-3 (0.44 g, 1.0 mmol) was dissolved in acetonitrile (20 mL), and S2-2 (0.52 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) were added sequentially with slow stirring. The mixture was stirred at room temperature for about 20 h. After the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E10-1 (0.64 g). The main data of the H NMR spectrum of E10-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.71-4.68 (m, 1H), 4.21 (t, 2H), 4.03 (t, 2H), 3.68-3.60 (t, 2H), 2.55-2.46 (m, 10H), 1.75-1.25 (m, 64H), 0.89 (t, 12H). MALDI-TOF analysis determined the molecular weight of E10-1 to be 782.72 Da.

[0379]

[0380] Example 11: Cationic lipid (E11-1)

[0381]

[0382] Corresponding to the general formula (1), in E11-1, R1 is R2 is B1 is a pentylene group, B2 is a hexylene group, L1 is an ester group (-OC(=O)-), L2 is an ester group (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 869 Da.

[0383] The preparation process is as follows:

[0384] Step a: 1,3-propylene glycol containing one TBS-protected hydroxyl group (S11-1, 9.50 g, 50 mmol) was dissolved in 400 mL of dichloromethane solution, and pyridinium chlorochromate (PCC, 16.13 g, 75.0 mmol) was added. After stirring at 15°C for at least 2 hours, the mixture was filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain 3-hydroxypropionic acid (S11-2, 6.02 g) with a TBS-protected hydroxyl group.

[0385] Step b: Dissolve the above compound S11-2 (5.64 g, 30.0 mmol) and 1-octanol (S11-3, 9.75 g, 75.0 mmol) in 200 mL of dichloromethane. Add p-toluenesulfonic acid monohydrate (TsOH·H2O, 1.14 g, 6.0 mmol) and anhydrous sodium sulfate (10.65 g, 75.0 mmol). Stir at 15°C for at least 24 hours, filter, and concentrate under reduced pressure. The crude product is purified by column chromatography to obtain the TBS-protected hydroxyl acetal (S11-4, 2.84 g).

[0386] Step c: Dissolve the above-mentioned product S11-4 (2.16 g, 5.0 mmol) in THF (50 mL) in a nitrogen-protected flask. Add tetrabutylammonium fluoride solution (TBAF, 50 mL, 1 M) and react overnight to remove the TBS protection. Dry over anhydrous sodium sulfate, filter, and concentrate the filtrate to obtain crude compound S11-5. Purify by column chromatography, concentrate, and pump dry to obtain the acetal S11-5 (1.40 g, 88.6%) containing exposed hydroxyl groups.

[0387] Step d: Under argon atmosphere, DCC (0.91 g, 4.4 mmol) was added to a round-bottom flask containing S11-5 (0.76 g, 2.4 mmol), S11-6 (0.39 g, 2.0 mmol) and DMAP (61.00 mg, 0.5 mmol) dissolved in dichloromethane (100 mL) in an argon atmosphere. The mixture was reacted at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, the filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography to give the bromoester S11-7 (0.81 g).

[0388] Step e: Under nitrogen, compound S1-6 (0.09 g, 1.0 mmol) was dissolved in acetonitrile (20 mL). S11-7 (0.62 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) were added sequentially with slow stirring. The mixture was stirred at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to afford compound S11-8 (0.42 g).

[0389] Step f: Under nitrogen protection, compound 11-8 (0.30 g, 0.6 mmol) was dissolved in acetonitrile (20 mL), and S1-5 (0.34 g, 0.8 mmol) and DIPEA (0.05 g, 0.6 mmol) were added sequentially with slow stirring and the reaction was stirred at room temperature for about 20 h. After the reaction, the reaction solution was concentrated and dissolved with dichloromethane, extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E11-1 (0.43 g). The main data of the H NMR spectrum of E11-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.64 (t, 1H), 4.06 (t, 4H), 3.64-3.61 (m, 4H), 3.52-3.36 (m, 4H), 3.02-2.81 (m, 10H), 2.32 (t, 4H), 1.80-1.21 (m, 64H), 0.87 (t, 12H). MALDI-TOF analysis determined the molecular weight of E11-1 to be 868.79 Da.

[0390]

[0391] Example 12: Cationic lipid (E12-1)

[0392]

[0393] Corresponding to the general formula (1), in E12-1, R1 is R2 is B1 and B2 are both heptylene, B2 is hexylene, L1 is an ester group (-OC(=O)-), L2 is an ester group (-C(=O)O-), X is N, L3 is a butylene group, R3 is a hydroxyl group, and the total molecular weight is about 855 Da.

[0394] The preparation process is as follows:

[0395] Step a: Under argon atmosphere, glycerol containing one TBS-protected hydroxyl group (S12-1, 3.09 g, 15.0 mmol), K2CO3 (6.21 g, 45.0 mmol), and bromohexane (S12-2, 2.71 g, 16.5 mmol) were dissolved in 100 mL of DMF. The mixture was stirred at 110°C for 16 h. After the reaction was confirmed to be complete by thin-layer chromatography, the reaction solution was poured into water (100 mL) for precipitation, filtered, and further separated and purified by column chromatography to obtain glycerol etherate S12-3 (3.35 g, 89.3%) with TBS-protected hydroxyl group.

[0396] Step b: Dissolve the above-mentioned product S12-3 (1.88 g, 5.0 mmol) in THF (50 mL) in a nitrogen-protected flask. Add tetrabutylammonium fluoride solution (TBAF, 50 mL, 1 M) and react overnight to remove the TBS protection. Dry over anhydrous sodium sulfate, filter, and concentrate the filtrate to obtain crude compound S12-4. Purify by column chromatography, concentrate, and pump dry to obtain the hydroxyl-containing glycerol ether S12-4 (1.14 g, 87.9%).

[0397] Step c: Under argon atmosphere, DCC (0.91 g, 4.4 mmol) was added to a round-bottom flask containing S12-4 (0.62 g, 2.4 mmol), 8-bromooctanoic acid (S12-5, 0.45 g, 2.0 mmol) and DMAP (61.00 mg, 0.5 mmol) dissolved in dichloromethane (50 mL) in argon atmosphere. The mixture was reacted at room temperature for 16 h. After the reaction, the precipitate was removed by filtration, the filtrate was concentrated, and the resulting residue was purified by silica gel column chromatography to give the bromoester S12-6 (0.76 g).

[0398] Step d: Under nitrogen, compound S1-6 (0.09 g, 1.0 mmol) was dissolved in acetonitrile (20 mL). S12-6 (0.58 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) were added sequentially with slow stirring. The mixture was stirred at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to afford compound S12-7 (0.39 g).

[0399] Step e: Under nitrogen protection, compound S12-7 (0.28 g, 0.6 mmol) was dissolved in acetonitrile (20 mL), and S4-2 (0.37 g, 0.8 mmol) and DIPEA (0.05 g, 0.6 mmol) were added sequentially with slow stirring, and the reaction was stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved in dichloromethane, and extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E12-1 (0.42 g). The main data of the H NMR spectrum of E12-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 5.15-5.07 (m, 1H), 4.03 (t, 2H), 3.70 (t, 2H), 3.58-3.50 (m, 4H), 3.48-3.36 (m, 4H), 3.22-2.91 (m, 10H), 2.35-2.28 (m, 2H), 1.96-1.47 (m, 20H), 1.38-1.23 (m, 44H), 0.87 (t, 12H). MALDI-TOF analysis determined the molecular weight of E12-1 to be 854.58 Da.

[0400]

[0401] Example 13: Cationic lipid (E13-1)

[0402]

[0403] Corresponding to the general formula (1), in E13-1, R1 is R2 is B1 and B2 are both hexylene, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is butylene, and R3 is The total molecular weight is approximately 794 Da.

[0404] The preparation process is as follows:

[0405] Step a: Under nitrogen, 4-dimethylaminobutylamine (S13-1, 0.09 g, 1.0 mmol) was dissolved in acetonitrile (50 mL). S5-2 (2.10 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were added sequentially with slow stirring. The mixture was stirred at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to afford compound S13-2 (1.51 g).

[0406] Step b: Under nitrogen protection, compound S13-2 (0.91 g, 2.0 mmol) was dissolved in acetonitrile (30 mL). S2-2 (1.05 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring and the mixture was stirred at room temperature for about 20 h. After the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E13-1 (1.28 g). The main data of the H NMR spectrum of E13-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.07-4.01 (m, 4H), 3.22-3.08 (m, 4H), 2.92-2.76 (m, 8H), 2.33-2.26 (m, 1H), 2.23 (s, 6H), 1.83-1.22 (m, 64H), 0.87 (t, 12H). MALDI-TOF analysis determined the molecular weight of E13-1 to be 793.74 Da.

[0407]

[0408] Example 14: Cationic lipid (E14-1)

[0409]

[0410] Corresponding to the general formula (1), in E14-1, R1 is R2 is B1 and B2 are both heptylene, L1 is an ester group (-OC(=O)-), L2 is an ester group (-C(=O)O-), X is N, L3 is a butylene group, and R3 is The total molecular weight is approximately 882 Da.

[0411] Referring to the preparation process of E13-1, cationic lipid E14-1 (1.43 g) was obtained using S13-1, S12-6, and S4-2 as raw materials in the same molar amounts. The main data of the H-NMR spectrum of E14-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 5.12-5.08 (m, 1H), 4.04 (t, 2H), 3.58-3.52 (m, 4H), 3.48-3.36 (m, 4H), 3.23-2.92 (m, 12H), 2.32-2.28 (m, 2H), 2.23 (s, 6H), 1.96-1.22 (m, 64H), 0.87 (t, 12H). MALDI-TOF analysis determined the molecular weight of E14-1 to be 881.82 Da.

[0412]

[0413] Example 15: Cationic lipid (E15-1)

[0414]

[0415] Corresponding to the general formula (1), in E15-1, R1 is R2 is B1 and B2 are both hexylene, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is butylene, and R3 is The total molecular weight is approximately 822 Da.

[0416] Referring to the preparation process of E13-1, cationic lipid E15-1 (1.33 g) was obtained using S13-1, S5-2, and S1-5 as starting materials in equal molar amounts. The main H NMR data of E15-1 are as follows: 4.06 (t, 2H), 4.01 (t, 2H), 3.63 (t, 2H), 3.20 (t, 2H), 3.02-2.81 (m, 12H), 2.26 (t, 1H), 2.20 (s, 6H), 1.92-1.21 (m, 64H), 0.83 (t, 12H). MALDI-TOF analysis determined the molecular weight of E15-1 to be 821.77 Da.

[0417]

[0418] Example 16: Cationic lipid (E16-1)

[0419]

[0420] Corresponding to the general formula (1), in E16-1, R1 is undecyl, R2 is B1 is pentylene, B2 is heptylene, L1 is an ester group (-OC(=O)-), L2 is an ester group (-C(=O)O-), X is N, L3 is butylene, and R3 is The total molecular weight is approximately 766 Da.

[0421] The preparation process is as follows:

[0422] Step a: Dissolve S3-1 (2.89 g, 12.0 mmol) in dichloromethane (60 mL), then add 7-bromoheptyl-N-succinimidyl carbonate (S16-1, 3.35 g, 10.0 mmol) and TEA (1.10 mL, 15.0 mmol) in the resulting mixture. Stir and react overnight at room temperature. After completion of the reaction, concentrate the reaction mixture to obtain the crude product. Purify the product by column chromatography, concentrate, and pump dry to obtain the bromoester S16-2 (3.65 g).

[0423] Step b: Under nitrogen, compound S13-1 (0.46 g, 4.0 mmol) was dissolved in acetonitrile (50 mL). S16-2 (2.31 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were added sequentially with slow stirring. The mixture was stirred at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to afford compound S16-3 (1.62 g).

[0424] Step c: Under nitrogen protection, compound S16-3 (1.00 g, 2.0 mmol) was dissolved in acetonitrile (30 mL), and 6-bromohexanoic acid undecyl ester (S16-4, 0.87 g, 2.5 mmol, wherein S16-4 is prepared by reacting 6-bromohexanoic acid and undecyl alcohol) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring and stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved in dichloromethane, and extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E16-1 (1.26 g). The main data of the H NMR spectrum of E16-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.03 (t, 4H), 3.22-3.09 (m, 4H), 2.90-2.79 (m, 8H), 2.30 (t, 2H), 2.23 (s, 6H), 1.76-1.19 (m, 62H), 0.87 (t, 9H). MALDI-TOF analysis determined the molecular weight of E16-1 to be 765.74 Da.

[0425]

[0426] Example 17: Cationic lipid (E17-1)

[0427]

[0428] Corresponding to the general formula (1), in E17-1, R1 is R2 is B1 and B2 are both hexamethylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is -CH2CH2OCH2CH2-, R3 is a hydroxyl group, and the total molecular weight is about 811 Da.

[0429] The preparation process is as follows:

[0430] Step a: Under nitrogen, diglycolamine (S17-1, 0.42 g, 4.0 mmol) was dissolved in acetonitrile (50 mL). S5-2 (2.10 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were added sequentially with slow stirring. The mixture was stirred at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to afford compound S16-3 (1.44 g).

[0431] Step b: Under nitrogen protection, compound S16-3 (0.89 g, 2.0 mmol) was dissolved in acetonitrile (30 mL), and S1-5 (0.87 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring, and the mixture was stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved in dichloromethane, and extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E17-1 (1.33 g). The main data of the H NMR spectrum of E17-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.06 (t, 2H), 4.00 (t, 2H), 3.70 (t, 2H), 3.65-3.63 (m, 6H), 3.20 (t, 2H), 3.02-2.81 (m, 8H), 2.65 (t, 2H), 2.25 (t, 1H), 1.80-1.19 (m, 60H), 0.83 (t, 12H). MALDI-TOF analysis determined the molecular weight of E17-1 to be 810.74 Da.

[0432]

[0433] Example 18: Cationic lipid (E18-1)

[0434]

[0435] Corresponding to the general formula (1), in E18-1, R1 is undecyl, R2 is B1 is a pentylene group, B2 is a butylene group, L1 is an ester group (-OC(=O)-), L2 is an ester group (-C(=O)O-), X is N, L3 is -CH2CH2OCH2CH2-, R3 is a hydroxyl group, and the total molecular weight is about 755 Da.

[0436] Referring to the preparation process of E13-1, cationic lipid E18-1 (1.24 g) was obtained using S16-2, S17-1, and S16-4 as raw materials in equal molar amounts. The main data of the H NMR spectrum of E18-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.03 (t, 4H), 3.71 (t, 2H), 3.63 (t, 4H), 3.22-2.81 (m, 8H), 2.65 (t, 2H), 2.30 (t, 2H), 1.77-1.19 (m, 58H), 0.87 (t, 9H). MALDI-TOF analysis determined the molecular weight of E18-1 to be 754.64 Da.

[0437]

[0438] Example 19: Cationic lipid (E19-1)

[0439]

[0440] Corresponding to the general formula (1), in E19-1, R1 is undecyl, R2 is B1 is a pentylene group, B2 is a heptylene group, L1 is an ester group (-OC(=O)-), L2 is an ester group (-C(=O)O-), X is N, L3 is an ethylene group, R3 is a hydroxyl group, and the total molecular weight is about 711 Da.

[0441] Referring to the preparation process of E13-1, cationic lipid E19-1 (1.15 g) was obtained using S16-2, S17-1, and S16-4 as raw materials in equal molar amounts. The main data of the H NMR spectrum of E19-1 are as follows: 1H NMR (400 MHz, CDCl3) δ: 4.03 (t, 4H), 3.86-3.78 (m, 2H), 3.22-3.09 (m, 4H), 2.98-2.81 (m, 6H), 2.30 (t, 2H), 1.79-1.20 (m, 58H), 0.88 (t, 9H). MALDI-TOF analysis determined the molecular weight of E19-1 to be 710.70 Da.

[0442]

[0443] Example 20: Cationic lipid (E20-1)

[0444]

[0445] Corresponding to the general formula (1), in E20-1, R1 is undecyl, R2 is B1 is a pentylene group, B2 is a heptylene group, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is an ethylene group, R3 is a hydroxyl group, and the total molecular weight is about 711 Da.

[0446] The preparation process is as follows:

[0447] Under nitrogen protection, compound S16-3 (0.89 g, 2.0 mmol) was dissolved in acetonitrile (50 mL), and 5-bromopentyl laurate (S20-1, 0.87 g, 2.5 mmol, wherein S20-1 is prepared by reacting lauric acid and 5-bromo-1-pentanol) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring, and the reaction was stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved with dichloromethane, and extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E20-1 (1.11 g). The main data of the H NMR spectrum of E20-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.03 (t, 4H), 3.86-3.78 (m, 2H), 3.22-3.09 (m, 4H), 2.98-2.83 (m, 6H), 2.32 (t, 2H), 1.76-1.22 (m, 58H), 0.86 (t, 9H). MALDI-TOF analysis determined the molecular weight of E20-1 to be 710.92 Da.

[0448]

[0449] Example 21: Cationic lipid (E21-1)

[0450]

[0451] Corresponding to the general formula (1), in E21-1, R1 is undecyl, R2 is B1 is a pentylene group, B2 is a heptylene group, L1 is a carbonate group (-OC(=O)O-), L2 is an ester group (-C(=O)O-), X is N, L3 is an ethylene group, R3 is a hydroxyl group, and the total molecular weight is about 727 Da.

[0452] The preparation process is as follows:

[0453] Step a: Under the protection of nitrogen, S10-1 (4.14 g, 12.0 mmol) was dissolved in dichloromethane (200 mL), and 1-undecanol (S21-1, 8.26 g, 48.0 mmol) was added dropwise under stirring at room temperature, followed by slow addition of pyridine (1.00 mL, 15.0 mmol) over 10 min, and then DMAP (0.29 g, 2.4 mmol) was added at one time. The reaction was stirred at room temperature for 16 h. After completion of the reaction, the mixture was extracted twice with dichloromethane, the organic phases were combined and washed with salt water, then dried over anhydrous magnesium sulfate, filtered and concentrated to give a crude product. Purified by silica gel column separation, concentrated to give 6-bromohexyl undecyl carbonate (S21-2, 1.18 g).

[0454] Step b: Under nitrogen, compound S1-7 (0.12 g, 2.0 mmol) was dissolved in acetonitrile (30 mL). S21-2 (1.08 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring. The mixture was stirred at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to afford compound S21-3 (0.60 g).

[0455] Step c: Under nitrogen protection, compound S21-3 (0.36 g, 1.0 mmol) was dissolved in acetonitrile (20 mL), and S16-2 (0.58 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) were added sequentially with slow stirring and the reaction was stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved with dichloromethane, and extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E21-1 (0.59 g). The main data of the H NMR spectrum of E21-1 are as follows: 1HNMR (400 MHz, CDCl3) δ: 4.19 (t, 4H), 4.03 (t, 2H), 3.86-3.78 (m, 2H), 3.22-3.09 (m, 4H), 2.96-2.81 (m, 6H), 1.76-1.23 (m, 58H), 0.87 (t, 9H). MALDI-TOF analysis determined the molecular weight of E21-1 to be 726.63 Da.

[0456]

[0457] Example 22: Cationic lipid (E22-1)

[0458]

[0459] Corresponding to the general formula (1), in E22-1, R1 is undecyl, R2 is B1 is a pentylene group, B2 is a heptylene group, L1 is an ester group (-OC(=O)-), L2 is an ester group (-C(=O)O-), X is N, L3 is an ethylene group, R3 is a hydroxyl group, and the total molecular weight is about 739 Da.

[0460] The preparation process is as follows:

[0461] Step a: Under nitrogen protection, compound S1-7 (0.12 g, 2.0 mmol) was dissolved in acetonitrile (30 mL), and S22-1 (1.23 g, 2.5 mmol, wherein S22-1 is prepared by 7-bromo-n-heptanol and The obtained compound was prepared by reaction. The specific experimental steps refer to Example 1.1, step b) and DIPEA (0.18 g, 2.0 mmol). The reaction was stirred at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound S22-2 (0.78 g).

[0462] Step b: Under nitrogen protection, compound S22-2 (0.47 g, 1.0 mmol) was dissolved in acetonitrile (20 mL), and S16-4 (0.44 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) were added sequentially with slow stirring. The mixture was stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E22-1 (0.59 g). The main data of the H NMR spectrum of E22-2 are as follows: 1H NMR (400 MHz, CDCl3) δ: 4.03 (t, 4H), 3.86-3.78 (m, 2H), 3.63 (t, 2H), 3.22-3.09 (m, 4H), 2.99-2.81 (m, 6H), 2.30 (t, 4H), 1.81-1.19 (m, 58H), 0.88 (t, 9H). MALDI-TOF analysis determined the molecular weight of E22-1 to be 738.65 Da.

[0463]

[0464] Example 23: Cationic lipid (E23-1)

[0465]

[0466] Corresponding to the general formula (1), in E23-1, R1 is a nonyl group, and R2 is B1 and B2 are both heptylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is ethylene, R3 is hydroxyl, and the total molecular weight is about 739 Da.

[0467] The preparation process is as follows:

[0468] Under nitrogen protection, compound S22-2 (0.94 g, 2.0 mmol) was dissolved in acetonitrile (20 mL), and S23-1 (0.87 g, 2.5 mmol, wherein S23-1 was prepared by reacting 7-bromo-n-heptanol with n-decanoic acid, the specific experimental steps refer to Example 1.1 step b) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring and the reaction was stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved with dichloromethane, and extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E23-1 (1.21 g). The main data of the H NMR spectrum of E23-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.03 (t, 4H), 3.86-3.76 (m, 2H), 3.64 (t, 2H), 3.22-3.09 (m, 4H), 2.98-2.81 (m, 6H), 2.31 (t, 4H), 1.75-1.21 (m, 58H), 0.87 (t, 9H). MALDI-TOF analysis determined the molecular weight of E23-1 to be 738.69 Da.

[0469]

[0470] Example 24: Cationic lipid (E24-1)

[0471]

[0472] Corresponding to the general formula (1), in E24-1, R1 is octyl, R2 is B1 and B2 are both heptylene groups, L1 is a carbonate group (-OC(=O)O-), L2 is an ester group (-C(=O)O-), X is N, L3 is ethylene, R3 is a hydroxyl group, and the total molecular weight is about 741 Da.

[0473] The preparation process is as follows:

[0474] Under nitrogen protection, compound S22-2 (0.94 g, 2.0 mmol) was dissolved in acetonitrile (20 mL), and S24-1 (0.88 g, 2.5 mmol, wherein S24-1 was prepared by reacting 7-bromoheptyl-4-nitrophenyl carbonate with octanol, the specific experimental steps refer to Example 10 step a) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring and the reaction was stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved with dichloromethane, and extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E24-1 (1.22 g). The main data of the H NMR spectrum of E24-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.19 (t, 4H), 4.03 (t, 2H), 3.85-3.78 (m, 2H), 3.63 (t, 2H), 3.22-3.09 (m, 4H), 2.98-2.83 (m, 6H), 2.30 (t, 2H), 1.79-1.19 (m, 56H), 0.87 (t, 9H). MALDI-TOF analysis determined the molecular weight of E24-1 to be 740.68 Da.

[0475]

[0476] Example 25: Cationic lipid (E25-1)

[0477]

[0478] Corresponding to the general formula (1), in E25-1, R1 and R2 are B1 and B2 are both hexamethylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, and L3 is R3 is a hydroxyl group, and the total molecular weight is about 908 Da.

[0479] The preparation process is as follows:

[0480] Under nitrogen protection, compound 2-(4-(2-aminoethyl)piperazin-1-yl)ethanol (S25-1, 0.35 g, 2.0 mmol) was dissolved in acetonitrile (100 mL), and S1-5 (2.24 g, 5.0 mmol) and DIPE A (0.18 g, 2.0 mmol) were added sequentially with slow stirring and stirred at room temperature for about 20 hours. After the reaction, the reaction solution was concentrated and dissolved with dichloromethane, extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence, and the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E25-1 (1.48 g). The main data of the H NMR spectrum of E25-1 are as follows: 1 HNMR (400 MHz, CDCl3) δ: 4.03 (t, 4H), 3.71 (t, 2H), 3.63 (t, 4H), 3.12-2.49 (m, 26H), 2.31 (t, 4H), 1.78-1.19 (m, 56H), 0.87 (t, 12H). MALDI-TOF analysis determined the molecular weight of E25-1 to be 907.83 Da.

[0481]

[0482] Example 26: Cationic lipid (E26-1)

[0483]

[0484] Corresponding to the general formula (1), in E26-1, R1 is R2 is B1 and B2 are hexamethylene groups, L1 is a carbonate group (-OC(=O)O-), L2 is an ester group (-C(=O)O-), X is N, and L3 is R3 is a hydroxyl group, and the total molecular weight is about 867 Da.

[0485] The preparation process is as follows:

[0486] Step a: Under nitrogen, compound S25-1 (0.69 g, 4.0 mmol) was dissolved in acetonitrile (50 mL). S10-2 (2.17 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were added sequentially with slow stirring. The mixture was stirred at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to afford compound S26-1 (1.67 g).

[0487] Step b: Under nitrogen protection, compound S26-1 (1.06 g, 2.0 mmol) was dissolved in acetonitrile (30 mL). S2-2 (1.05 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring and the mixture was stirred at room temperature for about 20 h. After the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E26-1 (1.39 g). The main data of the H NMR spectrum of E26-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.71-4.68 (m, 1H), 4.21 (t, 2H), 4.03 (t, 2H), 3.71 (t, 2H), 3.12-2.49 (m, 18H), 2.55-2.46 (m, 4H), 1.75-1.25 (m, 60H), 0.89 (t, 12H). MALDI-TOF analysis determined the molecular weight of E26-1 to be 866.75 Da.

[0488]

[0489] Example 27: Cationic lipid (E27-1)

[0490]

[0491] Corresponding to the general formula (1), in E27-1, R1 is R2 is B1 and B2 are both hexylene groups, L1 and L2 are both ester groups (-C(=O)O-), X is N, L3 is propylene group, R3 is azido group, and the total molecular weight is about 778 Da.

[0492] The preparation process is as follows:

[0493] Step a: Under nitrogen, 3-azidopropylamine (S27-1, 0.40 g, 4.0 mmol) was dissolved in acetonitrile (50 mL). S5-2 (2.09 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were added sequentially with slow stirring. The mixture was stirred at room temperature for approximately 20 h. After completion of the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted sequentially with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to afford compound S27-2 (1.41 g).

[0494] Step b: Under nitrogen protection, compound S27-2 (0.88 g, 2.0 mmol) was dissolved in acetonitrile (30 mL). S2-2 (1.05 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were added sequentially with slow stirring and the mixture was stirred at room temperature for about 20 h. After the reaction, the reaction solution was concentrated and dissolved in dichloromethane. The mixture was extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E27-1 (1.23 g). The main data of the H NMR spectrum of E27-1 are as follows: 1 H NMR (400 MHz, CDCl3) δ: 4.06 (t, 2H), 4.01 (t, 2H), 3.24-3.06 (m, 4H), 2.90-2.59 (m, 6H), 2.25 (t, 1H), 1.81-1.22 (m, 64H), 0.85 (t, 12H). MALDI-TOF analysis determined the molecular weight of E27-1 to be 777.72 Da.

[0495]

[0496] Example 28: Preparation of cationic liposome nucleic acid pharmaceutical composition

[0497] Table 1 - Formulations of various liposomes and physicochemical properties of liposome pharmaceutical compositions

[0498]

[0499] In this example, multiple groups of cationic liposomes were prepared for comparison. The neutral lipids contained in the composition of each group of cationic liposomes were all DSPC, the sterol lipids contained were all cholesterol, and the PEGylated lipids contained were all PEG2k-DMG (DMG for short). The only difference was the cationic lipids. Among them, control group 1: the cationic lipid was ALC-0315, prepared with reference to the method disclosed in document CN108368028A; control group 2: the cationic lipid was SM102, prepared with reference to the method disclosed in document CN110520409A; experimental group series (L-1 to L-31): the cationic lipids were the cationic lipids prepared in the examples of this application, as shown in Table 1.

[0500] Preparation of a cationic liposome nucleic acid pharmaceutical composition (LNP-mRNA): The cationic lipids, DSPC, cholesterol, and PEGylated lipids listed in Table 1 were dissolved in ethanol at appropriate molar ratios to obtain an ethanol phase solution; Fluc-mRNA was added to 50 mM citrate buffer (pH = 4) at an N / P ratio of 6:1 to obtain an aqueous phase solution; the aforementioned ethanol phase solution and the aqueous phase solution were mixed at a volume ratio of 1:3, and washed with DPBS ultrafiltration multiple times to remove ethanol and free molecules, and finally filtered through a 0.2 μm sterile filter to obtain a cationic liposome nucleic acid pharmaceutical composition.

[0501] Example 29: Physical and Chemical Property Testing of Cationic Liposome Nucleic Acid Pharmaceutical Compositions

[0502] Encapsulation efficiency determination: In this example, the encapsulation efficiency of cationic liposomes was determined using the Quant-it Ribogreen RNA quantitative assay kit. The results showed that the cationic liposomes of the present invention had a high encapsulation efficiency for nucleic acid drugs (mRNA), all within the range of 80%-95%, with most encapsulation efficiencies within the range of 85%-95%, as shown in Table 1. The results showed that the cationic lipids containing multiple nitrogen branches in the present application had an encapsulation efficiency higher or lower than that of the control group, and the encapsulation efficiency of lipid compounds with tertiary amines as nitrogen branches to introduce hydrophobic fatty tails was lower, for example, L-1, L-2, L-3, and L-12 all had relatively low encapsulation efficiencies, while the encapsulation efficiency of cationic lipids with amines in carbamate bonds as nitrogen branches to introduce hydrophobic fatty tails was higher, and the encapsulation effect of cationic lipids with a hydrophobic fatty tail introduced by an amine in a carbamate bond as a nitrogen branch at one end and a hydrophobic tail introduced by a carbon branch at the other end was even better, such as L-8, L-9, L-10, and L-16.

[0503] Particle diameter determination: In the present embodiment, the particle diameter of LNP-mRNA is measured by dynamic light scattering (DLS). The cationic liposome size uniformity measured is higher, and its PDI is all less than 0.3. The particle diameter of the cationic liposome prepared by the lipid composition of the application is within the range of 90-120nm, as specifically shown in Table 1.

[0504] Example 30: Biological Activity Test of Cationic Liposome Nucleic Acid Pharmaceutical Composition

[0505] (1) Cytotoxicity (biocompatibility) research

[0506] The cytotoxicity of the cationic liposome nucleic acid drug composition of the present invention was tested by MTT staining. The cationic liposome nucleic acid drug was dissolved in culture medium to prepare the required dose. 293T cells were used as a cell model at a seeding density of 4×10 4Cells / well, 100 μL / well of cell suspension was inoculated into a 96-well plate. After inoculation, the cells were incubated in a cell culture incubator for 24 hours, and then administered at a dose of 0.2 ug mRNA per well. The blank control group was added with a corresponding volume of fresh culture medium, with 3 replicates per group. After the composition preparation was incubated with 293T cells for 24 hours, 20 μL of PBS buffer of 5 mg / mL MTT was added to each well. After incubation with 293T cells for 4 hours, the mixture of culture medium and MTT buffer was discarded, 150 μL / well of DMSO was added, and after sufficient vibration, the absorbance was tested with a microplate reader. Calculation was performed based on the measured absorbance value. The results showed that, compared with the blank control group, the cell viability of the cationic liposome nucleic acid pharmaceutical composition prepared by the present invention was greater than 95%, indicating that the cationic liposome nucleic acid pharmaceutical composition of the present invention has good biocompatibility.

[0507] (2) Study on mRNA transfection rate at the cellular level

[0508] In order to investigate the mRNA transfection efficiency of some cationic liposome pharmaceutical compositions (L-CT1, L-CT2, L-1, L-8, L-9, L-10, L-11, L-12, L-16, L-22, and L-23) prepared in Example 28 of the present invention at the cellular level, Luciferase bioluminescence was used to test. The cationic liposome nucleic acid pharmaceutical preparations were dissolved in culture medium to prepare the required dose. 293T cells were used as a cell model at a seeding density of 4×10 4Cells / well, 100 μL / well of the cell suspension was inoculated into a 96-well plate with a black-edged transparent bottom. 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 the corresponding dose of free Fluc-mRNA. Each group had 3 replicates of each concentration. After 24 hours of transfection, the old culture medium was removed and replaced with a new culture medium containing D-fluorescein sodium (1.5 mg / mL) substrate. After incubation for 5 minutes, bioluminescence was detected using a microplate reader. The stronger the fluorescence, the more Fluc-mRNA was transported into the cytoplasm and translated into the corresponding fluorescent protein. The results are shown in Table 2, where the relative value of fluorescence intensity is the ratio of the fluorescence intensity value of each group to the fluorescence intensity value of the blank control group. The results show that compared with the blank group, the cationic liposome nucleic acid drug composition prepared by the present invention has an excellent in vitro transfection effect, and the transfection rate of most cationic liposome nucleic acid drug compositions is higher than that of the control group, which further shows that the more tertiary amines in the cationic lipids, the better. That is, the ability to ionize more positive charges does not necessarily result in better encapsulation efficiency and transfection efficiency. The position of the ionizable tertiary amine structure is particularly important for the overall performance of the cationic lipid. Cationic lipids with tertiary amines at the polar head of the short chain (e.g., L-8, L-9, L-10, L-11, L-16, L-23) rather than the hydrophobic long tail chain (e.g., L-1, L-12, L-26) are more conducive to the formation of cationic liposomes, can better encapsulate nucleic acid drugs, and also help nucleic acid drugs to be released from endosomes to the cytoplasm to exert their effects, thereby showing higher encapsulation efficiency and cell transfection efficiency.

[0509] Table 2 - Relative fluorescence values ​​of cell transfection

[0510]

[0511] The foregoing is only an embodiment of the present invention and does not limit the scope of the patent of the present invention. Any equivalent structure or equivalent process transformation made by utilizing the contents of the present invention specification, or directly or indirectly applied in other related technical fields, is similarly included in the scope of patent protection of the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without the need to perform unnecessary experiments, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that are out of the scope disclosed in this application and made with conventional techniques known in the art.

Claims

1. A cationic lipid, characterized in that The structure is shown in the general formula (1): Where X is N; L1 is -C(=O)O-, L2 is -OC(=O)- or -C(=O)O-; L3 is -(CH2) t -, t in L3 is an integer from 1 to 12; B1 and B2 are each independently any one of propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, and dodecylene; R1 is R2 is C 1-30 an aliphatic hydrocarbon group; and for Any one of the following, wherein t is 0; said C 1-30 The aliphatic hydrocarbon group is selected from any one of the following structures: Wherein, t is an integer from 0 to 12; R3 is -(CH2) t OH, -(CH2) t N(CH3)2 or -(CH2) t N(CH2CH3)2; t in R3 is an integer of 0-12.

2. The cationic lipid according to claim 1, wherein The B1 and B2 are each independently any one of pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene and dodecylene.

3. A cationic lipid, characterized in that The structure is shown in the general formula (1): Where X is N; L1 is -C(=O)O-, L2 is -C(=O)O-; L3 is -(CH2) t -, t in L3 is an integer from 1 to 12; B1 and B2 are each independently any one of propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, and dodecylene; R1 is R2 is and for Any of the following, where t is 0; R3 is -(CH2) t OH, -(CH2) t N(CH3)2 or -(CH2) t N(CH2CH3)2; t in R3 is an integer of 0-12.

4. The cationic lipid according to claim 1 or 3, wherein Its structure is selected from any one of the following structures:

5. A cationic lipid, characterized in that The structure is or 6. A cationic liposome, characterized in that The method comprises the cationic lipid according to any one of claims 1 to 5.

7. The cationic liposome according to claim 6, wherein It also contains one or more of neutral lipids, steroid lipids and PEGylated lipids; wherein the neutral lipids are phospholipids.

8. The cationic liposome according to claim 7, wherein It also contains three types of lipids: neutral lipids, steroid lipids and PEGylated lipids.

9. The cationic liposome according to claim 7, wherein The neutral lipid is selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-decanoyl-sn-glycero-3-phosphocholine, Octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diamidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn -glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-bisdocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dioleoylphosphatidylcholine Serine, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine, and combinations thereof.

10. The cationic liposome according to claim 7, characterized in that The steroid lipid is selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and mixtures thereof.

11. The cationic liposome according to claim 7, wherein The PEGylated lipid is selected from polyethylene glycol-1,2 dimyristin, polyethylene glycol-distearyl phosphatidylethanolamine, PEG-cholesterol, polyethylene glycol-diacylglycerol, polyethylene glycol-dialkoxypropyl, specifically including any one of polyethylene glycol 500-dipalmitoylphosphatidylcholine, polyethylene glycol 2000-dipalmitoylphosphatidylcholine, polyethylene glycol 500-stearoylphosphatidylethanolamine, polyethylene glycol 2000-distearoylphosphatidylethanolamine, polyethylene glycol 500-1,2-dioleoylphosphatidylethanolamine, polyethylene glycol 2000-1,2-dioleoylphosphatidylethanolamine and polyethylene glycol 2000-2,3-dimyristoylglycerol.

12. The cationic liposome according to any one of claims 7 to 11, characterized in that The invention comprises 20-80% cationic lipids, 5-15% neutral lipids, 25-55% steroid lipids and 0.5-10% PEGylated lipids, wherein the percentages are the molar percentages of each lipid to the total lipids in the solution including the solvent.

13. The cationic liposome according to any one of claims 7 to 11, characterized in that The molar percentage of the cationic lipid in the solution containing the solvent is 30-65% of the total lipids.

14. The cationic liposome according to any one of claims 7 to 11, characterized in that The molar percentage of the cationic lipid in the total lipids in the solution containing the solvent is 5%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, or 55%.

15. The cationic liposome according to any one of claims 7 to 11, characterized in that The molar percentage of the neutral lipids in the solution containing the solvent is 7.5-13% of the total lipids.

16. The cationic liposome according to any one of claims 7 to 11, characterized in that The molar percentage of the neutral lipids in the solution containing the solvent is 8%, 9%, 10%, 11% and 12% of the total lipids.

17. The cationic liposome according to any one of claims 7 to 11, characterized in that The molar percentage of the steroid lipid in the solution containing the solvent is 35-50% of the total lipids.

18. The cationic liposome according to any one of claims 7 to 11, characterized in that The molar percentage of the steroid lipid in the solution comprising a solvent is 0%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of the total lipids.

19. The cationic liposome according to any one of claims 7 to 11, characterized in that The molar percentage of the PEGylated lipid in the solution containing the solvent is 0.5-5% of the total lipids.

20. The cationic liposome according to any one of claims 7 to 11, characterized in that The molar percentage of the PEGylated lipids in the solution containing the solvent is 1-3% of the total lipids.

21. The cationic liposome according to any one of claims 7 to 11, characterized in that The molar percentage of the PEGylated lipid in the total lipid in the solution containing the solvent is 1.5%, 1.6%, 1.7%, 1.8% and 1.9%.

22. A cationic liposome pharmaceutical composition, characterized in that: Contains the cationic liposome according to any one of claims 7 to 21 and a drug, wherein the drug is selected from any one of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs.

23. The cationic liposome pharmaceutical composition according to claim 22, characterized in that The nucleic acid drug is selected from any one of DNA, antisense nucleic acid, plasmid, mRNA, interfering nucleic acid, aptamer, antagomir, miRNA, ribozyme and siRNA.

24. The cationic liposome pharmaceutical composition according to claim 22, characterized in that: The nucleic acid drug is selected from any one of DNA, mRNA, miRNA and siRNA.

25. The cationic liposome pharmaceutical composition according to claim 22, 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.

26. A cationic liposome pharmaceutical composition preparation, characterized in that: A cationic liposome pharmaceutical composition comprising the cationic liposome pharmaceutical composition according to any one of claims 22 to 25 and a pharmaceutically acceptable diluent or excipient, wherein the diluent or excipient is any one of deionized water, ultrapure water, phosphate buffer and physiological saline.

27. The cationic liposome pharmaceutical composition preparation according to claim 26, characterized in that: The diluent or excipient is phosphate buffer or physiological saline.

28. The cationic liposome pharmaceutical composition preparation according to claim 26, characterized in that: The diluent or excipient is physiological saline.

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