A cationic lipid containing a nitrogen heterocycle and its application

Abstract

The present invention provides a novel cationic lipid having a structure shown in general formula (1), specifically relates to a cationic lipid containing a nitrogen heterocycle and having a polar head group derived from the nitrogen heterocycle, also relates to a liposome containing the cationic lipid, a liposome pharmaceutical composition containing the cationic lipid, its preparation and application, and the definitions of each symbol in the formula are as defined herein. A cationic liposome containing the cationic lipid shown in formula (1) according to the present invention can improve the loading rate and transport rate of drugs, especially nucleic acid drugs. A foregoing cationic liposome nucleic acid pharmaceutical composition preparation according to the present invention has good gene complexing ability and high gene transfection ability, can improve the therapeutic and / or prophylactic efficacy of nucleic acid drugs, and provides more alternative cationic lipids for the field of drug delivery. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of drug delivery, and particularly relates to a cationic lipid for a pharmaceutical carrier, and more particularly to a cationic lipid containing a nitrogen heterocycle, a liposome containing the cationic lipid, a liposome nucleic acid drug composition containing the cationic lipid, its preparation and application. Background Art

[0002] Liposomes are widely used for delivering nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs. Especially with the approval of two messenger ribonucleic acid (mRNA) vaccines for vaccination to prevent COVID-19, 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 co-lipids, sterol lipids, and polyethylene glycolated lipids. Among them, cationic lipids interact with negatively charged mRNA through electrostatic interaction. Neutral co-lipids are generally phospholipids, which play a role in preventing lipid oxidation, connecting ligands to the surface of liposomes, or reducing the aggregation of lipid particles. Sterol lipids have strong membrane fusion properties, promoting the intracellular uptake and cytoplasmic entry of mRNA; 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 for preparing LNPs, the most critical one is the ionizable cationic lipid, which is not ionized and has a neutral charge under physiological conditions, and can be ionized to carry a partial positive charge under acidic conditions. For example, when cationic lipids are used as carriers to deliver nucleic acid drugs, at low pH, cationic lipids and nucleic acids (such as mRNA encoding antigens) are combined through electrostatic interaction and encapsulated into LNPs. After LNPs enter cells, nucleic acids (such as mRNA) escape from endosomes and are released into the cytoplasm, and mRNA is further translated into antigen molecules in the cytoplasm, ultimately achieving efficient delivery and transfection of mRNA molecules.

[0003] Although there have been recent advances 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. Document WO2021026358A1 reports that nitrogen-containing lipids can be protonated to carry a positive charge or a partial positive charge at physiological pH. The nitrogen heterocycle of the cationic lipid containing a nitrogen heterocycle disclosed in Document CN102421417B is in the middle part of the entire lipid structure, and a lipophilic long chain is led out by the nitrogen heterocycle. In this application, some novel cationic lipids containing a nitrogen heterocycle and having a polar head led out by the nitrogen heterocycle are designed. Summary of the Invention

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

[0005] The above object of the present invention is achieved by the following technical solutions:

[0006] An embodiment of the present invention:

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

[0008]

[0009] Wherein, X is N;

[0010] represents a divalent nitrogen-containing heterocyclic group, and the nitrogen-containing heterocycle is a heterocycle or a substituted heterocycle in which the ring-forming atoms contain one or more nitrogen atoms; the nitrogen-containing heterocycle is a six-membered ring or a heterocycle with six or more members;

[0011] L1 and L2 are each independently a divalent linking group, selected from -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR c R c ) s O-, -S-, -S-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 -, -NR c C(=O)S-, -C(R c )=N-NR c - and -NR c -N=C(R c )-, where R c is independently a hydrogen atom or a C 1-12 alkyl each time it appears, and s is 2, 3 or 4;

[0012] L3 is a divalent linking group; selected from any one, any two or any combination of two or more of the divalent linking groups of L4, L5, and Z; wherein, the L4 and L5 are carbon chain linking groups, and each is independently -(CR aR b ) t -(CR a R b ) o -(CR a R b ) p -, t, o, p are each independently an integer from 0 to 12, and t, o, p are not simultaneously 0, R a and R b each independently represents a hydrogen atom or a C 1-12 alkyl group each time it appears; Z each time it appears 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-; R c each independently represents H or C 1-12 alkyl group each time it appears;

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

[0014] R1 and R2 are each independently a C 1-30 hydrocarbon group or a C 1-30 hydrocarbon derivative residue;

[0015] R3 is -L 01 -R 01 wherein, L 01 is a divalent linking group selected from -(CR c R c ) m -, -NR c C(=O)-, -C(=O)NR c -, -NR c C(=O)NR c -, -OC(=O)NR c -, -NR c C(=O)O-, -SC(=O)NR c -, -NR c C(=O)S-, -C(R c )=N-NR c-, -NR c -N=C(R c )- or any one of them, or any combination of two or more of them; R 01 is a functional group capable of reacting with a biologically related substance; R c is independently a hydrogen atom or C 1-12 alkyl each time it appears; m is an integer from 1 to 12;

[0016] The alkyl, alkylene, alkoxy, aliphatic hydrocarbon group and aliphatic hydrocarbon derivative residue are each independently substituted or unsubstituted.

[0017] The present invention also provides another embodiment:

[0018] A lipid composition comprising a cationic lipid having a structure as shown in formula (1).

[0019] The present invention also provides another embodiment:

[0020] A lipid composition containing the aforementioned lipid composition and a drug, and the lipid composition contains a cationic lipid having a structure as shown in formula (1).

[0021] The present invention also provides another embodiment:

[0022] A lipid drug composition preparation containing the aforementioned lipid drug composition and a pharmaceutically acceptable diluent or excipient.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The novel cationic lipid compound of the present invention is a novel cationic lipid containing a nitrogen heterocycle and having a polar head group derived from the nitrogen heterocycle, which enriches the types of cationic lipids and provides more choices for the selection of lipid delivery materials. The novel cationic lipid of the present invention has strong gene complexing ability, and the lipid nucleic acid drug composition prepared therefrom shows high gene transfection effect in vivo. 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, etc., so as to improve the therapeutic and / or diagnostic effects of these drugs as prophylactic agents 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 drug composition containing the cationic lipid can have both fluorescence or targeting functions, further improving the gene therapy and / or diagnostic effects of the drug. Embodiment

[0025] Term Explanation

[0026] In the present invention, unless otherwise specified, each term has the following meanings.

[0027] In the present invention, when the structure involved has isomers, without special designation, any one of the isomers may be used. For example, for a structure having cis-trans isomers, it may be either the cis structure or the trans structure; for a structure having E / Z isomers, it may be either the E structure or the Z structure; when there is optical activity, it may be either the left-handed or the right-handed form.

[0028] In the present invention, the interpretation of a numerical range includes both the numerical range marked with a short dash (such as 1-6) and the numerical range marked with a tilde (such as 1~6). In the present invention, without special explanation, an integer range marked in interval form can represent the set composed of all integers within the range of the interval, and this range includes both endpoints. For example, the integer range 1-6 represents the set composed of 1, 2, 3, 4, 5, and 6. The numerical ranges in the present invention include, but are not limited to, numerical ranges represented by integers, non-integers, percentages, and fractions. Without special explanation, both endpoints are included.

[0029] In the present invention, when a numerical value involves "about" or "around", it generally refers to a numerical range of ±10%, and in some cases, it can be enlarged to ±15%, but not exceeding ±20%, based on the preset numerical value. For example, when the molar percentage of steroid lipid in the total lipid in a solution containing a solvent is about 40%, it is generally considered to include the case where the molar percentage of steroid lipid is 30%-50%.

[0030] In the present invention, unless otherwise specified, the terms "comprising", "including", "containing" and similar expressions shall be interpreted in an open and inclusive sense as "including but not limited to" in this specification and the claims.

[0031] In the present invention, when two or more objects are "each independently preferably", when there are multiple levels of preferred cases, it is not required that they are all selected from the same level of preferred groups. One can be a preferred case in a large range, one can be a preferred case in a small range, or one can be the maximum range and the other can be any preferred case, or they can be selected from the same level of preferred cases.

[0032] In the present invention, for the divalent linking group, such as alkylene, alkylidene, arylene, amide bond, etc., without special limitation, when connecting other groups, either of the two connecting ends can be selected. For example, when using an amide bond 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.

[0033] In the structural formula of the present invention, when the end group of the linking group is likely to be confused with the substituent contained in the linking group, is used to mark the position where the linking group connects other groups. For example, in the structural formula In [it], the is used to label the two positions connecting other groups in the divalent linking group. The above two structural formulas respectively represent -CH(CH2CH2CH3)2- and -CH2CH2CH(CH3)2-CH2CH2-.

[0034] In the present invention, the number of carbon atoms in a group is marked in subscript form at the subscript position of C, indicating the number of carbon atoms in the group. For example, C 1-12 represents "having 1 to 12 carbon atoms", and C 1-30 represents "having 1 to 30 carbon atoms". "Substituted C 1-12 alkyl" refers to a compound obtained by substituting the hydrogen atoms of C 1-12 alkyl. "C 1-12 substituted alkyl" refers to a compound having 1 - 12 carbon atoms obtained after substituting the hydrogen atoms of the alkyl. For another example, when a group can be selected from C 1-12 alkylene, it can be selected from any alkylene having the number of carbon atoms within the range indicated by the subscript, that is, it can be selected from C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 any one of the alkylene. In the present invention, unless otherwise specified, the subscripts marked in interval form all represent any integer within the range, and this range includes both endpoints.

[0035] In the present invention, a heterocycle refers to a cyclic group containing 3 - 12 ring-forming atoms, in which one or more ring-forming carbon atoms are replaced by one or more heteroatoms preferably selected from oxygen, sulfur, and nitrogen. Heterocycles include but are not limited to any one of monocyclic, polycyclic, spirocyclic, bridged cyclic, fused cyclic, carbocyclic, heterocyclic, heterocycloaliphatic, heteromonocyclic, heteropolycyclic, heterospirocyclic, heterobridged cyclic, heterocycloaliphatic structures or a combined structure of any two or more cyclic types. A nitrogen-containing heterocycle is a heterocycle or substituted heterocycle in which the ring-forming atoms contain one or more nitrogen atoms.

[0036] In the present invention, ring-forming atoms refer to the atoms located on the ring, which are carbon atoms or heteroatoms.

[0037] In the present invention, the heteroatoms 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 heteroatoms used for substitution are called "substituting atoms", and any group used for substitution is called a "substituent".

[0039] In the present invention, "substituted" means that at least one hydrogen atom in any group (e.g., aliphatic hydrocarbon group, hydrocarbon group, alkyl group or alkylene group) is replaced by a bond connected to a non-hydrogen atom, and the non-hydrogen atom is, for example, but not limited to: halogen atoms such as F, Cl, Br and I; oxo group (=O); hydroxyl group (-OH); alkoxy group (-OR d , where R d is C 1-12 alkyl); carboxyl group (-COOH,); amine group (-NR c R c , and two R c are each independently H, C 1-12 alkyl); C 1-12 alkyl and cycloalkyl. In some embodiments, the substituent is C 1-12 alkyl. In other embodiments, the substituent is cycloalkyl. In other embodiments, the substituent is a halogenated group, such as fluoro. 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 main chain atoms are carbon atoms, and the side chain part allows heteroatoms or groups containing heteroatoms to replace the hydrogen atoms of the main chain carbon. When the "main chain atom" is a heteroatom, it is also called "main chain heteroatom", such as A-S-CH2-B, A-O-CH2-B, (the atomic interval is recorded as 4) is regarded as containing a main chain heteroatom. The carbon chain linking group can be divided into alkylene group and carbon chain linking group with heteroatoms in the side chain; the carbon chain linking group with heteroatoms in the side chain includes but is not limited to oxo (=O), thio (=S), amino (connected to the main chain carbon through a carbon-nitrogen double bond), oxoalkyl group in the form of an ether bond, thioalkyl group in the form of a thioether bond, azaalkyl group in the form of a tertiary amino group, etc. The "carbon chain linking group" has a main chain entirely composed of carbon atoms, and the side chain of the carbon chain is allowed to contain heteroatoms. That is, it is connected by methylene or substituted methylene. The substituted methylene can be replaced by a monovalent substituent, two monovalent substituents or a divalent substituent (such as divalent oxygen, such as forming a three-membered ring together with divalent methylene ). The substituted methylene can be that one hydrogen atom is replaced (such as -CH(CH3)-), or two hydrogen atoms are respectively replaced (such as -(CH3)C(OCH3)-), or two hydrogen atoms are simultaneously replaced (such as carbonyl, thiocarbonyl, -C(=NH)-, -C(=N + H2)-), or a cyclic side group (such as the atomic interval is recorded as 1).

[0041] In the present invention, the secondary amine bond and hydrazine bond refer to those with both ends of “-NH-” capped by alkylene groups, such as -CH2-NH-CH2-; while -C(=O)-NH- is called an amide bond and is not regarded as containing a secondary amine bond.

[0042] In the present invention, a compound, a group or an atom can be simultaneously substituted and hybridized. For example, a nitro phenyl group substitutes a hydrogen atom, or -CH2-CH2-CH2- is replaced by -CH2-S-CH(CH3)-.

[0043] In the present invention, the “linking bond” only serves as a connecting function and does not contain any atoms. When a certain group is defined as a linking bond, it means that this group can be absent.

[0044] In the present invention, “each occurrence is independently” not only means that different groups can independently be any option in the definition, but also means that when appearing at different positions in the same group, they can also independently be any option in the definition. For example, in -Z-L4-Z-, “Z each occurrence 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- and any one of them, where R c each occurrence is independently a hydrogen atom or C 1-12 alkyl”. In the group “-Z-L4-Z-”, the two Z groups can be the same or different. In the group “-NR c C(=O)NR c -”, the two R c can be the same or different and are independently a hydrogen atom or C 1-12 alkyl.

[0045] The “group” in the present invention contains at least 1 atom and refers to a radical formed by a compound losing one or more atoms. Relative to a compound, the group formed after losing some groups is also called a residue. The valence state of the group is not particularly limited. As examples, it can be divided into monovalent groups, divalent groups, trivalent groups, tetravalent groups,..., 100-valent groups, etc. Among them, groups with a valence state of 2 or more are collectively called linking groups. A linking group can also contain only one atom, such as an oxygen group or a sulfur group.

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

[0047] In the present invention, according to the type of hydrocarbon group, hydrocarbons are divided into two types: aliphatic hydrocarbons and aromatic hydrocarbons. Hydrocarbons that do not contain any benzene ring or benzene ring substituted by a hydrocarbon group are defined as aliphatic hydrocarbons. Hydrocarbons that contain at least one benzene ring or benzene ring substituted by a hydrocarbon group are defined as aromatic hydrocarbons. And aromatic hydrocarbons may contain aliphatic hydrocarbon group structures, such as toluene, diphenylmethane, indane, etc.

[0048] In the present invention, according to the saturation situation, hydrocarbons are divided into two types: saturated hydrocarbons and unsaturated hydrocarbons. 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. By way of example, and without limitation, include olefins (containing double bonds), alkynes (containing triple bonds), dienes (containing two conjugated double bonds), etc. When the aliphatic hydrocarbon part in an aromatic hydrocarbon is a saturated structure, it is also called an arylalkane, such as toluene.

[0049] In the present invention, there is no particular limitation on the structure of hydrocarbons, which can 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, etc. In the absence of a special definition, a straight-chain structure without side groups, a branched-chain structure with side groups, and a cyclic structure are preferably selected, corresponding to straight-chain hydrocarbons, branched-chain hydrocarbons, and cycloalkanes respectively. Among them, hydrocarbons without cyclic structures are collectively referred to as open-chain hydrocarbons, including but not limited to a straight-chain structure without side groups and a branched-chain structure with side groups. Open-chain hydrocarbons belong to aliphatic hydrocarbons. Therefore, straight-chain hydrocarbons can also be called straight-chain aliphatic hydrocarbons. Branched-chain hydrocarbons can also be called branched-chain aliphatic hydrocarbons.

[0050] In the present invention, compounds formed by substituting any carbon atom in a hydrocarbon with a heteroatom are collectively referred to as hetero-hydrocarbons.

[0051] In the present invention, "hydrocarbon group" refers to a residue formed after a hydrocarbon loses at least one hydrogen atom. According to the number of hydrogen atoms lost, it can be divided into monovalent hydrocarbon groups (losing one hydrogen atom), divalent hydrocarbon groups (losing two hydrogen atoms, also called alkylene groups), trivalent hydrocarbon groups (losing three hydrogen atoms), and so on. By analogy, when losing n hydrogen atoms, the valence state of the formed hydrocarbon group is n. In the absence of a special designation, the hydrocarbon groups in the present invention specifically refer to monovalent hydrocarbon groups.

[0052] There is no particular limitation on the source of the hydrocarbon groups in the present invention. For example, they can be derived from aliphatic hydrocarbons or aromatic hydrocarbons, can also be derived from saturated hydrocarbons or unsaturated hydrocarbons, can also be derived from straight-chain hydrocarbons, branched-chain hydrocarbons or cycloalkanes, and can also be derived from hydrocarbons or hetero-hydrocarbons, etc. From the perspective of saturation, for example, they can be derived from alkanes, olefins, alkynes, dienes, etc.; for cycloalkanes, for example, they can be derived from alicyclic hydrocarbons or aromatic hydrocarbons, monocyclic hydrocarbons or polycyclic hydrocarbons; for heterocyclic hydrocarbons, for example, they can be derived from alicyclic heterocyclic hydrocarbons or aromatic heterocyclic hydrocarbons.

[0053] In the present invention, "aliphatic hydrocarbon group" refers to a residue formed by an aliphatic hydrocarbon after losing at least one hydrogen atom. Unless otherwise specified, the aliphatic hydrocarbon group in the present invention specifically refers to a monovalent aliphatic hydrocarbon group. The aliphatic hydrocarbon group includes a saturated aliphatic hydrocarbon group and an unsaturated aliphatic hydrocarbon group.

[0054] In the present invention, "alkyl group" 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, which can be straight-chain or branched-chain, and can be substituted or unsubstituted. Specifically, for example, propyl group refers to either n-propyl or isopropyl, and propylene group refers to any one of 1,3-propylene group, 1,2-propylene group, and isopropylene group.

[0055] In the present invention, "unsaturated hydrocarbon group" refers to a hydrocarbon group formed by an unsaturated hydrocarbon losing a hydrogen atom. The hydrocarbon group formed by an unsaturated hydrocarbon losing a hydrogen atom on an unsaturated carbon can be classified into alkenyl group, alkynyl group, diene group, etc. For example, propenyl group and propynyl group. The hydrocarbon group formed by an unsaturated hydrocarbon losing a hydrogen atom on a saturated carbon is called an olefinic group, an alkyne, a diene group, etc. according to the different unsaturated bonds. Specifically, for example, allyl group and propargyl group.

[0056] In the present invention, "alkenyl group" or "alkenyl group" means a substituted or unsubstituted straight-chain or branched-chain alkenyl group including two or more carbon atoms (for example, 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 label "C 2-15 alkenyl" means a substituted or unsubstituted straight-chain or branched-chain alkenyl group including 2-15 carbon atoms and at least one carbon-carbon double bond, that is, the alkenyl group can include 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] In the present invention, "alkynyl group" or "alkynyl group" means an optionally substituted straight-chain or branched-chain hydrocarbon including two or more carbon atoms (for example, 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. The label "C 2-15 alkynyl" means a substituted or unsubstituted straight-chain or branched-chain alkynyl group including 2-15 carbon atoms and at least one carbon-carbon triple bond. The alkynyl group can include 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 to 2 ether bonds, and more preferably an aliphatic hydrocarbon derivative containing 2 ether bonds.

[0059] In the present invention, "molecular weight" characterizes the mass of a compound molecule. When not specifically stated, the measurement unit of "molecular weight" is Dalton, Da.

[0060] In the present invention, for percentages, "about" generally means ±0.5%.

[0061] In the present invention, "stable existence" and "degradability" of a group are relative concepts.

[0062] In the present invention, "be degradable or can be degraded" means the breaking of chemical bonds of the invention, and the breaking results in at least two residues independently of each other. If the structure is changed after chemical change, but the whole linking group is still only one complete linking group, then the linking group still belongs to the category of "can exist stably". The conditions for degradation are not particularly limited, and can be either in vivo physiological conditions or in vitro simulated physiological environments or other conditions, preferably in vivo physiological conditions and in vitro simulated physiological conditions. The physiological conditions are not particularly limited, including but not limited to serum, heart, liver, spleen, lung, kidney, bone, muscle, fat, brain, lymph node, small intestine, gonad and other parts, which can refer to intracellular or extracellular matrix, and can refer to normal physiological tissues or pathological physiological tissues (such as tumors, inflammations, etc.). The in vitro simulated environments are not particularly limited, including but not limited to physiological saline, buffer solution, culture medium, etc. The rate of degradation is not particularly limited. For example, it can be rapid degradation under the action of enzymes or slow hydrolysis under physiological conditions, etc. The in vivo physiological conditions include physiological conditions during treatment, such as ultraviolet irradiation, hyperthermia, etc. It can be degraded under conditions including but not limited to light, heat, low temperature, enzymes, redox, acidic, alkaline, physiological conditions, in vitro simulated environments, etc., preferably under conditions of light, heat, enzymes, redox, acidic, alkaline, etc. The degradable means degradation occurs under the stimulation of any of the above conditions. The light conditions include but not limited to illumination conditions such as visible light, ultraviolet light, infrared light, near-infrared light, mid-infrared light, etc. The heat conditions refer to temperatures higher than normal physiological temperature, usually temperatures higher than 37 °C, and usually lower than 45 °C, preferably lower than 42 °C. The low temperature conditions refer to temperatures lower than human physiological temperature, preferably lower than 25 °C, more preferably ≤10 °C. Specific examples include refrigeration temperature, freezing temperature, liquid nitrogen treatment temperature, 2-10 °C, 4-8 °C, 4 °C, 0 °C, -20±5 °C, etc. The enzyme conditions are not particularly limited, and all enzymes that can be generated under physiological conditions are included. For example, peptidases, proteases, lyases, etc. The redox conditions are not particularly limited, such as the redox transformation between sulfhydryl and disulfide bond, hydrogenation reduction transformation. The acidic and alkaline conditions mainly refer to the pH conditions of internal parts of the body such as normal tissues, pathological tissues, organs or tissues during treatment, etc. For example, the stomach is in an acidic condition, and the tumor site also tends to be acidic. The degradable here means that it can be degraded through in vivo metabolic actions (such as physiological actions, such as enzymes, such as redox, etc.), degraded due to microenvironment stimulation at specific sites in vivo (such as acidic, alkaline), or degraded under clinical treatment stimulation (such as light, such as heat, such as low temperature), etc. It should be noted that the bond breaking under some extreme conditions in organic chemistry relative to organisms, such as strong acids, strong bases, high temperatures (such as above 100 °C), etc., is not included in the scope of the degradation conditions of the present invention.For another example, although ether bonds can be cleaved under strong acid conditions such as hydrobromic acid, they are always classified as linkers that can stably exist in the present invention.

[0063] In the present invention, "can stably exist" means that the linker can remain as a complete linker (a linker is stably covalently connected to its adjacent groups), and is defined as "can be stable or remain stable". Among them, chemical changes that can maintain the integrity of the linker are allowed. The chemical changes are not particularly limited, including but not limited to isomerization transformation, oxidation, reduction, ionization, protonation, deprotonation, substitution reaction, etc. The conditions for stable existence are not particularly limited, including but not limited to stable existence under conditions such as light, heat, low temperature, enzymes, redox, neutral, acidic, alkaline, physiological conditions, in vitro simulated environment, etc., and preferably stable existence under conditions such as light, heat, enzymes, redox, acidic, alkaline, etc. Stable existence here means that under the condition of no special stimulation (such as the pH condition of a special site, light, heat, low temperature, etc. during treatment), a stable connection can be maintained in the in vivo metabolic cycle, and the molecular weight will not decrease due to chain cleavage (as long as the integrity can still be maintained).

[0064] In the present invention, for the same linker, "can stably exist" is not an absolute concept. For example, an amide bond is much more stable than an ester bond under acidic or alkaline conditions, and the linker of "can stably exist" in the present invention includes an amide bond. However, for example, a peptide bond, an amide bond formed by dehydration condensation of the α-carboxyl group of one molecule of amino acid and the α-amino group of one molecule of amino acid, can be cleaved when acted upon by a specific enzyme, and thus is also included in the "degradable" linker. Similarly, a carbamate group, a thiocarbamate group, etc. can be either a linker that can stably exist or a degradable linker. More generally, carbamate groups, thiocarbamate groups, etc. tend to degrade slowly, while amide bonds other than peptide bonds can stably exist during the in vivo circulation process. Another example is that common ester bonds can be degraded under acidic and basic conditions, and ester bonds contained in special structures can also be degraded under ultraviolet light conditions. Also, for example, even though some chemical bonds can be degraded under the action of specific enzymes, if their circulation path does not pass through or basically does not pass through the specific enzyme environment during clinical use (such as in the case of site-specific drug delivery), the corresponding chemical bonds can still be regarded as stably existing.

[0065] In the present invention, in order to more clearly define the degradable properties of the compound structure, a reference judgment criterion is provided, that is, within a limited time interval, the chemical bond connection is examined with a specific percentage (such as 90%) as the boundary. Taking 90% as an example, usually, the pharmacokinetic curve of the functionalized polyethylene glycol modified product is used as a reference, and the dose percentage meeting the clinical evaluation criteria is used as a benchmark. For example, for PEGylated drugs administered intravenously, when the blood drug concentration (calculated based on the active drug ingredient, including the PEGylated drug and the non-PEGylated component after degradation) is lower than 15% of the initial concentration (or other ratios more in line with the clinical evaluation of the drug), taking the remaining 85% as the base, if the proportion of a linker maintaining the chemical bond connection exceeds 90%, it belongs to the group that can stably exist in the present invention; conversely, if it is lower than 90%, it belongs to the degradable group. The hydrolysis stability, enzyme degradation, etc. reported in the published literature are also included in the present invention. Taking hydrolysis stability as an example, it includes the hydrolysis rate when hydrolysis stability is reported in the published literature, preferably referring to the hydrolysis rate under physiological conditions being lower than 1-2% per day (usually taking 2%), in terms of mass or molar amount. The hydrolysis rate of typical chemical bonds can be referred to in most standard chemical manuals.

[0066] In the present invention, the "hydroxy protecting group" includes all groups that can be used as the protecting group of the usual hydroxyl group. The hydroxy protecting group is preferably an alkanoyl group (such as acetyl group, tert-butylcarbonyl group), an aralkanoyl group (such as benzoyl group), 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 are the ammonolysis of NH3 / MeOH and the methanolysis catalyzed by methoxide anion; the benzyl group can be easily removed by palladium-catalyzed hydrogenolysis at room temperature in a neutral solution, and it can also be reductively cleaved with metallic sodium in ethanol or liquid ammonia; the trityl group is generally removed by catalytic hydrogenolysis; the trimethylsilyl group is usually removed using a fluoride ion-containing reagent (such as tetrabutylammonium fluoride / anhydrous THF, etc.); the tert-butyldimethylsilyl ether is relatively stable and can withstand the ester hydrolysis conditions of alcoholic potassium hydroxide and mild reduction conditions (such as Zn / CH3OH, etc.), and can be removed with fluoride ions (such as Bu4N + F - ) in a tetrahydrofuran solution, or can be removed with acetic acid containing water at room temperature.

[0067] In the present invention, a "carboxyl protecting group" refers to a protecting group that can be converted into a carboxyl group through hydrolysis or a deprotection reaction of the carboxyl protecting group. The carboxyl protecting group is preferably an alkyl group (such as methyl, ethyl, tert-butyl) or an aralkyl group (such as benzyl), more preferably tert-butyl (tBu), methyl (Me) or ethyl (Et). In the present invention, a "protected carboxyl group" refers to a group formed after the carboxyl group is protected by a suitable carboxyl protecting group, preferably methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl. The carboxyl protecting group can be removed by hydrolysis under the catalysis of an acid or a base, and occasionally can also be removed by a pyrolysis reaction. For example, tert-butyl can be removed under mild acidic conditions, and benzyl can be removed by hydrogenolysis. The reagent for removing the carboxyl protecting group is selected from TFA, H2O, LiOH, NaOH, KOH, 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 protected carboxyl group is deprotected to produce the corresponding free acid, and the deprotection is carried out in the presence of a base, and the base and the free acid formed by the deprotection form a pharmaceutically acceptable salt.

[0068] In the present invention, an "amino protecting group" includes all groups that can be used as a protecting group for a normal amino group, such as aryl C 1-6 alkyl, C 1-6 alkoxy C 1-6 alkyl, C 1-6 alkoxycarbonyl, aryloxycarbonyl, C 1-6 alkylsulfonyl, arylsulfonyl or silyl, etc. The amino protecting group is preferably Boc (tert-butoxycarbonyl), Moz (p-methoxybenzyloxycarbonyl) and Fmoc (9-fluorenylmethoxycarbonyl). The reagent for removing the amino protecting group is selected from TFA, H2O, LiOH, MeOH, EtOH and combinations thereof, preferably a combination of TFA and H2O, a combination of LiOH and MeOH, or a combination of LiOH and EtOH. The reagent for removing the Boc protecting group is TFA or HCl / EA; preferably TFA. The deprotecting agent used for the reaction of removing the Fmoc protecting group is a solution of N,N-dimethylformamide (DMF) containing 20% piperidine.

[0069] In the present invention, "carboxyl activation" refers to the activation treatment of a carboxyl group with a carboxyl activating agent. After the carboxyl group is activated, it can promote the better progress of the condensation reaction, such as inhibiting the generation of racemic impurities in the condensation reaction, catalyzing and accelerating the reaction rate, etc. A "carboxyl activating group" is a residue of a carboxyl activating agent. The carboxyl activating agent is one or a combination of more than one 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, it is a combination of NHS / EDCI, NHS / DCC, or HONb / DCC, and most preferably a combination of NHS / EDCI.

[0070] In the present invention, a "cation" means that the corresponding structure permanently or non-permanently carries a positive charge in response to certain conditions (such as pH). Therefore, a cation includes both a permanent cation and a cationizable one. A permanent cation means that the corresponding compound, group, or atom carries a positive charge at any pH value or hydrogen ion activity in its environment. Typically, a positive charge is generated due to the presence of a quaternary nitrogen atom. When a compound carries multiple such positive charges, it can be called a permanent cation. A cationizable one means that a compound, group, or atom carries a positive charge at a lower pH and does not carry a charge at a higher pH in its environment. Additionally, in a non-aqueous environment where the pH value cannot be measured, a cationizable compound, group, or atom carries a positive charge at a high hydrogen ion concentration and does not carry a charge at a low hydrogen ion concentration or activity. It depends on the individual properties of the cationizable or polycationizable compound, especially the pKa of the corresponding cationizable group or atom, at which it carries a charge or does not carry a charge at the said pH or hydrogen ion concentration. In a dilute aqueous environment, the so-called Henderson-Hasselbalch equation can be used to estimate the fraction of the cationizable compound, group, or atom carrying a positive charge, which is well-known to those skilled in the art. For example, in some embodiments, if a certain compound or moiety is cationizable, preferably, it carries a positive charge at a pH value of about 1 to 9, preferably 4 to 9, 5 to 8, or even 6 to 8, more preferably at a pH value equal to or lower than 9, equal to or lower than 8, equal to or lower than 7, and most preferably at a physiological pH value (such as about 7.3 to 7.4), i.e., under physiological conditions, especially under the physiological salt conditions of cells in vivo. In other embodiments, preferably, the cationizable compound or moiety is mainly neutral at a physiological pH value (such as about 7.0 - 7.4), but becomes positively charged at a lower pH value. In some embodiments, the preferred range of the pKa of the cationizable compound or moiety is about 5 to about 7.

[0071] In the present invention, "cationic lipid" refers to a lipid that contains a positive charge or an ionizable lipid as a whole. In addition to those shown in the general structural formula (1) of the present invention, cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 3-(dilauryldimethylammonio)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(dilauryldimethylammonio)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-bis(tridecyl)-15,18,21,24-tetraaza-38-ane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 4-(dimethylamino)butyric acid heptatriaconta-6,9,28,31-tetraen-19-yl ester (DLin-MC3-DMA), and 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), any one of them and their mixtures.

[0072] In the present invention, "PEGylated lipid" refers to a molecule containing a lipid moiety and a polyethylene glycol moiety. In addition to those shown in the general structural formula (2) of the present invention, PEGylated lipids 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), etc.

[0073] In the present invention, "neutral lipid" refers to any one of many 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-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (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), dioleoyl phosphatidylserine (DOPS), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine (LPE), and any combination thereof. Neutral lipids can be synthetic or of natural origin.

[0074] In the present invention, the "steroid lipid" is selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and mixtures thereof.

[0075] In the present invention, the "amino acid residue" includes an amino acid 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 mercapto group and / or the amino group is protected and / or the carboxyl group is protected and / or the mercapto group is protected. Loosely speaking, the amino acid residue can be referred to as an amino acid. The source of the amino acid in the present invention is not particularly limited without special indication, and can be either a natural source, a non-natural source, or a mixture of both. The structural type of the amino acid in the present invention is not particularly limited without special indication, and can refer to either the L-form, the D-form, or a mixture of both.

[0076] The "functional group source" in the present invention refers to having reactive activity or having potential reactive activity, having photosensitive properties or having potential photosensitive properties, having targeting properties or having potential targeting properties. The term "potential" means that it can be transformed into a reactive group through a chemical process selected from including but not limited to functionalization modification (such as grafting, substitution, etc.), deprotection, salt complexation and dissociation, ionization, protonation, deprotonation, changing the leaving group, etc., and can emit light or generate targeting properties under external stimuli such as light, heat, enzymes, specific binding molecules, and the in vivo microenvironment. The emission of light is not particularly limited, including but not limited to visible light, fluorescence, phosphorescence, etc.

[0077] The variant form in the present invention refers to a structural form that can be transformed into a target reactive group through any one of chemical change processes such as oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and dissociation, ionization, protonation, deprotonation, substitution, deprotection, changing the leaving group, etc.

[0078] The "variant form of the reactive group" in the present invention refers to a form that remains active (is still a reactive group) after at least one chemical change process such as oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and dissociation, ionization, protonation, deprotonation, substitution, deprotection, changing the leaving group, etc. for a reactive group, or a non-active form after being protected.

[0079] The "micro-modification" in the present invention refers to a chemical modification process that can be completed through a simple chemical reaction process. The said simple chemical reaction process mainly refers to chemical reaction processes such as deprotection, salt complexation and dissociation, ionization, protonation, deprotonation, and transformation of leaving groups. The "micro-variant form" corresponds to the "micro-modification", and refers to a structural form that can form a target reactive group after undergoing simple chemical reaction processes such as deprotection, salt complexation and dissociation, ionization, protonation, deprotonation, and transformation of leaving groups. The transformation of the leaving group, such as the transformation from an ester form to an acyl chloride form.

[0080] In the present invention, the "N / P ratio" refers to the molar ratio of nitrogen atoms in the cationic lipid to phosphoric acid in the nucleic acid.

[0081] In the present invention, "nucleic acid" refers to DNA or RNA or its modified forms, which contain purine or pyrimidine bases present in DNA (adenine "A", cytosine "C", guanine "G", thymine "T") or purine or pyrimidine bases present in RNA (adenine "A", cytosine "C", guanine "G", uracil "U").

[0082] In the present invention, "RNA" refers to ribonucleic acid that may be naturally occurring or non-naturally occurring. For example, RNA can include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. RNA can include a cap structure, chain-terminating nucleosides, stem-loops, polyadenylation sequences, and / or polyadenylation signals. RNA can have a nucleotide sequence encoding a polypeptide of interest. For example, RNA can be messenger RNA (mRNA). Translating mRNA encoding a specific polypeptide, such as translating mRNA inside mammalian cells in vivo, can produce the encoded polypeptide. RNA can 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.

[0083] In the present invention, antisense oligonucleotides or small interfering RNA (siRNA) can inhibit the expression of target genes and target proteins in vitro or in vivo.

[0084] In the present invention, FLuc mRNA can express luciferase protein, which emits bioluminescence in the presence of luciferin substrate, so FLuc is commonly used in mammalian cell culture to measure gene expression and cell viability.

[0085] In the present invention, "inhibiting the expression of a target gene" refers to the ability of nucleic acids to silence, reduce or inhibit the expression of a target gene. To test the degree of gene silencing, a test sample (e.g., a cell sample in a culture medium expressing the target gene) is contacted with a nucleic acid that inhibits the expression of the target gene. The expression of the target gene in the test sample or test animal is compared with the expression of the target gene in a control sample (e.g., a cell sample in a culture medium expressing the target gene) that has not been contacted or administered the nucleic acid. The expression of the target gene in the control sample can be assigned a value of 100%. In certain embodiments, inhibition of the expression of the target gene is achieved when the level of expression of the target gene in the test sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% relative to the level of expression of the target gene in the control sample or control mammal.

[0086] In the present invention, methods for determining the level of expression of a target gene include, but are not limited to, dot blot, northern blot, in situ hybridization, ELISA, immunoprecipitation, enzymatic action, and phenotypic assays.

[0087] In the present invention, "transfection" refers to the introduction of a substance (e.g., RNA) into a cell. Transfection can occur, for example, in vitro, ex vivo or in vivo.

[0088] In the present invention, an "antigen" typically refers to a substance that can be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, such as by forming antibodies and / or antigen-specific T cells as part of the adaptive immune response. Typically, an antigen can be or can comprise a peptide or protein that can be presented by MHC to T cells. In the context of the present invention, an antigen can be the translation product of the provided nucleic acid molecule (preferably mRNA as defined herein). Peptides and protein fragments, variants and derivatives that contain at least one epitope are also understood as antigens in this context.

[0089] In the present invention, "delivery" refers to the provision of an entity to a target. For example, delivering a drug and / or therapeutic agent and / or prophylactic agent to a subject, which is a tissue and / or cell of a human and / or other animal.

[0090] As used herein, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle administered together with a therapeutic agent, and which, within the scope of reasonable medical judgment, is suitable for contact with the tissues of humans and / or other animals without undue toxicity, irritation, allergic response 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, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is an exemplary carrier. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, especially for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene glycol, water, ethanol, etc. The composition may also optionally contain small amounts of wetting agents, emulsifying agents or pH buffering agents. Oral preparations 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-adhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), demulcents, emulsifiers, fillers (diluents), film formers or coatings, flavoring agents, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweetening agents, and water for hydration. More specifically, excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, croscarmellose sodium, crospovidone, citric acid, cross-linked polyvinylpyrrolidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, phenylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, xylitol.

[0091] The pharmaceutical compositions of the present invention can act systemically and / or locally. For this purpose, they can be administered by suitable routes, such as by injection (e.g., intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including infusion) or transdermal administration; or by oral, buccal, nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation. For these routes of administration, the pharmaceutical compositions of the present invention can be administered in suitable dosage forms. The dosage forms include, but are not limited to, tablets, capsules, lozenges, troches, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups.

[0092] In the present invention, a vaccine is a prophylactic or therapeutic material that provides at least one antigen or antigenic function. The antigen or antigenic function can stimulate the adaptive immune system of the body to provide an adaptive immune response.

[0093] 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 mitigating 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.

[0094] 1. Cationic lipid

[0095] 1.1. A cationic lipid, characterized in that its structure is shown in the general formula (1):

[0096]

[0097] Wherein, X is N;

[0098] represents a divalent nitrogen-containing heterocyclic group, and the nitrogen-containing heterocycle is a heterocycle or a substituted heterocycle in which the ring-forming atoms contain one or more nitrogen atoms; the nitrogen-containing heterocycle is a six-membered ring or a heterocycle with six or more members;

[0099] L1 and L2 are each independently a divalent linking group, selected from -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CR c R c ) s O-, -S-, -S-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)NRc -, -NR c C(=O)S-, -C(R c )=N-NR c - and -NR c -N=C(R c )-, where R c is independently a hydrogen atom or C 1-12 alkyl each time it appears, and s is 2, 3 or 4;

[0100] L3 is a divalent linking group; a divalent linking group selected from any one, any two or any combination of two or more of L4, L5, and Z divalent linking groups; wherein, the 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 from 0 to 12, and t, o, p are not all 0 at the same time, and R a and R b are each independently a hydrogen atom or C 1-12 alkyl each time they appear; the Z is each 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-; R c is each independently H or C 1-12 alkyl each time it appears;

[0101] B1 and B2 are each independently C 1-30 alkylene;

[0102] R1 and R2 are each independently C 1-30 aliphatic hydrocarbon group or C 1-30 residue of aliphatic hydrocarbon derivative;

[0103] R3 is -L 01 -R 01 , where L01 is a divalent linking group selected from -(CR c R c ) m -, -NR c C(=O)-, -C(=O)NR c -, -NR c C(=O)NR c -, -OC(=O)NR c -, -NR c C(=O)O-, -SC(=O)NR c -, -NR c C(=O)S-, -C(R c )=N-NR c -, -NR c -N=C(R c )-, any one of them, or any combination of two or more; R 01 is a functional group capable of reacting with a biologically related substance; R c is independently a hydrogen atom or C 1-12 alkyl each time it appears; m is an integer from 1 to 12;

[0104] The alkyl, alkylene, alkoxy, aliphatic hydrocarbon group and aliphatic hydrocarbon derivative residue are each independently substituted or unsubstituted.

[0105] In a specific embodiment of the present invention, preferably the aforementioned substitution means that the group is substituted by C 1-12 hydrocarbon group, cycloalkyl group, heterocyclic group, F, Cl, Br, I, =O, -OH, -OR d -, -COOH, -NR c R c any one or more of them; wherein, R c is independently H or C 1-12 alkyl each time it appears; R d is C 1-12 alkyl.

[0106] 1.1.1.L1, L2, L3, L4, L5, L 01 , Z

[0107] In the present invention, L1, L2, L3, L4, L5, L 01, the number of non-hydrogen atoms of Z is not particularly limited, and each is independently preferably 1 to 50 non-hydrogen atoms; more preferably 1 to 20 non-hydrogen atoms; still 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, etc. When the number of non-hydrogen atoms is 1, the non-hydrogen atom can be a carbon atom or a heteroatom. When the number of non-hydrogen atoms is greater than 1, the types of non-hydrogen atoms are not particularly limited; it can be 1 type, or 2 types or more; when the number of non-hydrogen atoms is greater than 1, it can be any combination of carbon atoms with carbon atoms, carbon atoms with heteroatoms, and heteroatoms with heteroatoms.

[0108] In the present invention, two identical or different reactive groups can form a divalent linking group through reaction. The reaction conditions are related to the type of the divalent linking group formed by the reaction, and the existing publicly available technologies can be adopted. 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, isothiocyanates to obtain amide groups, urethane groups, amino groups, imino groups (which can be further reduced to secondary amino groups), amino groups, amide groups, amino alcohols, urea bonds, thiourea bonds, etc. divalent linking groups; mercapto groups react with those containing active esters, formic acid active esters, sulfonic acid esters, mercapto groups, maleimides, aldehydes, α,β-unsaturated bonds, carboxylic acid groups, iodoacetamides, acid anhydrides to obtain thioester groups, thiocarbonate groups, thioethers, disulfides, thioethers, thiohemiacetals, thioethers, thioesters, thioethers, imides, etc. divalent linking groups; unsaturated bonds react with mercapto groups to obtain thioether groups; carboxyl groups or acyl halides react with mercapto groups, amino groups to obtain thioester groups, amide groups, etc. groups; hydroxyl groups react with carboxyl groups, isocyanates, epoxides, chloromethoxy groups to obtain ester groups, carbamate groups, ether bonds, carbonate groups, etc. divalent linking groups; carbonyl groups or aldehyde groups react with amino groups, hydrazines, acylhydrazines to obtain imine bonds, hydrazones, acylhydrazones, etc. divalent linking groups; azides, alkynyl groups, alkenyl groups, mercapto groups, azides, dienes, maleimides, 1,2,4-triazoline-3,5-diones, dithioesters, hydroxylamines, acylhydrazines, acrylate esters, allyloxy groups, isocyanates, tetrazoles and other reactive groups undergo click chemical reactions to generate various divalent linking groups including but not limited to structures such as triazoles, isoxazoles, and thioether bonds.

[0109] L1, L2, L3, L4, L5, L 01 , the stability of Z is not particularly limited, and any one of the divalent linking groups or any divalent linking group formed by combining with adjacent heteroatom groups is independently a linking group STAG that can stably exist or a linking group DEGG that can be degraded.

[0110] 1.1.1.1.L1, L2

[0111] In the present invention, L1 and L2 are each independently a divalent linking group selected from -O(C═O)-, -(C═O)O-, -O(C═O)O-, -C(═O)-, -O-, -O(CR c R c ) s O-, -S-, -S-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 -, -NR c C(═O)S-, -C(R c )═N-NR c - and -NR c -N═C(R c )-, where R c is independently a hydrogen atom or a C 1-12 alkyl each time it appears, and s is 2, 3 or 4.

[0112] In a specific embodiment of the present invention, it is more preferred that L1 and L2 are each independently selected from -O(C═O)-, -(C═O)O-, -O(C═O)O-, -C(═O)-, -O-, -O(CH2) s O-, -S-, -S-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-; 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 L1 and L2 are selected from any one of the following situations:

[0114] (1) One of them is -O(C═O)O-, and the other is -O(C═O)- or -(C═O)O-;

[0115] (2) One is -O(C═O)-, and the other is -(C═O)O-;

[0116] (3) Both are -O(C═O)-, or both are -(C═O)O-, or both are -O(C═O)O-.

[0117] In a specific embodiment of the present invention, R c is preferably a hydrogen atom; or R c is preferably a C 1-12 alkyl group, more preferably a C 1-8 alkyl group, more preferably any one of methyl, ethyl, propyl, butyl, pentyl, hexyl.

[0118] 1.1.1.2.L3

[0119] In the present invention, L3 is a divalent linking group, selected from any one, any two or any combination of two or more of L4, L5, and Z divalent linking groups; 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 from 0 to 12, and t, o, p are not simultaneously 0, R a and R b are each independently a hydrogen atom or a C 1-12 alkyl group each time they appear; Z is each 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- each time it appears, wherein R c is each independently H or a C 1-12 alkyl group each time it appears. C 1-12 alkyl group refers to substituted or unsubstituted, preferably any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl.

[0120] In a specific embodiment of the present invention, R c in the aforementioned L3 is preferably a hydrogen atom.

[0121] In a specific embodiment of the present invention, the aforementioned L3 is more preferably -(CH2) t -, -(CH2) t Z-, -Z(CH2) t -, -(CH2) t Z(CH2) t -, -Z(CH2) t Z- is any one of them, where t is an integer from 1 to 12; any one or more H in -(CH2) t - are each independently substituted or unsubstituted, and the substitution means being substituted by C 1-6 alkyl or -OH; 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 -, -NR c C(=O)S- is any one of them.

[0122] In a specific embodiment of the present invention, L3 is more preferably -(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) tOC(=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-.

[0123] 1.1.1.3.L 01

[0124] In the present invention, L 01 is a divalent linking group selected from -(CR c R c ) m -, -NR c C(=O)-, -C(=O)NR c -, -NR c C(=O)NR c -, -OC(=O)NR c -, -NR c C(=O)O-, -SC(=O)NR c -, -NR c C(=O)S-, -C(R c )=N-NR c -, -NR c -N=C(R c )-, any one of them, or any combination of any two or more than two; R 01 is a functional group capable of reacting with a biologically related substance; R c is independently a hydrogen atom or a C 1-12 alkyl each time it appears; m is an integer from 1 to 12.

[0125] In a specific embodiment of the present invention, L 01 is preferably selected from -(CH2) m -, -(CH2) m O-, -(CH2) m C(=O)-, -(CH2)m OC(=O)O-, -O(CH2) m -, -C(=O)(CH2) m -, -C(=O)O(CH2) m -, -OC(=O)(CH2) m -, -OC(=O)O(CH2) m -, -(CH2) m C(=O)O(CH2) m -, -(CH2) m O(CH2) m -, -(CH2) m C(=O)(CH2) m -, -(CH2) m OC(=O)(CH2) m -, -O(CH2) m O-, -C(=O)(CH2) m C(=O)-, -C(=O)O(CH2) m C(=O)O-, -OC(=O)(CH2) m OC(=O)-, -C(=O)O(CH2) m OC(=O)-, -OC(=O)(CH2) m C(=O)O-, -OC(=O)O(CH2) m OC(=O)O-, -C(=O)(CH2) m O-, -C(=O)(CH2) m C(=O)O-, -C(=O)(CH2) m OC(=O)-, -C(=O)(CH2) m OC(=O)O-, -(CH2) m OC(=O)O(CH2) m -, -(CH2) m C(=O)O- and -(CH2) m Any one of OC(=O)-, where m is independently an integer from 1 to 3 each time it appears, -(CH2) m Any one or more of the H in - is substituted or unsubstituted, and the substitution means being substituted by C 1-6 alkyl or -OH; preferably L3, L 01 Each independently selected from -(CH2) m -, -(CH2) m C(=O)- and -C(=O)(CH2) m Any one of -.

[0126] 1.1.2. Description of Stable and Degradable Groups

[0127] The stable linking group STAG or degradable linking group DEGG in the present invention can be present in any of the divalent linking groups L1, L2, L3, L4, L5, L 01 , Z, or in a divalent linking group composed of any divalent linking group and an adjacent heteroatom group.

[0128] 1.1.2.1. The stable divalent linking group STAG in the present invention

[0129] There are no particular restrictions on the conditions under which the stable divalent linking group STAG can stably exist. It can stably exist under any condition including but not limited to light, heat, low temperature, enzymes, redox, acidic, basic conditions, physiological conditions, in vitro simulated environments, etc. Preferably, it can stably exist under any condition such as light, heat, enzymes, redox, acidic, and basic conditions.

[0130] There are no particular restrictions on the type of the stable divalent linking group STAG, including but not limited to alkylene, divalent heteroalkyl, double bond, triple bond, divalent diene, divalent cycloalkyl, divalent cycloalkenyl, divalent cycloalkenylene, divalent cycloalkynyl, aromatic ring, heteroalicyclic ring, heteroaromatic ring, arylheterocyclic ring, hetero-fused heterocyclic ring, substituted alkylene, substituted heteroalkyl, substituted divalent heteroalkyl, substituted double bond, substituted triple bond, substituted diene, substituted divalent cycloalkyl, substituted divalent cycloalkenyl, substituted divalent cycloalkenylene, substituted divalent cycloalkynyl, substituted aromatic ring, substituted heteroalicyclic ring, substituted heteroaromatic ring, substituted arylheterocyclic ring, substituted hetero-fused heterocyclic ring, ether bond, thioether bond, urea bond, thiourea bond, carbamate group, thiocarbamate group, -P(=O)-, divalent silicon group without active hydrogen, divalent linking group containing boron atom, secondary amino group, tertiary amino group, carbonyl group, thiocarbonyl group, amide group, thioamide group, sulfonamide group, enamine group, triazole, 4,5-dihydroisoxazole, any divalent linking group in the amino acid and its derivative skeleton, and stable divalent linking groups composed of any two or any two or more groups.

[0131] Specifically, the stable divalent linking group STAG includes but is not limited to the structures described and listed in documents CN104530413A, CN104530415A, and CN104530417A. Taking CN104530417A as an example, corresponding to paragraphs

[0627] to

[0704] . There are no particular restrictions on the way of combining two or more stable divalent linking groups into STAG. It includes but is not limited to paragraph

[704] of CN104530417A.

[0132] 1.1.2.2. The degradable divalent linking group DEGG in the present invention

[0133] There are no particular restrictions on the conditions for the degradation of the degradable divalent linker DEGG. It can be degraded under any condition including but not limited to light, heat, low temperature, enzymes, redox, acidic, alkaline, physiological conditions, in vitro simulated environment, etc., and is preferably degradable under any condition such as light, heat, enzymes, redox, acidic, alkaline, etc.

[0134] The divalent linker composed of any degradable divalent linker DEGG and any stably existing divalent linker STAG is still a degradable linker. For the degradable divalent linker containing an aromatic ring, it can also be composed of an aromatic ring and a degradable divalent linker.

[0135] There are no particular restrictions on the type of the degradable divalent linker DEGG, including but not limited to containing a disulfide bond, vinyl ether bond, ester group, thioester group, thiodiester group, dithiocarbonate group, carbonate group, thiocarbonate group, dithiocarbonate group, trithiocarbonate group, carbamate group, thiocarbamate group, dithiocarbamate group, acetal, cyclic acetal, thioacetal, azaacetal, azacyclic acetal, azathioacetal, dithioacetal, hemiacetal, thiohemiacetal, azahemiacetal, ketal, thioketal, azaketal, azacyclic ketal, azathioketal, imine bond, hydrazone bond, acylhydrazone bond, oxime bond, thiooxime ether group, semicarbazone bond, thiosemicarbazone bond, hydrazino group, acylhydrazino group, thiosemicarbazide group, azidocarbonylhydrazide group, thioazidocarbonylhydrazide group, hydrazinylformate group, hydrazinylthiocarbonate group, carbohydrazide, thiosemicarbazide, azo group, isourea group, isothiourea group, urethane group, thiourethane group, guanidine group, amidine group, aminoguanidine group, aminoamidine group, imino acid group, imino acid thioester group, sulfonate group, sulfinate group, sulfonylhydrazide group, sulfonylurea group, maleimide, orthoester group, phosphate group, phosphite group, hypophosphite group, phosphonate group, phosphosilane ester group, silane ester group, carbamide, thioamide, sulfonamide group, polyamide, phosphoramide, phosphite amide, pyrophosphoramide, cyclophosphamide, ifosfamide, thiophosphoramide, aconityl group, polypeptide fragment, nucleotide and its derivative backbone, deoxynucleotide and its derivative backbone, any divalent linker, or a combination of any two or more divalent linkers.

[0136] Here, the carbamate group, thiocarbamate group, carbamide, phosphoramide, etc. can be used as both stably existing linkers and degradable linkers, depending on the environmental characteristics of their use.

[0137] Specifically, the degradable divalent linker DEGG includes but is not limited to the structures described and listed in documents CN104530413A, CN104530415A, and CN104530417A. Taking CN104530417A as an example, refer to paragraphs

[0705] to

[0725] .

[0138] 1.1.3.B1, B2

[0139] In the present invention, B1 and B2 are each independently C 1-30 alkylene group.

[0140] In a specific embodiment of the present invention, B1 and B2 are each independently C 1-20 alkylene group. 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 icosylene; more preferably, B1 and B2 are each independently C 5-20 alkylene group.

[0141] 1.1.4.Divalent nitrogen-containing heterocyclic group

[0142] In the present invention, represents a divalent nitrogen-containing heterocyclic group, and the nitrogen-containing heterocycle is a heterocyclic or substituted heterocyclic structure in which the ring-forming atoms contain one or more nitrogen atoms.

[0143] In a specific embodiment of the present invention, the nitrogen-containing heterocycle can be a monocyclic structure or a polycyclic structure; it can be substituted or unsubstituted.

[0144] In a specific embodiment of the present invention, the divalent nitrogen-containing heterocyclic group is selected from any one of the following structures:

[0145]

[0146] and wherein, the asterisk end in the structure is connected to R3; more preferably selected from and any one of them.

[0147] 1.1.5.R1, R2

[0148] In the present invention, R1 and R2 are each independently C 1-30 aliphatic hydrocarbon group or C 1-30 residue of aliphatic hydrocarbon derivative.

[0149] In a specific embodiment of the present invention, R1 and R2 are preferably selected from any one of the following situations:

[0150] Situation (1): Both R1 and R2 are C 1-30 aliphatic hydrocarbon group;

[0151] Case (2): Both R1 and R2 are C 1-30 Residue of aliphatic hydrocarbon derivative;

[0152] Case (3): Optionally, one of R1 and R2 is C 1-30 aliphatic hydrocarbon group, and the other is C 1-30 residue of aliphatic hydrocarbon derivative.

[0153] In a specific embodiment of the present invention, the C 1-30 aliphatic hydrocarbon group is preferably any one of linear alkyl, branched alkyl, linear alkenyl, branched alkenyl, linear alkynyl and branched alkynyl; preferably linear alkyl, linear alkenyl or linear alkynyl, each independently represented as R g ; more preferably each independently is C 1-25 linear alkyl.

[0154] In a specific embodiment of the present invention, the C 1-30 aliphatic hydrocarbon group is preferably branched alkyl, branched alkenyl or branched alkynyl, each independently represented as wherein t is an integer from 0 to 12; R e , R f are each independently C1-C 15 alkyl, C2-C 15 alkenyl and C2-C 15 alkynyl; more preferably each independently selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, vinyl, propenyl, allyl, butenyl, butenyl, pentenyl, pentenyl, hexenyl, hexenyl, heptenyl, heptenyl, octenyl, octenyl, nonenyl, nonenyl, decenyl, decenyl, ethynyl, propynyl, propynyl, butynyl, butynyl, pentynyl, pentynyl, hexynyl, hexynyl, heptynyl, heptynyl, octynyl, octynyl, nonynyl, nonynyl, decynyl and decynyl; more preferably each independently selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl and their substituted forms; most preferably selected from any one of the following structures:

[0155]

[0156] In a specific embodiment of the present invention, the C 1-30 residue of aliphatic hydrocarbon derivative is preferably wherein t is independently an integer from 0 to 12 each time it appears; R e , R f are each independently C1-C 15 alkyl, C2-C15 any one of alkenyl and C2-C 15 alkynyl; more preferably selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, vinyl, propenyl, allyl, butenyl, butenyl, pentenyl, pentenyl, hexenyl, hexenyl, heptenyl, heptenyl, octenyl, octenyl, nonenyl, nonenyl, decenyl, decenyl, ethynyl, propynyl, propargyl, butynyl, butynyl, pentynyl, pentynyl, hexynyl, hexynyl, heptynyl, heptynyl, octynyl, octynyl, nonynyl, nonynyl, decynyl and decynyl, and substituted forms thereof; more preferably selected from any one of methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl and substituted forms thereof; further, the is preferably any one of; still further, the is preferably selected from any one of the following structures:

[0157] and

[0158] 1.1.6.R3

[0159] In the present invention, R3 is -L 01 -R 01 , wherein L 01 is a divalent linking group selected from -(CR c R c ) m -, -NR c C(=O)-, -C(=O)NR c -, -NR c C(=O)NR c -, -OC(=O)NR c -, -NR c C(=O)O-, -SC(=O)NR c -, -NR c C(=O)S-, -C(R c )=N-NR c -, -NR c -N=C(R c )- any one of, or any two or any combination of two or more; R 01 is a functional group capable of reacting with a biologically relevant substance; R c is independently a hydrogen atom or C 1-12 alkyl each time it appears; m is an integer of 1-12.

[0160] In a specific embodiment of the present invention, preferably R 01 is a functional group with therapeutic targeting properties; preferably it is a residue of any one of folic acid, N-acetylgalactosamine or a residue of a functional derivative of any one of them; more preferably it is any one of the following structures:

[0161]

[0162] In a specific embodiment of the present invention, preferably R3 is selected from -(CH2) m OH, -(CH2) m OC(=O)-(CH2) m CH3, -(CH2) m SH, -(CH2) m SC(=O)-(CH2) m CH3, -(CH2) m NH2, -(CH2) m NHC(=O)-(CH2) m CH3, -(CH2) m C(=O)OH, -(CH2) m C(=O)O-(CH2) m CH3, -(CH2) m N3, -(CH2) m N(CH3)2, -(CH2) m N(CH2CH3)2 and -(CH2) m CHO, where m is independently an integer from 1 to 3 each time it appears.

[0163] 1.1.7.

[0164] In a specific embodiment of the present invention, is selected from any one of the following structures:

[0165] and

[0166] 1.1.7. Specific examples of the structural general formula

[0167] In a specific embodiment of the present invention, when L1 and L2 in the structural general formula (1) are each independently a linking bond, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -C(=O)-, -O-, -O(CH2) sWhen it is 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-, the structure of the cationic lipid of the present invention preferably satisfies any one of the following structural formulas:

[0168]

[0169]

[0170]

[0171] and

[0172] In a specific embodiment of the present invention, preferably, R1 and R2 are preferably each independently R g , and any one of, said R e , R f each independently represents C1-C 15 alkyl, C2-C 15 alkenyl and C2-C 15 alkynyl any one of, said t each independently represents an integer from 0 to 12, said R g is a straight-chain alkyl, straight-chain alkenyl or straight-chain alkynyl; L1 and L2 each independently represent a linking bond, -O(C=O)-, -(C=O)O-, -O(C=O)O- any one of; further, the structure of the cationic lipid preferably satisfies any one of the following structural formulas:

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] and

[0179] 1.1.8. Specific structural examples

[0180] Some specific embodiments of the present invention finally obtained cationic lipids with the following structures, including but not limited to any one of the following structures:

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188] 2. Preparation of cationic lipids

[0189] In the present invention, the preparation of any of the foregoing cationic lipids can be carried out by methods including but not limited to the following:

[0190] Method 1:

[0191] Step 1: React one molecule of A-1 with one molecule of A-2 to generate an intermediate A-3 containing a divalent linking group L1, with a reactive group F at one end N and R1 at the other end; wherein, starting material A-1 contains a reactive gene F1, and starting material A-2 contains a pair of hetero-functional groups F2 and F N , F2 is a reactive group and can react with F1 to form a divalent linking group L1, and 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, etc.;

[0192] Step 2: Alkylate two molecules of intermediate A-3 containing one F N with a primary amine derivative A-4 containing a nitrogen source end group and a nitrogen heterocycle to obtain a cationic lipid A-5’, wherein the R3’ end contains a reactive group R 01 or a slightly modified form containing R 01 ; the slightly modified form refers to a group that can be converted into R 01 through any one of the chemical processes of deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group;

[0193] When R3’ is equal to R3, the resulting structure A-5’ corresponds to the structure shown in general formula (1);

[0194] When R3’ is not equal to R3, A-5’ is subjected to terminal micro-modification to obtain A-5 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, changing the leaving group; wherein, R1 and R2 are the same, B1 and B2 are the same, and L1 and L2 are the same;

[0195] Wherein, The definitions of L1, L2, L3, B1, B2, R3, R1 and R2 are the same as those described in general formula (1), and will not be elaborated here.

[0196] The aforementioned small molecule raw materials A-1, A-2, A-4, etc. can be obtained by purchase or by self-synthesis;

[0197] Step 1

[0198]

[0199] Step 2

[0200]

[0201] Method 2:

[0202] Step 1: React one molecule of B-1 with one molecule of B-2 to generate an intermediate B-3 containing a divalent linking group L1, with a hydroxyl group at one end and R1 at the other end; wherein, the raw material B-1 contains a reactive gene F1, and the raw material B-2 contains a hetero-functional group pair F2 and a hydroxyl group (OH), and F2 is a reactive group that can react with F1 to generate a divalent linking group L1;

[0203] Step 2: Oxidize the hydroxyl group of the intermediate B-3 to an aldehyde group to obtain an intermediate B-4 containing an aldehyde group, wherein B1’ is an alkylene group with one less methylene group than B1;

[0204] Step 3: Perform an addition reaction on two molecules of the intermediate B-4 containing an aldehyde group and a primary amine derivative B-5 containing a nitrogen source end group and a nitrogen heterocycle to obtain a cationic lipid B-6’, wherein the R3’ end contains a reactive group R 01 or contains R 01 or a micro-variation form of R; the micro-variation form refers to a group that can be transformed into R through any one of the chemical processes of deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group 01 ;

[0205] When R3’ is equal to R3, the resulting structure B-6’ corresponds to the structure shown in general formula (1);

[0206] When R3’ is not equal to R3, B-6’ is subjected to terminal micro-modification to obtain the structure shown in the general formula (1) of B-6; the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and dissociation, ionization, protonation, deprotonation, changing the leaving group, wherein, R1 and R2 are the same, B1 and B2 are the same, and L1 and L2 are the same;

[0207] Wherein, the definitions of L1, L2, L3, B1, B2, R3, R1 and R2 are the same as those described in the general formula (1), and will not be elaborated here.

[0208] The aforementioned small molecule raw materials B-1, B-2, B-5, etc. can be obtained by purchase or by self-synthesis;

[0209] Step 1

[0210]

[0211] Step 2

[0212]

[0213] Step 3

[0214]

[0215] Method 3:

[0216] Step 1: React one molecule of C-1 with one molecule of C-2 to form an intermediate C-3 containing a divalent linking group L1, with a reactive group F at one end N and R1 at the other end; react one molecule of C-1’ with one molecule of C-2’ to form an intermediate C-3’ containing a divalent linking group L2, with a reactive group F at one end NN and R2 at the other end; wherein, the raw material C-1 contains a reactive group F1; the raw material C-2 contains a pair of hetero-functional groups F2 and F N , F2 is a reactive group that can react with F1 to form a divalent linking group L1, and F N is a reactive group that can react with an amino group or a secondary amino group, preferably -OMs, -OTs, -CHO, -F, -Cl, -Br; the raw material C-1’ contains a reactive group F3; the raw material C-2’ contains a pair of hetero-functional groups 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 that can react with an amino group or a secondary amino group, preferably -OMs, -OTs, -CHO, -F, -Cl, -Br, -COOH, -COCl or an activated carboxyl group, and the activated carboxyl group refers to a group obtained by activating a carboxyl group with a carboxyl activating agent;

[0217] Step 2: One molecule of intermediate C-3 undergoes an alkylation reaction with a primary amine derivative C-4 containing a nitrogen source end group and a nitrogen heterocycle to obtain a secondary amine derivative C-5;

[0218] Step 3: React the secondary amine derivative C-5 with intermediate C-3' to generate a cationic lipid C-6', where the R3' end contains a reactive group R 01 or contains R 01 or a slightly modified form thereof; the slightly modified form refers to a group that can be converted into R through any one of the chemical processes of deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group 01 ;

[0219] When R3' is equal to R3, the resulting structure C-6' corresponds to the structure shown in general formula (1);

[0220] When R3' is not equal to R3, perform terminal micro-modification on C-6' to obtain the structure shown in general formula (1) for 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;

[0221] Among them, the definitions of L1, L2, L3, B1, B2, R3, R1, and R2 are the same as those described in general formula (1), and will not be elaborated here.

[0222] The aforementioned various raw materials C-1, C-1', C-2, C-2', C-4, etc. can be obtained by purchase or can be synthesized independently;

[0223] Step 1

[0224]

[0225] Step 2

[0226]

[0227] Step 3

[0228]

[0229] 2.2. Specific preparation method

[0230] Method 1:

[0231] Step 1: React one molecule of alcohol derivative I-1 with one molecule of I-2 to generate a bromide I-3 or I-3' containing a divalent linking group, with a reactive group at one end and R1 at the other end; among them, when I-2 is a small molecule acid derivative, bromide I-3 is obtained ( ), when I-2 is the bromide of 4-nitrophenyl carbonate, the bromide I-3’ is obtained ); wherein, each occurrence of t is independently an integer from 0 to 12;

[0232] Step 2: Two molecules of the bromide I-3 or I-3’ react with the primary amine derivative I-4 containing a nitrogen source end group and a nitrogen heterocycle through an alkylation reaction to obtain the cationic lipid I-5 or I-5’, wherein the R3’ end contains a reactive group R 01 or contains R 01 a slightly modified form; the slightly modified form refers to a group that can be converted into R 01 through any one of the chemical processes of deprotection, salt complexation and dissociation, ionization, protonation, deprotonation, and leaving group modification;

[0233] When R3’ is equal to R3, the obtained structure I-5 or I-5’ corresponds to the structure shown in the general formula (1);

[0234] When R3’ is not equal to R3, the I-5 or I-5’ is subjected to terminal micro-modification to obtain I-6 or I-6’ corresponding to the structure shown in the general formula (1); the terminal micro-modification is selected from the following chemical reactions: deprotection, salt complexation and dissociation, ionization, protonation, deprotonation, and leaving group modification; wherein, R1 and R2 are the same;

[0235] Among them, the definitions of L3, R3, R1, and R2 are the same as those described in the general formula (1), and will not be elaborated here.

[0236] The foregoing small molecule raw materials I-1, I-2, I-2’, I-4, etc. can be obtained by purchase or by self-synthesis.

[0237] Step 1

[0238]

[0239] Or

[0240]

[0241] Step 2

[0242]

[0243] Or

[0244]

[0245] 2.2. Method 2:

[0246] Step 1: React one molecule of alcohol derivative II-1 with one molecule of small molecule II-2 with a protected hydroxyl group at one end, and then deprotect to form an intermediate II-3 containing a divalent linking group, a hydroxyl group at one end, and R1 at the other end; wherein, each t independently represents an integer from 0 to 12 when it appears;

[0247] Step 2: Oxidize the hydroxyl group of intermediate II-3 to an aldehyde group to obtain intermediate II-4 containing an aldehyde group, wherein t’ is an integer 1 less than t;

[0248] Step 3: Perform an addition reaction on two molecules of intermediate II-4 containing an aldehyde group and a primary amine derivative II-5 containing a nitrogen source end group and a nitrogen heterocycle to obtain a cationic lipid II-6’, wherein the R3’ end contains a reactive group R 01 or contains R 01 or a micro-variant form of R; the micro-variant form refers to a group that can be transformed into R through any one of the chemical processes of deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group 01 ;

[0249] When R3’ is equal to R3, the obtained structure II-6’ corresponds to the structure shown in general formula (1);

[0250] When R3’ is not equal to R3, perform terminal micro-modification on II-6’ to obtain the structure II-6 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, changing the leaving group, wherein R1 and R2 are the same;

[0251] Wherein, the definitions of L3, R3, R1, and R2 are the same as those described in general formula (1), and will not be elaborated here.

[0252] The aforementioned various small molecule raw materials II-1, II-2, II-5, etc. can be obtained by purchase or by self-synthesis.

[0253] Step 1

[0254]

[0255] Step 2

[0256]

[0257] Step 3

[0258]

[0259] 2.3. Method 3:

[0260] Step 1: One molecule of alcohol derivative III-1 reacts with one molecule of acid derivative III-2 to form a bromide III-3 containing an ester group, with Br at one end and R1 at the other end; One molecule of alcohol derivative III-1' reacts with one molecule of 4-nitrophenyl carbonate bromide III-2' to form a bromide III-3' containing a carbonate group, with Br at one end and R2 at the other end; wherein, each t, when it appears, is independently an integer from 0 to 12;

[0261] Step 2: One molecule of bromide III-3 reacts with a primary amine derivative III-4 containing a nitrogen source end group and a nitrogen heterocycle to undergo an alkylation reaction to obtain a secondary amine derivative III-5;

[0262] Step 3: The secondary amine derivative III-5 reacts with bromide III-3' to form a cationic lipid III-6', wherein the R3' end contains a reactive group R 01 or contains R 01 a micro-variant form; the micro-variant form refers to a group that can be transformed into R 01 through any one of the chemical processes of deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group;

[0263] When R3' is equal to R3, the resulting structure III-6' is the structure corresponding to the general formula (1);

[0264] When R3' is not equal to R3, III-6' is subjected to terminal micro-modification to obtain the structure III-6 corresponding to the 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;

[0265] wherein, the definitions of L3, R3, R1, and R2 are the same as those described in the general formula (1), and will not be elaborated here.

[0266] The foregoing various raw materials III-1, III-1', III-2, III-2', III-4, etc. can be obtained by purchase or can be obtained by self-synthesis.

[0267] Step 1

[0268]

[0269] Or

[0270]

[0271] Step 2

[0272]

[0273] Step Three

[0274]

[0275] In the reaction raw material R1-OH in the foregoing preparation method, R1 may be a residue of an etherified aliphatic hydrocarbon derivative wherein, each time t appears, it is independently an integer from 0 to 12; R e , R f are each independently C1-C 15 alkyl, C2-C 15 alkenyl, and C2-C 15 alkynyl. More specifically, R1-OH may be It can be obtained by purchase or synthesized independently. When synthesized independently, aldol addition can be used. For example, one molecule reacts with two molecules of R e -OH to obtain At this time, R e and R f are the same; R1-OH may also be It can be obtained by purchase or synthesized independently. When synthesized independently, it can be obtained by reacting with a related alkylating agent. The alkylating agent is preferably a halide. For example, in Example 5, S5-2( ), it can be obtained by reacting one molecule of glycerol with a hydroxyl group protected by TBS and two molecules of bromohexane and then deprotecting.

[0276] 2.3. Description of related raw materials and / or steps in the preparation process

[0277] 2.3.1. Carboxyl activating agent, condensing agent, oxidizing agent, reducing agent

[0278] In the present invention, "carboxyl activation" refers to activating the carboxyl group with a carboxyl activating agent. After the carboxyl group is activated, it can promote the better progress of the condensation reaction, such as inhibiting the generation of racemic impurities in the condensation reaction and catalyzing and accelerating the reaction rate. The "carboxyl activation group" is the residue of the carboxyl activating agent. The carboxyl activating agent is one or a combination of N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2,3-dicarboximide (HONb), and N,N-dicyclohexylcarbodiimide (DCC). The preferred combinations are NHS / EDCI, NHS / DCC, and HONb / DCC, and the most preferred combination is NHS / EDCI.

[0279] In the present invention, the condensing agent used in the reaction is not limited, but N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) are preferred, and most preferably DCC. Generally, the amount of the condensing agent is 1 to 20 times the molar equivalent of the carboxylic acid, preferably 5 to 10 times. An appropriate catalyst (such as 4-dimethylaminopyridine) can be added to this reaction.

[0280] In the present invention, the oxidizing agent used in the reaction is not particularly limited, as long as it is a compound or a combination of multiple compounds that can increase the valence of the substrate. Preferred are phenyl iodide bis(trifluoroacetate), 1,4-benzoquinone, benzyltrimethylammonium tribromide, pyridinium dichromate, potassium dichromate, ozone, oxygen, hypofluorous acid, sodium hypochlorite, cobalt(III) acetate, cobalt(II) acetate, manganese(II) acetate, palladium(II) acetate, copper(II) acetate, monoperoxyphthalic acid, iodine, N-iodosuccinimide, iodobenzene dichloride, 2-iodoxybenzoic acid, dimethyldioxirane, dimethyl sulfoxide-oxalyl chloride, dimethyl sulfoxide-acetic anhydride, DDQ, dichloro(tris(triphenylphosphine))ruthenium, manganese dioxide, diacetoxyiodobenzene, periodic acid, sodium periodate, sodium periodate-osmium tetroxide, potassium permanganate, sodium metaborate, peroxybenzoic acid, benzoyl peroxide, nickel peroxide, hydrogen peroxide, cumene hydroperoxide, tert-butyl hydroperoxide, peracetic acid, m-chloroperoxybenzoic acid, N-chlorosuccinimide, pyridinium chlorochromate, palladium(II) chloride-copper(II) chloride, urea hydrogen peroxide complex, triphenylmethyl tetrafluoroborate, tributyltin oxide, cobalt(III) fluoride, vanadium oxytrifluoride, chromium(III) oxide, manganese(III) acetate, TEMPO, ammonium cerium(IV) nitrate, bromine, N-oxidopyridine, silver oxide, O-ethyl peroxycarbonate, manganese(III) acetylacetonate, vanadyl acetylacetonate, aluminum isopropoxide, potassium peroxymonosulfate, dichloroiodobenzene, etc., or a combination thereof. More preferably, it is a combination of one or more of oxygen, sodium hypochlorite, hydrogen peroxide, dichloroiodobenzene, potassium peroxymonosulfate, etc. The amount of the oxidizing agent is 1 to 50 times the molar equivalent of the hydroxyl group in the intermediate compound, preferably 1 to 20 times, more preferably 5 to 10 times.

[0281] In the present invention, the reducing agent used in the reaction is not particularly limited, as long as it can reduce the Schiff base formed by ammonia and aldehyde or ketone to an amino group; preferred are sodium borohydride, sodium cyanoborohydride, lithium aluminum hydride, borane, diborane, diisobutylaluminum hydride, diisopinocampheylborane, lithium borohydride, zinc borohydride, borane-pyridine, borane-methyl sulfide, borane-tetrahydrofuran, etc., or a combination thereof; more preferably sodium cyanoborohydride. The equivalent of the reducing agent is 1 to 50 times the molar equivalent of the amino group to be modified, preferably 1 to 20 times, more preferably 5 to 10 times.

[0282] In the present invention, the reaction temperature is 0 to 200 °C, preferably 0 to 100 °C, more preferably 0 to 25 °C, and the reaction time is preferably 10 minutes to 48 hours, more preferably 30 minutes to 24 hours. The obtained product can be purified by purification methods such as extraction, recrystallization, adsorption treatment, precipitation, anti-precipitation, thin film dialysis or supercritical extraction.

[0283] In the present invention, the reaction solvent can be solvent-free or aprotic solvents. Aprotic solvents include toluene, benzene, xylene, acetonitrile, ethyl acetate, ether, methyl tert-butyl ether, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, preferably tetrahydrofuran, dichloromethane, dimethyl sulfoxide, dimethylformamide.

[0284] In the present invention, the base used in the reaction is generally an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine), preferably triethylamine, pyridine. The amount of the base is 1 to 50 times the molar equivalent of the carboxylic acid, preferably 1 to 10 times, more preferably 2 to 3 times.

[0285] 2.3.2. "Protection" and "deprotection" of related groups involved in the reaction process

[0286] In the present invention, the reaction process also involves the "protection" and "deprotection" processes of related groups. To prevent the functional group from affecting the reaction, the functional group is usually protected. And when there are two or more functional groups, only the target functional group is selectively reacted, so other functional groups are protected. The protecting group not only stably protects the target functional group, but also needs to be easily removed as needed. Therefore, in organic synthesis, it is important to deprotect only the protecting group bonded to the specified functional group under appropriate conditions.

[0287] In the present invention, the "carboxyl protecting group" refers to a protecting group that can be converted into a carboxyl group through hydrolysis or a deprotection reaction of the carboxyl protecting group. The carboxyl protecting group is preferably an alkyl group (such as methyl, ethyl, tert-butyl) or an aralkyl group (such as benzyl), more preferably tert-butyl (tBu), methyl (Me) or ethyl (Et). In the present invention, the "protected carboxyl group" refers to a group formed after the carboxyl group is protected by a suitable carboxyl protecting group, preferably methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl. The carboxyl protecting group can be removed by hydrolysis under the catalysis of an acid or a base, and occasionally can also be removed by a pyrolysis reaction. 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 their combinations, preferably the combination of TFA and H2O, the combination of LiOH and MeOH, or the combination of LiOH and EtOH. The deprotection of the protected carboxyl group generates the corresponding free acid, and the deprotection is carried out in the presence of a base, and the base and the free acid formed by the deprotection form a pharmaceutically acceptable salt.

[0288] In the present invention, the "amino protecting group" includes all groups that can be used as the protecting group of a normal amino group, such as aryl C 1-6 alkyl, C 1-6 alkoxy C 1-6 alkyl, C 1-6 alkoxycarbonyl, aryloxycarbonyl, C 1-6 alkylsulfonyl, arylsulfonyl or silyl, etc. The amino protecting group is preferably Boc tert-butoxycarbonyl, Moz p-methoxybenzyloxycarbonyl and Fmoc 9-fluorenylmethoxycarbonyl. The reagent for removing the amino protecting group is selected from TFA, H2O, LiOH, MeOH, EtOH and their combinations, preferably the combination of TFA and H2O, the combination of LiOH and MeOH, or the combination of LiOH and EtOH. The reagent for removing the Boc protecting group is TFA or HCl / EA; preferably TFA. The deprotecting agent used for the reaction of removing the Fmoc protecting group is a solution of N,N-dimethylformamide (DMF) containing 20% piperidine.

[0289] In the present invention, the hydroxyl group protected by the hydroxyl protecting group is not particularly limited, and can be, for example, a hydroxyl group such as an alcohol hydroxyl group or a phenolic hydroxyl group. Among them, the amino group of the amino protecting group is not particularly limited, and can come from, for example, a primary amine, a secondary amine, a hydrazine, an amide, etc. In the present invention, the amino group is not particularly limited, including but not limited to a primary amino group, a secondary amino group, a tertiary amino group, and a quaternary ammonium ion.

[0290] In the present invention, the deprotection of the protected hydroxyl group is related to the type of the hydroxyl protecting group. The type of the hydroxyl protecting group is not particularly limited. Taking the protection of the terminal hydroxyl group with benzyl, silyl ether, acetal, tert-butyl as an example, the corresponding deprotection methods are as follows:

[0291] A: Deprotection of Benzyl Group

[0292] The deprotection of benzyl group can be achieved by the hydrogenation 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.

[0293] There is no limitation on the hydrogenation reduction catalyst, preferably palladium and nickel. However, there is no limitation on the carrier, but alumina or carbon is preferred, and carbon is more preferred. The dosage of palladium is 1 to 100 wt% of the hydroxy compound to be protected, preferably 1 to 20 wt% of the hydroxy compound to be protected.

[0294] There is no particular limitation on the reaction solvent as long as both the raw materials and the products can be dissolved in the solvent. However, methanol, ethanol, ethyl acetate, tetrahydrofuran, and acetic acid are preferred; methanol is more preferred. There is no particular limitation on the hydrogen donor, but hydrogen, cyclohexene, 2-propanol, ammonium formate, etc. are preferred. The reaction temperature is preferably 25 to 40 °C. There is no particular limitation on the reaction time, and the reaction time is negatively correlated with the dosage of the catalyst, preferably 1 to 5 hours.

[0295] B: Deprotection of Acetals and Ketals

[0296] The acetal or ketal compounds used for this type of hydroxy protection preferably include ethyl vinyl ether, tetrahydropyran, acetone, 2,2-dimethoxypropane, benzaldehyde, etc. The deprotection of these acetals and ketals is achieved under acidic conditions, and the solution pH is preferably 0 to 4. There is no particular limitation on the acid, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and nitric acid are preferred, and hydrochloric acid is more preferred. There is no particular limitation on the reaction solvent as long as it can dissolve the reactants and products, and water is preferred. The reaction temperature is preferably 0 to 30 °C.

[0297] C: Deprotection of Silyl Ethers

[0298] The compounds used for this type of hydroxy protection include trimethylsilyl ether, triethylsilyl ether, dimethyl tert-butylsilyl ether, tert-butyldiphenylsilyl ether, etc. The deprotection of these silyl ethers is carried out by a fluoride ion-containing compound, preferably tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid, potassium fluoride, and more preferably tetrabutylammonium fluoride and potassium fluoride. The dosage of the fluorine-containing reagent is 5 to 20 times the molar equivalent of the hydroxy group to be protected, preferably 8 to 15 times the initiator. If the dosage of fluorine is less than 5 times the molar equivalent of the hydroxy group to be protected, incomplete deprotection will occur; when the dosage of the deprotection reagent is greater than 20 times the molar equivalent of the hydroxy group to be protected, the excess reagent or compound will cause trouble in purification and may be mixed into subsequent steps, thus causing side reactions. There is no particular limitation on the reaction solvent as long as it can dissolve the reactants and products, and an aprotic solvent is preferred, and tetrahydrofuran and dichloromethane are more preferred. The reaction temperature is preferably 0 to 30 °C. When the temperature is lower than 0 °C, the reaction rate is slow and the protecting group cannot be completely removed.

[0299] D: Deprotection of tert-butyl

[0300] The deprotection of tert-butyl is carried out under acidic conditions, and the pH of the solution is preferably 0 to 4. The acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, and nitric acid are preferred, and hydrochloric acid is 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.

[0301] In the terminal functionalization method, it is preferred that q = 0, q1 = 1, and Z1 is 1,2-methylene. When q is not 0 and there is a linker such as an amino acid or succinyl group between A and R 01 The existing technologies in the technical field that can generate Z2 or Z1 (including but not limited to alkylation, condensation, click reaction, etc.) can be adopted, and the preparation can be carried out with reference to the following linear functionalization method.

[0302] 2.3.3. Alkylation reaction

[0303] The alkylation reaction of the present invention is preferably a reaction based on the alkylation of hydroxyl, mercapto or amino groups, corresponding to the formation of ether bonds, thioether bonds, secondary amino groups or tertiary amino groups in turn. Examples are as follows:

[0304] 2.3.3.1. Alkylation of substrate alcohol with sulfonate ester and halide

[0305] In the presence of a base, an amine intermediate is obtained by nucleophilic substitution of a substrate alcohol with a sulfonate ester derivative or a halide. Among them, the molar equivalent of the sulfonate ester and the halide is 1 to 50 times that of the substrate alcohol, preferably 1 to 5 times. When the molar equivalent of the sulfonate ester and the halide is less than 1 times the molar equivalent of the substrate alcohol, the reaction substitution is incomplete and difficult to purify. When the molar equivalent of the sulfonate ester and the halide is greater than 50 times that of the substrate alcohol, the excess reagent causes trouble in purification, may be mixed into the subsequent steps, resulting in an increase in side reactions in the next step and an increase in purification difficulty.

[0306] The obtained product is a mixture of an ether intermediate and excess sulfonate ester and halide, which can be purified by anion exchange resin, osmosis, ultrafiltration, etc. Among them, the anion exchange resin is not particularly limited as long as the target product can undergo ion exchange and adsorption on the resin, and an ion exchange resin of a tertiary amine or quaternary ammonium salt with a backbone such as dextran, agarose, polyacrylate, polystyrene, polydiphenylethylene, etc. is preferred. The solvent for osmosis and ultrafiltration is not limited, generally water or an organic solvent, and the organic solvent is not particularly limited as long as the product can be dissolved in it, and dichloromethane, chloroform, etc. are preferred.

[0307] The reaction solvent is not limited, and aprotic solvents are preferred, such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, and dimethylformamide, dichloromethane, dimethyl sulfoxide or tetrahydrofuran are more preferred.

[0308] The base includes organic bases (such as triethylamine, pyridine, 4-dimethylaminopyridine, imidazole or diisopropylethylamine) or inorganic bases (such as sodium carbonate, sodium hydroxide, sodium bicarbonate, sodium acetate, potassium carbonate or potassium hydroxide), and organic bases are preferred, and triethylamine and pyridine are more preferred. The molar amount of the base is 1 to 50 times the molar equivalent of the sulfonate or halide, preferably 1 to 10 times, and more preferably 3 to 5 times.

[0309] 2.3.3.2. Alkylation of the substrate amine with sulfonate and halide

[0310] A. Alkylation of the substrate amine with sulfonate and halide

[0311] In the presence of a base, an amine intermediate is obtained by nucleophilic substitution of the substrate amine with a sulfonate derivative or halide. Among them, the molar equivalent of the sulfonate or halide is 1 to 50 times that of the substrate amine, preferably 1 to 5 times. When the molar equivalent of the sulfonate or halide is less than 1 times the molar equivalent of the substrate amine, the reaction substitution is incomplete and difficult to purify. When the molar equivalent of the sulfonate or halide is greater than 50 times that of the substrate amine, the excess reagent causes trouble in purification and may be mixed into the subsequent steps, resulting in an increase in side reactions in the next step and an increase in the purification difficulty.

[0312] The obtained product is a mixture of an amine intermediate and excess sulfonate and halide, and it can be purified by anion exchange resin, osmosis, ultrafiltration, etc. Among them, the anion exchange resin is not particularly limited as long as the target product can undergo ion exchange and adsorption on the resin, and ion exchange resins of tertiary amines or quaternary ammonium salts with skeletons such as dextran, agarose, polyacrylate, polystyrene, and polydiphenylethylene are preferred. The solvents for osmosis and ultrafiltration are not limited, and generally water or organic solvents can be used. The organic solvents are not particularly limited as long as the product can be dissolved in them, and dichloromethane, chloroform, etc. are preferred.

[0313] The reaction solvent is not limited, and aprotic solvents are preferred, such as toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, and dimethylformamide, dichloromethane, dimethyl sulfoxide or tetrahydrofuran are more preferred.

[0314] 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 sulfonate or halide, preferably 1 to 10 times, more preferably 3 to 5 times.

[0315] 2.3.3.3. Alkylation reaction of the substrate amine with aldehyde derivatives

[0316] After obtaining an imine intermediate from the reaction of the substrate amine with aldehyde derivatives, an intermediate is obtained under the action of a reducing agent. Among them, the molar equivalent of the aldehyde derivative is 1 to 20 times that of the substrate amine, preferably 1 to 2 times, 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 causes trouble in purification, may be mixed into subsequent steps, and increases the purification difficulty. When the molar equivalent of the aldehyde derivative is less than 1 times that of the substrate amine, the reaction is incomplete and the purification difficulty increases. Among them, the reaction product can be purified by means such as cation exchange resin, osmosis, ultrafiltration, etc. to obtain the intermediate. The cation exchange resin has no particular limitation as long as it can exchange with quaternary ammonium cations to achieve a separation effect. The solvents for osmosis and ultrafiltration are not limited. Generally, water or organic solvents can be used. The organic solvents have no particular limitation as long as the product can be dissolved in them. Preferred are dichloromethane, chloroform, etc.

[0317] The reaction solvent is not restricted. Preferred are organic solvents such as methanol, ethanol, water, toluene, benzene, xylene, acetonitrile, ethyl acetate, tetrahydrofuran, chloroform, dichloromethane, dimethyl sulfoxide, dimethylformamide or dimethylacetamide, etc.; more preferred are water and methanol.

[0318] The reducing agent has no particular limitation as long as it can reduce the imine to an amine. Preferred are sodium borohydride, lithium aluminum hydride, sodium cyanoborohydride, Zn / AcOH, etc., more preferably sodium cyanoborohydride. Generally, the dosage of the reducing agent is 0.5 to 50 times the amount of the aldehyde derivative substance, more preferably 1 - 10 times.

[0319] 2.3.4. Linear functionalization at the R3 terminus

[0320] The method for linear functionalization at the R3 terminus has no particular limitation and is related to the type of the final functional group or its protected form. It mainly includes the functionalization of the terminal hydroxyl group and the transformation from the reactive group to the target functional group or its protected form.

[0321] The preparation method for the functionalization of terminal hydroxyl groups starts from hydroxyl groups and obtains functional groups of Class A to Class J or their protected forms through functionalization. The specific preparation method is as described in paragraphs

[0960] to

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

[0322]

[0323] Among them, q and q1 are each independently 0 or 1; Z1 and Z2 are each independently divalent linking groups; R 01 is a functional group capable of reacting with a biologically relevant substance.

[0324] Based on the transformation of reactive groups into the target functional group or its protected form, it can be achieved by any of the following methods:

[0325] Method 1: Direct modification. Based on the direct modification of reactive groups, the target functional group or its protected form is obtained. As an example, the transformation of a carboxyl group into an acyl halide, acyl hydrazide, ester, thioester, dithioester, and the transformation of hydroxyl, mercapto, alkynyl, amino, carboxyl, etc. into their corresponding protected structures, etc. Another example is the modification of hydroxyl, amino, etc. by acid anhydrides.

[0326] Method 2: Coupling reaction between two reactive groups. Using a hetero-functionalized reagent containing one type of reactive group and the target functional group or its protected form as the raw material, through the reaction between one of the reactive groups and the reactive group at the A end, the target functional group or its protected form is introduced. There are no particular restrictions on the reaction mode and method between the two reactive groups. Its reaction conditions are related to the type of divalent linking group generated by the reaction, and existing publicly available technologies can be used. Such as alkylation, alkenyl addition reaction, alkynyl addition reaction, Schiff base reaction combined with reduction reaction, condensation reaction, etc. Among them, the alkylation reaction is preferably an alkylation reaction based on mercapto or amino, corresponding to the formation of a thioether bond, secondary amino or tertiary amino in sequence. Among them, the condensation reaction includes but is not limited to condensation reactions for generating ester groups, thioester groups, amide groups, imine bonds, hydrazone bonds, carbamate groups, etc. Another example is using a hetero-functionalized reagent containing groups such as azide, alkynyl, alkenyl, trithioester group, mercapto, dienyl, furyl, 1,2,4,5-tetrazinyl, cyanate, etc. and the target functional group or its protected form as the raw material, and introducing the target functional group or its protected form through a click reaction. The reaction between the two reactive groups is accompanied by the formation of a new bond. Typical representatives of the newly formed divalent linking group are amide bonds, urethane bonds, ester groups, secondary amine bonds, thioether bonds, triazole groups, etc.

[0327] Method 3: Obtaining the target functional group or its protected form through a combination of direct modification and coupling reaction.

[0328] In the present invention, the raw materials used in each preparation method can be obtained by purchase or self-synthesis.

[0329] In the present invention, the intermediates and end products prepared can be purified by purification methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, antprecipitation, thin film dialysis or supercritical extraction, etc. For the characterization and confirmation of the structure and molecular weight of the end product, characterization methods including but not limited to nuclear magnetic resonance, electrophoresis, ultraviolet-visible spectrophotometer, FTIR, AFM, GPC, HPLC, MALDI-TOF, ESI-MS, circular dichroism spectrometry, etc. can be used.

[0330] 3. Lipid composition

[0331] 3.1. Lipid composition

[0332] In one embodiment of the present invention, a lipid composition contains any one of the cationic lipids having the structure as shown in the general formula (1) described above.

[0333] In a specific embodiment of the present invention, preferably, the lipid composition, in addition to containing the cationic lipid having the structure as shown in the general formula (1), further contains one or more of phospholipids, steroid lipids and polyethylene glycolated lipids, selected from any one of the following situations:

[0334] Situation (1): further contains phospholipids;

[0335] Situation (2): further contains steroid lipids;

[0336] Situation (3): further contains polyethylene glycolated lipids;

[0337] Situation (4): further contains phospholipids and steroid lipids;

[0338] Situation (5): further contains phospholipids and polyethylene glycolated lipids;

[0339] Situation (6): further contains steroid lipids and polyethylene glycolated lipids;

[0340] Situation (7): further contains phospholipids, steroid lipids and polyethylene glycolated lipids;

[0341] More preferably, it further contains three lipids, namely neutral lipids, steroid lipids and polyethylene glycolated lipids, at the same time.

[0342] In a specific embodiment of the present invention, the phospholipids in the aforementioned lipid composition 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-doundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenoyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (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), dioleoyl phosphatidylserine (DOPS), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine (LPE), any one of them and their compositions.

[0343] In a specific embodiment of the present invention, the sterol lipid in the lipid composition is preferably any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and mixtures thereof.

[0344] In a specific embodiment of the present invention, the polyethylene glycolated lipid in the aforementioned lipid composition is preferably 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 any one of polyethylene glycol 500-dipalmitoyl phosphatidylcholine, polyethylene glycol 2000-dipalmitoyl phosphatidylcholine, polyethylene glycol 500-stearoyl phosphatidylethanolamine, polyethylene glycol 2000-distearoyl phosphatidylethanolamine, polyethylene glycol 500-1,2-oleoyl phosphatidylethanolamine, polyethylene glycol 2000-1,2-oleoyl phosphatidylethanolamine, and polyethylene glycol 2000-2,3-dimyristoyl glycerol (PEG-DMG).

[0345] In a specific embodiment of the present invention, the structure of the polyethylene glycolated lipid in the lipid composition is selected from any one of the following structural formulas:

[0346]

[0347]

[0348] and

[0349] In a specific embodiment of the present invention, it is preferred that any one of the aforementioned lipid compositions contains 20-80% of the cationic lipid shown in formula (1), 5-15% of phospholipids, 25-65% of sterol lipids, and 0.5-10% of polyethylene glycolated lipids, and the percentages are the molar percentages of each lipid in the total lipids in the solution containing the solvent.

[0350] In a specific embodiment of the present invention, it is preferred that in any one of the aforementioned lipid compositions, the molar percentage of the cationic lipid in the total lipids in the solution containing the solvent is 30-65%; more preferably about 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 55%.

[0351] In a specific embodiment of the present invention, it is preferred that in any one of the aforementioned lipid compositions, the molar percentage of phospholipids in the total lipids in the solution containing the solvent is 7.5-13%; more preferably about 8%, 9%, 10%, 11%, 12%.

[0352] In a specific embodiment of the present invention, preferably in any of the aforementioned lipid compositions, the molar percentage of the steroid lipid in the total lipid in the solution containing the solvent is 35 - 50%, more preferably about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.

[0353] In a specific embodiment of the present invention, preferably in any of the aforementioned lipid compositions, the molar percentage of the polyethylene glycolated lipid in the total lipid in the solution containing the solvent is 0.5 - 5%; preferably 1 - 3%; more preferably about 1.5%, 1.6%, 1.7%, 1.8%, 1.9%.

[0354] 3.2. Lipid pharmaceutical compositions and their formulations

[0355] 3.2.1 Lipid pharmaceutical compositions

[0356] In one embodiment of the present invention, a lipid pharmaceutical composition contains any of the aforementioned lipid compositions and a drug, and the drug is selected from any one of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs.

[0357] In a specific embodiment of the present invention, preferably the nucleic acid drug is selected from any one of RNA, DNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir and ribozyme, and the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA and siRNA; preferably any one of DNA, mRNA, miRNA and siRNA.

[0358] In a specific embodiment of the present invention, preferably, the drug includes but is not limited to doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cytarabine, anthracycline, nitrogen mustard, thiotepa, chlorambucil, razoxane, melphalan, carmustine, lomustine, busulfan, dibromomannitol, mitomycin C, cis-dichlorodiamine platinum (II), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, dibucaine, chlorpromazine, propranolol, timolol, labetalol, clonidine, hydralazine, imipramine, amitriptyline, doxepin, phenytoin, diphenhydramine, chlorpheniramine, promethazine, gentamicin, ciprofloxacin, cefoxitin, miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, naftifine, amphotericin B, antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma drugs, vitamins, sedatives, and imaging agents, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, colchicine, daunorubicin, mitoxantrone, mithramycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, puromycin, maytansinoids.

[0359] In a specific embodiment of the present invention, preferably, the lipid drug composition is used as a drug, and is selected from any one of the following drugs: antitumor agents, antiviral agents, antifungal agents, and vaccines.

[0360] In a specific embodiment of the present invention, the drug in the aforementioned lipid drug composition is a nucleic acid drug, and the N / P ratio is (0.1 - 100):1, preferably (0.2 - 30):1, more preferably (0.5 - 20):1.

[0361] 3.2.1 Lipid drug composition preparation

[0362] In a specific embodiment of the present invention, the drug in the lipid drug composition is a nucleic acid drug, and the working solution of the lipid drug composition preparation is deionized water, ultrapure water, phosphate buffer solution, or physiological saline, more preferably phosphate buffer solution or physiological saline, and most preferably physiological saline; preferably, the lipid drug composition: working solution = (0.05 - 20) g: 100 mL, more preferably (0.1 - 10) g: 100 mL, and most preferably (0.2 - 5) g: 100 mL.

[0363] In a specific embodiment of the present invention, a lipid drug composition preparation contains any one of the aforementioned lipid drug compositions and a pharmaceutically acceptable diluent or excipient. The diluent or excipient is preferably any one of deionized water, ultrapure water, phosphate buffer solution, and physiological saline, more preferably phosphate buffer solution or physiological saline, and most preferably physiological saline.

[0364] In the present invention, the preparation of the lipid drug composition preparation comprises the following steps:

[0365] (1) Equilibrate the lipid composition in the diluent or excipient;

[0366] (2) Add the drug to the mixture of the equilibrated lipid composition and the diluent or excipient for complexation;

[0367] Preferably, the equilibration time is 0.1 - 12 h, preferably 0.2 - 6 h, more preferably 0.5 - 3 h; preferably, the complexation time is 0.1 - 12 h, preferably 0.2 - 5 h, more preferably 0.5 - 2 h.

[0368] 4. Liposomes or lipid nanoparticles and their preparation

[0369] 4.1. Liposomes or lipid nanoparticles

[0370] In a specific embodiment of the present invention, a liposome or lipid nanoparticle contains any one of the aforementioned lipid drug compositions.

[0371] In a specific embodiment of the present invention, preferably, the aforementioned lipid nanoparticle is an LNP - drug composition, an LPP - drug composition, or a PNP drug composition; preferably an LNP - drug composition; more preferably an LNP - nucleic acid drug composition; more preferably an LNP - mRNA composition.

[0372] 4.2. Preparation of liposomes or lipid nanoparticles

[0373] In a specific embodiment of the present invention, liposomes can be prepared by the following methods, including but not limited to the thin - film dispersion method, the ultrasonic dispersion method, the reverse - phase evaporation method, the freeze - drying method, the freeze - thaw method, the double - emulsion method, and / or the injection method, preferably the thin - film dispersion method, the ultrasonic dispersion method, and / or the reverse - phase evaporation method.

[0374] In a specific embodiment of the present invention, lipid nanoparticles can be prepared by the following methods, including but not limited to the micro - emulsion method, the double - emulsion method, the high - shear homogenization and ultrasonic method, the thin - film hydration and extrusion method, the microfluidic method.

[0375] In a specific embodiment of the present invention, liposomes are prepared by the thin - film dispersion method, and the thin - film dispersion method comprises the following steps:

[0376] (1) Weigh the cationic lipid, steroid lipid, neutral lipid, and polyethylene glycolylated lipid, dissolve them thoroughly in an organic solvent, shake well, remove the organic solvent by rotary evaporation under reduced pressure to form an oil film, and dry it with a vacuum pump to remove the organic solvent;

[0377] (2) Add a phosphate buffer solution containing a cryoprotectant, and perform ultrasonic treatment in a water bath to form a translucent emulsion;

[0378] (3) Add the emulsion to a high-pressure homogenizer for overpressure treatment, and then add the overpressure-treated emulsion to a liposome extruder for membrane filtration to form liposomes;

[0379] (4) Optionally, dry the liposomes in a freeze dryer to form liposome powder;

[0380] Among them, preferably, the organic solvent is dichloromethane, chloroform, and / or methanol, more preferably chloroform and methanol; preferably, the rotation speed of the rotary evaporation under reduced pressure is 30 - 300 rpm, more preferably 50 - 200 rpm, and most preferably 100 - 170 rpm; preferably, the temperature of the rotary evaporation under reduced pressure is 10 - 200 °C, more preferably 20 - 100 °C, and most preferably 40 - 80 °C;

[0381] Preferably, the drying time with the vacuum pump is 1 - 72 h, more preferably 5 - 48 h, and most preferably 15 - 36 h;

[0382] Preferably, the mass concentration of the cryoprotectant dissolved in the phosphate buffer solution is 0.1 - 80%, preferably 1 - 50%, and more preferably 5 - 20%;

[0383] Preferably, the frequency of the ultrasonic treatment in the water bath is 10 - 300, more preferably 30 - 200, and most preferably 60 - 150;

[0384] Preferably, the time of the ultrasonic treatment in the water bath is 0.1 - 5 h, more preferably 0.2 - 2 h, and most preferably 0.25 - 1 h;

[0385] Preferably, the pressure of the high-pressure homogenizer is 50 - 240 MPa, more preferably 80 - 200 MPa, and most preferably 100 - 150 MPa;

[0386] Preferably, the number of overpressure treatments with the high-pressure homogenizer is any integer between 1 and 50, more preferably any integer between 3 and 20, and most preferably any integer between 5 and 10;

[0387] Preferably, the pressure of the liposome extruder is 50 - 300 MPa, more preferably 80 - 250 MPa, and most preferably 120 - 200 MPa;

[0388] Preferably, the number of times of passing through the membrane of the liposome extruder is any integer between 1 and 50, more preferably any integer between 3 and 30, and most preferably any integer between 5 and 20;

[0389] Preferably, the drying time of the freeze dryer is 1 to 120 h, more preferably 5 to 72 h, and most preferably 10 to 36 h.

[0390] In a specific embodiment of the present invention, in the method for preparing liposomes, the ratio of liposomes to phosphate buffer solution dissolved with cryoprotectant can be 1 mg:(0.1 - 100) mL, preferably 1 mg:(0.3 - 50) mL, and more preferably 1 mg:(0.5 - 5) mL.

[0391] In a specific embodiment of the present invention, preferably, lipid nanoparticles are prepared by a microfluidic method, and the steps are as follows:

[0392] (1) Dissolve each lipid component in an organic solvent to obtain a lipid composition dissolved in the organic phase; the organic phase is preferably ethanol;

[0393] (2) Add the nucleic acid drug to a buffer solution to obtain an aqueous solution; the aqueous phase is preferably citrate buffer salt or sodium acetate buffer solution;

[0394] (3) Mix the organic phase solution and the aqueous solution through a microfluidic device to obtain a lipid nanoparticle composition, and purify it by ultrafiltration or the like to remove the organic solvent and free molecules.

[0395] The preparation of cationic lipids, lipid compositions, lipid nucleic acid drug compositions and the bioactivity test of lipid nucleic acid drug compositions will be further described below in conjunction with some specific examples. The specific examples are for further detailed description of the present invention and do not limit the protection scope of the present invention. Among them, in the examples for preparing cationic lipids, the final product is characterized by nuclear magnetic resonance for its structure, or the molecular weight is confirmed by MALDI-TOF or ESI-MS.

[0396] Example 1: Cationic Lipid (E1-1)

[0397]

[0398] Corresponding to the general formula (1), in E1-1, both R1 and R2 are Both B1 and B2 are hexamethylene groups, both L1 and L2 are ester groups (-C(=O)O-), X is N, L3 is ethylene group, and N ring is R3 is -CH2CH2OH, and the total molecular weight is about 850 Da.

[0399] The preparation process is as follows:

[0400] Step a: Under nitrogen protection, to a round-bottom flask containing 2-hexyldecanoic acid (S1-1, 2.05 g, 8.0 mmol), 6-bromohexanol (S1-2, 1.74 g, 9.6 mmol), and 4-(dimethylamino)pyridine (DMAP, 0.24 g, 2.0 mmol) dissolved in dichloromethane (80 mL), dicyclohexylcarbodiimide (DCC, 3.63 g, 17.6 mmol) was added, and the reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration. The filtrate was concentrated, and the residue obtained was purified by column chromatography to obtain 6-bromohexyl 2-hexyldecanoate (S1-3, 2.77 g).

[0401] Step b: Under nitrogen protection, 2-[4-(2-aminoethyl)-1-piperazinyl]ethanol (S1-4, 0.35 g, 2.0 mmol) was dissolved in acetonitrile (50 mL). Under slow stirring, S1-3 (2.10 g, 5.0 mmol) and N,N-diisopropylethylamine (DIPEA, 0.36 g, 4.0 mmol) were successively added, and the reaction was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated and then dissolved in dichloromethane. It was successively extracted with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E1-1 (1.39 g). 1 1H NMR (400 MHz, CDCl3) δ: 4.02 (t, 4H), 3.60 - 3.58 (brm, 2H), 2.77 - 2.36 (m, 18H), 2.27 (tt, 2H), 1.62 - 1.14 (m, 62H), 0.84 (t, 12H). MS (ESI): m / z = 850.88 ([M + H] + )

[0402]

[0403] Example 2: Cationic lipid (E2-1)

[0404]

[0405] Corresponding to the general formula (1), in E2-1, R1 and R2 are both B1 and B2 are both hexamethylene groups, L1 and L2 are both ester groups (-OC(=O)-), X is N, L3 is ethylene group, and N ring is R3 is -CH2CH2OH, and the total molecular weight is about 850 Da.

[0406] The preparation process is as follows:[[]]END]]

[0407] Step a: Under nitrogen protection, DCC (3.63 g, 17.6 mmol) was added to a round-bottom flask containing 7-bromoheptanoic acid (S2-1, 1.67 g, 8.0 mmol), 7-pentadecanol (S2-2, 2.19 g, 9.6 mmol) and DMAP (0.24 g, 2.0 mmol) dissolved in dichloromethane (80 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration. The filtrate was concentrated, and the residue obtained was purified by column chromatography to obtain 7-bromoheptanoic acid 1-hexylnonyl ester (S2-3, 2.79 g).

[0408] Step b: Under nitrogen protection, S1-4 (0.35 g, 2.0 mmol) was dissolved in acetonitrile (50 mL). S2-3 (2.10 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were successively added under slow stirring. The reaction was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, and extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E2-1 (1.40 g). 1 1H NMR (400 MHz, CDCl3) δ: 4.87 - 4.80 (m, 2H), 3.63 - 3.61 (m, 2H), 2.93 - 2.40 (m, 18H), 2.26 (t, 4H), 1.65 - 1.15 (m, 64H), 0.85 (t, 12H). MS (ESI): m / z = 850.88 ([M + H] + )

[0409]

[0410] Example 3: Cationic lipid (E3-1)

[0411]

[0412] Corresponding to the general formula (1), in E3-1, both R1 and R2 are Both B1 and B2 are hexamethylene groups, both L1 and L2 are carbonate groups (-OC(=O)O-), X is N, L3 is ethylene, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 882 Da.

[0413] The preparation process is as follows:[[]]

[0414] Step a: Under the protection of nitrogen, dissolve 6-bromohexyl 4-nitrophenyl carbonate (S3-1, 3.46 g, 10.0 mmol, where S3-1 is prepared by reacting p-nitrophenyl chloroformate with 6-bromohexanol) in DCM (100 mL). While stirring at room temperature, add S2-2 (9.12 g, 40.0 mmol) dropwise, and then slowly add pyridine (1.0 mL, 12.5 mmol) dropwise over 10 min. Then, add DMAP (0.24 g, 2.0 mmol) all at once. Stir the reaction at room temperature for 16 h. After the reaction is completed, extract twice with DCM and water. Combine the organic phases, wash with brine, dry with anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. The crude product is separated and purified by column chromatography. Collect the target eluate and concentrate to obtain S3-2 (1.48 g).

[0415] Step b: Under the protection of nitrogen, dissolve S1-4 (0.17 g, 1.0 mmol) in acetonitrile (20 mL). While stirring slowly, add S3-2 (1.09 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) in sequence. Stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. Combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain the cationic lipid E3-1 (0.72 g). 1 H NMR (400 MHz, CDCl3) δ: 4.71 - 4.68 (m, 2H), 4.07 (t, 4H), 3.45 (t, 2H), 2.74 - 2.49 (m, 18H), 1.72 - 1.25 (m, 64H), 0.89 (t, 12H). MS (ESI): m / z = 882.77 ([M + H] + )。

[0416]

[0417] Example 4: Cationic Lipid (E4-1)

[0418]

[0419] Corresponding to the general formula (1), in E4-1, both R1 and R2 are Both B1 and B2 are hexamethylene groups, both L1 and L2 are carbonate groups (-OC(=O)O-), X is N, L3 is ethylene group, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 910 Da.

[0420] The preparation process is as follows:[[]]

[0421] Step a: Under the protection of nitrogen, dissolve S3-1 (3.46 g, 10.0 mmol) in DCM (100 mL), add 2-hexyl-1-decanol (S4-1, 9.12 g, 40.0 mmol) dropwise under stirring at room temperature, and then slowly add pyridine (1.0 mL, 12.5 mmol) over 10 min. Then, add DMAP (0.24 g, 2.0 mmol) all at once. Stir the reaction at room temperature for 16 h. After the reaction is completed, extract twice with DCM and water, combine the organic phases, wash with brine, dry with anhydrous sodium sulfate, filter and concentrate to obtain the crude product. The crude product is purified by column chromatography, collect the target eluate, and concentrate to obtain S4-2 (1.52 g).

[0422] Step b: Under the protection of nitrogen, dissolve S1-4 (0.17 g, 1.0 mmol) in acetonitrile (20 mL), and successively add S4-2 (1.13 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) under slow stirring. Stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain the cationic lipid E4-1 (0.74 g). 1 H NMR (400 MHz, CDCl3) δ: 4.03 (t, 4H), 3.94 (d, 4H), 3.78 (brs, 2H), 3.16 - 2.95 (m, 6H), 2.93 - 2.87 (m, 6H), 2.86 - 2.72 (m, 6H), 1.81 - 1.12 (m, 66H), 0.80 (t, 12H). MS (ESI): m / z = 910.94 ([M + H] + )。

[0423]

[0424] Example 5: Cationic Lipid (E5-1)

[0425]

[0426] Corresponding to the general formula (1), in E5-1, both R1 and R2 are Both B1 and B2 are heptamethylene groups, both L1 and L2 are ester groups (-OC(=O)-), X is N, L3 is ethylene group, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 942 Da.

[0427] The preparation process is as follows:

[0428] Step a: Under nitrogen protection, to a round-bottom flask containing 8-bromooctanoic acid (S5-1, 1.78 g, 8.0 mmol) dissolved in dichloromethane (80 mL), S5-2 (2.50 g, 9.60 mmol, where S5-2 is obtained by reacting glycerol with TBS-protected hydroxyl group and bromohexane and then deprotecting), and DMAP (0.24 g, 2.0 mmol), add DCC (3.63 g, 17.6 mmol). React at room temperature for 16 h. After the reaction is completed, remove the precipitate by filtration. Concentrate the filtrate, and purify the resulting residue by column chromatography to obtain S5-3 (3.10 g).

[0429] Step b: Under nitrogen protection, dissolve S1-4 (0.35 g, 2.0 mmol) in acetonitrile (50 mL). Sequentially add S5-3 (2.33 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) under slow stirring, and stir at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence. Combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E5-1 (1.55 g). 1 H NMR (400 MHz, CDCl3) δ: 5.17 - 5.02 (m, 2H), 3.71 (t, 2H), 3.58 - 3.52 (m, 8H), 3.48 - 3.40 (m, 8H), 3.12 - 2.49 (m, 18H), 2.33 (t, 4H), 1.64 - 1.27 (m, 52H), 0.88 (t, 12H). MS (ESI): m / z = 942.85 ([M + H] + )。

[0430]

[0431] Example 6: Cationic Lipid (E6-1)

[0432]

[0433] Corresponding to the general formula (1), in E6-1, R1 and R2 are both B1 and B2 are both pentamethylene groups, L1 and L2 are both ester groups (-OC(=O)-), X is N, L3 is ethylene group, and N ring is R3 is -CH2CH2OH, and the total molecular weight is about 998 Da.

[0434] The preparation process is as follows:[[]]END]]

[0435] Step a: Under nitrogen protection, to a flask containing S6-2 (3.04 g, 9.6 mmol) dissolved in dichloromethane (80 mL), which is obtained from (obtained by aldol condensation of heptanol followed by deprotection), 6-bromohexanoic acid (S6-1, 1.56 g, 8.0 mmol) and DMAP (0.24 g, 2.0 mmol) were added to a round-bottom flask containing DCC (3.63 g, 17.6 mmol), and the reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration. The filtrate was concentrated, and the residue obtained was purified by column chromatography to give S6-3 (3.29 g).

[0436] Step b: Under nitrogen protection, S1-4 (0.35 g, 2.0 mmol) was dissolved in acetonitrile (50 mL), and S6-3 (2.47 g, 5.0 mmol) and DIPEA (0.36 g, 4.0 mmol) were sequentially added with slow stirring. The reaction was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, and extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to give cationic lipid E6-1 (1.65 g). 1 H NMR (400 MHz, CDCl3) δ: 4.64 (t, 2H), 4.07 (t, 4H), 3.52 - 3.36 (m, 10H), 2.85 - 2.35 (m, 18H), 2.30 (t, 4H), 1.70 - 1.25 (m, 62H), 0.89 (t, 12H). MS (ESI): m / z = 998.84 ([M + H] + )

[0437]

[0438] Example 7: Cationic Lipid (E7-1)

[0439]

[0440] Corresponding to the general formula (1), in E7-1, R1 is R2 is B1 is heptylene, B2 is pentylene, L1 and L2 are both ester groups (-OC(=O)-), X is N, L3 is ethylene, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 970 Da.

[0441] The preparation process is as follows:[[]]

[0442] Step a: Under nitrogen protection, dissolve S1-4 (0.35 g, 2.0 mmol) in acetonitrile (50 mL). Under slow stirring, sequentially add S5-3 (1.17 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound S7-1 (0.94 g).

[0443] Step b: Under nitrogen protection, dissolve S7-1 (0.56 g, 1.0 mmol) in acetonitrile (20 mL). Under slow stirring, sequentially add S6-3 (0.62 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E7-1 (0.81 g). 1 H NMR (400 MHz, CDCl3) δ: 5.23 - 5.18 (m, 1H), 4.64 (t, 1H), 4.08 (t, 2H), 3.52 - 3.36 (m, 14H), 2.78 - 2.64 (m, 18H), 2.30 (t, 4H), 1.70 - 1.21 (m, 58H), 0.86 (t, 12H). MS (ESI): m / z = 970.84 ([M + H] + )。

[0444]

[0445] Example 8: Cationic Lipid (E8-1)

[0446]

[0447] Corresponding to general formula (1), in E8-1, R1 is undecyl, R2 is B1 is pentamethylene, B2 is heptamethylene, L1 and L2 are both ester groups (-OC(=O)-), X is N, L3 is ethylene, and N ring is R3 is -CH2CH2OH, and the total molecular weight is about 822 Da.

[0448] The preparation process is as follows:

[0449] Step a: Under nitrogen protection, DCC (3.63 g, 17.6 mmol) was added to a round-bottom flask containing 8-bromooctanoic acid (S8-1, 1.78 g, 8.0 mmol), 1-octadecanol (S8-2, 2.46 g, 9.6 mmol) and DMAP (0.24 g, 2.0 mmol) dissolved in dichloromethane (80 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration. The filtrate was concentrated, and the residue obtained was purified by column chromatography to obtain 1-octylnonyl 8-bromooctanoate (S8-3, 3.08 g).

[0450] Step b: Under nitrogen protection, S1-4 (0.35 g, 2.0 mmol) was dissolved in acetonitrile (50 mL). S8-3 (1.16 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were successively added with slow stirring. The reaction was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, and extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound S8-4 (0.93 g).

[0451] Step c: Under nitrogen protection, S8-4 (0.55 g, 1.0 mmol) was dissolved in acetonitrile (20 mL). S8-5 (0.44 g, 1.3 mmol, where S8-5 was prepared by the reaction of 6-bromohexanoic acid and 1-undecanol) and DIPEA (0.09 g, 1.0 mmol) were successively added with slow stirring. The reaction was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, and extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E8-1 (0.67 g). 1 H NMR (400 MHz, CDCl3) δ: 4.87 - 4.80 (m, 1H), 4.03 (t, 2H), 3.70 - 3.55 (m, 2H), 2.92 - 2.47 (m, 18H), 2.27 (dt, 4H), 1.68 - 1.16 (m, 62H), 0.87 - 0.83 (m, 9H). MS (ESI): m / z = 822.79 ([M + H] + )

[0452]

[0453] Example 9: Cationic Lipid (E9-1)

[0454]

[0455] Corresponding to general formula (1), in E9-1, R1 is undecyl, and R2 is B1 is pentamethylene, B2 is heptamethylene, L1 is an ester group (-C(=O)O-), L2 is an ester group (-OC(=O)-), X is N, L3 is ethylene, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 822 Da.

[0456] The preparation process is as follows:

[0457] Under nitrogen protection, dissolve S8-4 (0.55 g, 1.0 mmol) in acetonitrile (20 mL). Under slow stirring, sequentially add S9-1 (0.44 g, 1.3 mmol, where S9-1 is prepared by the reaction of lauric acid and 5-bromo-1-pentanol) and DIPEA (0.09 g, 1.0 mmol), and stir and react at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain the cationic lipid E9-1 (0.68 g). 1 H NMR (400 MHz, CDCl3) δ: 4.91 - 4.83 (m, 1H), 4.06 (t, 2H), 3.46 (t, 2H), 2.85 - 2.44 (m, 18H), 2.30 (t, 4H), 1.67 - 1.25 (m, 62H), 0.89 (t, 9H). MS (ESI): m / z = 822.74 ([M + H] + )。

[0458]

[0459] Example 10: Cationic lipid (E10-1)

[0460]

[0461] Corresponding to general formula (1), in E10-1, R1 is undecyl, and R2 is B1 is pentamethylene, B2 is heptamethylene, L1 is an ester group (-OC(=O)-), L2 is an ester group (-C(=O)O-), X is N, L3 is ethylene, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 822 Da.

[0462] The preparation process is as follows:

[0463] Step a: Under nitrogen protection, DCC (3.63 g, 17.6 mmol) was added to a round-bottom flask containing 2-octyldecanoic acid (S10-2, 2.27 g, 8.0 mmol), 7-bromo-1-heptanol (S10-1, 1.87 g, 9.6 mmol) and DMAP (0.24 g, 2.0 mmol) dissolved in dichloromethane (80 mL). The reaction was carried out at room temperature for 16 h. After the reaction was completed, the precipitate was removed by filtration. The filtrate was concentrated, and the residue obtained was purified by column chromatography to obtain 7-bromoheptyl 2-octyldecanoate (S10-3, 3.10 g).

[0464] Step b: Under nitrogen protection, S1-4 (0.35 g, 2.0 mmol) was dissolved in acetonitrile (50 mL). S10-3 (1.16 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were successively added under slow stirring, and the reaction was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, and extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound S10-4 (0.92 g).

[0465] Step c: Under nitrogen protection, S10-4 (0.55 g, 1.0 mmol) was dissolved in acetonitrile (20 mL). S8-5 (0.44 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) were successively added under slow stirring, and the reaction was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, and extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E10-1 (0.69 g). 1 H NMR (400 MHz, CDCl3) δ: 4.09 (t, 2H), 4.05 (t, 2H), 3.45 (t, 2H), 2.82 - 2.45 (m, 18H), 2.32 - 2.27 (m, 3H), 1.69 - 1.25 (m, 62H), 0.89 (t, 9H). MS (ESI): m / z = 822.78 ([M + H] + )。

[0466]

[0467] Example 11: Cationic Lipid (E11-1)

[0468]

[0469] Corresponding to the general formula (1), in E11-1, R1 is undecyl, and R2 is B1 is a pentamethylene group, B2 is a heptamethylene group, L1 is an ester group (-OC(=O)-), L2 is a carbonate group (-OC(=O)O-), X is N, L3 is an ethylene group, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is approximately 838 Da.

[0470] The preparation process is as follows:

[0471] Step a: Under the protection of nitrogen, dissolve 7-bromoheptyl 4-nitrophenyl carbonate (S11-1, 3.60 g, 10.0 mmol, where S11-1 is prepared by reacting p-nitrophenyl chloroformate with 7-bromoheptanol) in DCM (150 mL). While stirring at room temperature, add S8-2 (10.24 g, 40.0 mmol) dropwise, and then slowly add pyridine (1.0 mL, 12.5 mmol) dropwise over 10 min. Then, add DMAP (0.24 g, 2.0 mmol) all at once. Stir the reaction at room temperature for 16 h. After the reaction is completed, extract twice with DCM and water, combine the organic phases, wash with brine, dry with anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. The crude product is separated and purified by column chromatography, collect the target eluate, and concentrate to obtain S11-2 (1.57 g).

[0472] Step b: Under the protection of nitrogen, dissolve S1-4 (0.35 g, 2.0 mmol) in acetonitrile (50 mL). While stirring slowly, add S11-2 (1.20 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) in sequence, and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound S11-3 (0.95 g).

[0473] Step c: Under the protection of nitrogen, dissolve S11-3 (0.57 g, 1.0 mmol) in acetonitrile (20 mL). While stirring slowly, add S8-5 (0.44 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) in sequence, and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain the cationic lipid E11-1 (0.70 g). 11H NMR (400 MHz, CDCl3) δ: 4.71 - 4.68 (m, 1H), 4.21 (t, 2H), 4.07 (t, 2H), 3.45 (t, 2H), 2.69 - 2.36 (m, 18H), 2.27 (t, 2H), 1.72 - 1.25 (m, 62H), 0.90 (t, 9H). MS (ESI): m / z=838.73 ([M + H] + )。

[0474]

[0475] Example 12: Cationic Lipid (E12 - 1)

[0476]

[0477] Corresponding to the general formula (1), in E12 - 1, R1 is undecyl, R2 is B1 is pentamethylene, B2 is heptamethylene, L1 is carbonate group (-OC(=O)O-), L2 is ester group (-OC(=O)-), X is N, L3 is ethylene, N ring is R3 is -CH2CH2OH, and the total molecular weight is about 838 Da.

[0478] The preparation process is as follows:

[0479] Step a: Under the protection of nitrogen, dissolve 5 - bromopentyl 4 - nitrophenyl carbonate (S12 - 1, 3.32 g, 10.0 mmol, where S12 - 1 is prepared by reacting p - nitrophenyl chloroformate with 5 - bromopentanol) in DCM (100 mL). Dropwise add 1 - undecanol (S12 - 2, 6.88 g, 40.0 mmol) under stirring at room temperature, and then slowly dropwise add pyridine (1.0 mL, 12.5 mmol) over 10 min. Then add DMAP (0.24 g, 2.0 mmol) at one time. Stir the reaction at room temperature for 16 h. After the reaction is completed, extract twice with DCM and water, combine the organic phases, wash with brine, then dry with anhydrous sodium sulfate, filter and concentrate to obtain the crude product. The crude product is separated and purified by column chromatography, collect the target eluate, and concentrate to obtain S12 - 3 (1.21 g).

[0480] Step b: Under nitrogen protection, dissolve S1-4 (0.35 g, 2.0 mmol) in acetonitrile (50 mL). Sequentially add S12-3 (0.91 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) under slow stirring, and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound S12-4 (0.76 g).

[0481] Step c: Under nitrogen protection, dissolve S12-4 (0.45 g, 1.0 mmol) in acetonitrile (20 mL). Sequentially add S8-3 (0.58 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) under slow stirring, and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E12-1 (0.72 g). 1 H NMR (400 MHz, CDCl3) δ: 4.91 - 4.82 (m, 1H), 4.15 (t, 4H), 3.45 (t, 2H), 2.80 - 2.53 (m, 18H), 2.29 (t, 2H), 1.72 - 1.25 (m, 62H), 0.89 (t, 9H). MS (ESI): m / z = 838.76 ([M+H] + )。

[0482]

[0483] Example 13: Cationic Lipid (E13-1)

[0484]

[0485] Corresponding to general formula (1), in E13-1, R1 is undecyl, R2 is B1 is pentamethylene, B2 is heptamethylene, L1 and L2 are both carbonate groups (-OC(=O)O-), X is N, L3 is ethylene, and N ring is R3 is -CH2CH2OH, and the total molecular weight is about 854 Da.

[0486] The preparation process is as follows:

[0487] Under nitrogen protection, S12-4 (0.45 g, 1.0 mmol) was dissolved in acetonitrile (20 mL). Under slow stirring, S11-2 (0.60 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) were added successively. The mixture was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, and extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the cationic lipid E13-1 (0.72 g). 1 HNMR(400MHz,CDCl3)δ:4.71-4.68(m,1H),4.21(t,2H),4.16(t,4H),3.45(t,2H),2.81-2.33(m,18H),1.72-1.25(m,62H),0.89(t,9H). MS(ESI):m / z=854.77([M+H] + )。

[0488]

[0489] Example 14: Cationic Lipid (E14-1)

[0490]

[0491] Corresponding to the general formula (1), in E14-1, R1 is undecyl, R2 is B1 is pentamethylene, B2 is heptamethylene, L1 and L2 are both ester groups (-OC(=O)-), X is N, L3 is ethylene, and N ring is R3 is -CH2CH2OH, and the total molecular weight is about 854 Da.

[0492] The preparation process is as follows:[[]]END]]

[0493] Under nitrogen protection, S17-1 (0.56 g, 1.0 mmol) was dissolved in acetonitrile (20 mL). Under slow stirring, S8-5 (0.44 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) were added successively. The mixture was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, and extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the cationic lipid E14-1 (0.72 g). 11H NMR (400 MHz, CDCl3) δ: 5.23 - 5.18 (m, 1H), 4.06 (t, 2H), 3.56 - 3.43 (m, 10H), 2.74 - 2.38 (m, 18H), 2.30 (t, 4H), 1.67 - 1.25 (m, 50H), 0.89 (t, 9H). MS (ESI): m / z = 854.73 ([M + H] + ).

[0494]

[0495] Example 15: Cationic Lipid (E15 - 1)

[0496]

[0497] Corresponding to the general formula (1), in E15 - 1, R1 is undecyl, R2 is Both B1 and B2 are pentamethylene groups, both L1 and L2 are ester groups (-OC(=O)-), X is N, L3 is ethylene group, and the N - ring is R3 is -CH2CH2OH, and the total molecular weight is about 854 Da.

[0498] The preparation process is as follows:

[0499] Step a: Under nitrogen protection, dissolve S1 - 4 (0.35 g, 2.0 mmol) in acetonitrile (50 mL). Sequentially add S6 - 3 (1.24 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) under slow stirring, and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound S15 - 1 (0.98 g).

[0500] Step b: Under nitrogen protection, dissolve S15 - 1 (0.59 g, 1.0 mmol) in acetonitrile (20 mL). Sequentially add S8 - 3 (0.44 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) under slow stirring, and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E15 - 1 (0.71 g). 11H NMR (400 MHz, CDCl3) δ: 4.64 (t, 1H), 4.20 (t, 2H), 4.06 (t, 2H), 3.56 - 3.43 (m, 6H), 2.63 - 2.37 (m, 18H), 2.28 (t, 4H), 1.89 (m, 2H), 1.67 - 1.25 (m, 54H), 0.89 (t, 9H). MS (ESI): m / z=854.76 ([M+H] + ).

[0501]

[0502] Example 16: Cationic Lipid (E6 - 1)

[0503]

[0504] Corresponding to the general formula (1), in E16 - 1, both R1 and R2 are Both B1 and B2 are hexamethylene groups, L1 is an ester group (-C(=O)O-), L2 is a carbonate group (-OC(=O)O-), X is N, L3 is an ethylene group, and the N - ring is R3 is -CH2CH2OH, and the total molecular weight is approximately 866 Da.

[0505] The preparation process is as follows:[[]]

[0506] Step a: Under nitrogen protection, dissolve S1 - 4 (0.35 g, 2.0 mmol) in acetonitrile (50 mL). With slow stirring, successively add S3 - 2 (1.09 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound S16 - 1 (0.88 g).

[0507] Step b: Under nitrogen protection, dissolve S16 - 1 (0.53 g, 1.0 mmol) in acetonitrile (20 mL). With slow stirring, successively add S1 - 3 (0.52 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E16 - 1 (0.72 g). 11H NMR (400 MHz, CDCl3) δ: 4.71 - 4.68 (m, 1H), 4.21 (t, 2H), 4.09 (t, 2H), 3.45 (t, 2H), 2.84 - 2.32 (m, 18H), 2.25 (tt, 1H), 1.70 - 1.25 (m, 64H), 0.89 (t, 12H). MS (ESI): m / z = 866.78 ([M+H] + )。

[0508]

[0509] Example 17: Cationic Lipid (E17-1)

[0510]

[0511] Corresponding to the general formula (1), in E17-1, both R1 and R2 are Both B1 and B2 are hexamethylene, L1 is an ester group (-OC(=O)-), L2 is a carbonate group (-OC(=O)O-), X is N, L3 is ethylene, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 866 Da.

[0512] The preparation process is as follows:[[]]

[0513] Under nitrogen protection, dissolve S16-1 (0.53 g, 1.0 mmol) in acetonitrile (20 mL). Slowly stir and sequentially add S2-3 (0.52 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain the cationic lipid E17-1 (0.73 g). 1 1H NMR (400 MHz, CDCl3) δ: 4.86 (t, 2H), 4.71 - 4.68 (m, 1H), 4.21 (t, 2H), 3.46 (t, 2H), 2.91 - 2.36 (m, 18H), 2.27 (tt, 1H), 1.68 - 1.25 (m, 64H), 0.89 (t, 12H). MS (ESI): m / z = 866.80 ([M+H] + )。

[0514]

[0515] Example 18: Cationic Lipid (E18-1)

[0516]

[0517] For the general formula (1), in E18-1, both R1 and R2 are Both B1 and B2 are hexamethylene groups, both L1 and L2 are ester groups (-C(=O)O-), X is N, L3 is ethylene group, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 849 Da.

[0518] Under nitrogen protection, 2-[4-(aminoethyl)-1-piperidinyl]ethanol (18-1, 0.17 g, 1.0 mmol) was dissolved in acetonitrile (20 mL). Under slow stirring, S1-3 (1.05 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were added successively, and the mixture was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, and extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain the cationic lipid E18-1 (0.69 g). 1 H NMR (400 MHz, CDCl3) δ: 4.09 (t, 4H), 3.51 (t, 2H), 2.52 - 2.35 (m, 12H), 2.25 (tt, 2H), 1.68 - 1.25 (m, 71H), 0.86 (t, 12H). MS (ESI): m / z = 849.81 ([M + H] + )。

[0519]

[0520] Example 19: Cationic lipid (E19-1)

[0521]

[0522] For the general formula (1), in E19-1, both R1 and R2 are Both B1 and B2 are hexamethylene groups, both L1 and L2 are ester groups (-OC(=O)-), X is N, L3 is ethylene group, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 849 Da.

[0523] The preparation process is as follows:

[0524] Under nitrogen protection, compound 18-1 (0.17 g, 1.0 mmol) was dissolved in acetonitrile (20 mL). Under slow stirring, S1-3 (1.05 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were added successively, and the mixture was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain cationic lipid E19-1 (0.70 g). 1 H NMR (400 MHz, CDCl3) δ: 4.87 (t, 2H), 3.50 (t, 2H), 2.57 - 2.36 (m, 12H), 2.31 (t, 4H), 1.68 - 1.25 (m, 71H), 0.88 (t, 12H). MS (ESI): m / z = 849.82 ([M+H] + )。

[0525]

[0526] Example 20: Cationic lipid (E20-1)

[0527]

[0528] Corresponding to the general formula (1), in E20-1, both R1 and R2 are Both B1 and B2 are hexamethylene groups, L1 is an ester group (-C(=O)O-), L2 is a carbonate group (-OC(=O)O-), X is N, L3 is an ethylene group, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 865 Da.

[0529] The preparation process is as follows:[[]]END]]

[0530] Step a: Under nitrogen protection, S18-1 (0.34 g, 2.0 mmol) was dissolved in acetonitrile (50 mL). Under slow stirring, S3-2 (1.09 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) were added successively, and the mixture was stirred at room temperature for about 20 h. After the reaction was completed, the reaction solution was concentrated, dissolved in dichloromethane, extracted successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound S20-1 (0.88 g).

[0531] Step b: Under nitrogen protection, dissolve S20-1 (0.53 g, 1.0 mmol) in acetonitrile (20 mL). While stirring slowly, sequentially add S1-3 (0.52 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E20-1 (0.70 g). 1 1H NMR (400 MHz, CDCl3) δ: 4.71 - 4.68 (m, 1H), 4.21 (t, 2H), 4.09 (t, 2H), 3.50 (t, 2H), 2.52 - 2.36 (m, 12H), 2.26 (tt, 1H), 1.70 - 1.25 (m, 71H), 0.89 (t, 12H). MS (ESI): m / z = 865.76 ([M+H] + )。

[0532]

[0533] Example 21: Cationic lipid (E21-1)

[0534]

[0535] Corresponding to the general formula (1), in E21-1, both R1 and R2 are Both B1 and B2 are hexamethylene groups, L1 is an ester group (-OC(=O)-), L2 is a carbonate group (-OC(=O)O-), X is N, L3 is an ethylene group, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 865 Da.

[0536] The preparation process is as follows:[[]]END]]

[0537] Under nitrogen protection, dissolve S20-1 (0.53 g, 1.0 mmol) in acetonitrile (20 mL). While stirring slowly, sequentially add S2-3 (0.52 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E21-1 (0.71 g). 1HNMR (400 MHz, CDCl3) δ: 4.87 (m, 1H), 4.71 - 4.66 (m, 1H), 4.22 (t, 2H), 3.52 (t, 2H), 2.52 - 2.36 (m, 12H), 2.31 (tt, 2H), 1.72 - 1.25 (m, 71H), 0.88 (t, 12H). MS (ESI): m / z = 865.80 ([M + H] + ).

[0538]

[0539] Example 22: Cationic Lipid (E22-1)

[0540]

[0541] Corresponding to the general formula (1), in E22-1, both R1 and R2 are Both B1 and B2 are hexamethylene groups, both L1 and L2 are carbonate groups (-OC(=O)O-), X is N, L3 is ethylene group, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 881 Da.

[0542] The preparation process is as follows:[[]]

[0543] Under nitrogen protection, dissolve compound 18-1 (0.17 g, 1.0 mmol) in acetonitrile (20 mL), and successively add S3-2 (1.09 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) under slow stirring. Stir and react at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E22-1 (0.73 g). 1 H NMR (400 MHz, CDCl3) δ: 4.73 - 4.68 (m, 2H), 4.22 (t, 4H), 3.50 (t, 2H), 2.52 - 2.34 (m, 12H), 1.73 - 1.25 (m, 71H), 0.87 (t, 12H). MS (ESI): m / z = 881.82 ([M + H] + ).

[0544]

[0545] Example 23: Cationic Lipid (E23-1)

[0546]

[0547] Corresponding to the general formula (1), in E23-1, R1 is undecyl, and R2 is B1 is pentamethylene, B2 is heptamethylene, L1 and L2 are both ester groups (-OC(=O)-), X is N, L3 is ethylene, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 821 Da.

[0548] The preparation process is as follows:

[0549] Step a: Under nitrogen protection, dissolve S18-1 (0.34 g, 2.0 mmol) in acetonitrile (50 mL). While stirring slowly, add S8-3 (1.16 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) in sequence, and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound S23-1 (0.92 g).

[0550] Step b: Under nitrogen protection, dissolve S23-1 (0.55 g, 1.0 mmol) in acetonitrile (20 mL). While stirring slowly, add S8-5 (0.44 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) in sequence, and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain the cationic lipid E23-1 (0.67 g). 1 H NMR (400 MHz, CDCl3) δ: 4.87 (m, 1H), 4.08 (t, 2H), 3.50 (t, 2H), 2.55 - 2.37 (m, 12H), 2.30 (t, 4H), 1.68 - 1.25 (m, 69H), 0.88 (t, 9H). MS (ESI): m / z = 821.75 ([M + H] + )。

[0551] Example 24: Cationic lipid (E24-1)

[0552]

[0553] Corresponding to the general formula (1), in E24-1, R1 is undecyl, and R2 is B1 is pentamethylene, B2 is heptamethylene, L1 is an ester group (-C(=O)O-), L2 is an ester group (-OC(=O)-), X is N, L3 is ethylene, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 821 Da.

[0554] The preparation process is as follows:

[0555] Under nitrogen protection, dissolve S23-1 (0.55 g, 1.0 mmol) in acetonitrile (20 mL), and successively add S9-1 (0.44 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) under slow stirring. Stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E24-1 (0.68 g). 1 H NMR (400 MHz, CDCl3) δ: 4.87 (t, 1H), 4.09 (t, 2H), 3.50 (t, 2H), 2.54 - 2.37 (m, 12H), 2.30 (t, 4H), 1.68 - 1.25 (m, 69H), 0.87 (t, 9H). MS (ESI): m / z = 821.73 ([M+H] + )

[0556]

[0557] Example 25: Cationic Lipid (E25-1)

[0558]

[0559] Corresponding to the general formula (1), in E25-1, R1 is undecyl, R2 is B1 is pentamethylene, B2 is heptamethylene, L1 is an ester group (-OC(=O)-), L2 is an ester group (-C(=O)O-), X is N, L3 is ethylene, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 821 Da.

[0560] The preparation process is as follows:

[0561] Step a: Under nitrogen protection, dissolve S18-1 (0.34 g, 2.0 mmol) in acetonitrile (50 mL). While stirring slowly, sequentially add S8-5 (0.87 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound S25-1 (0.73 g).

[0562] Step b: Under nitrogen protection, dissolve S25-1 (0.44 g, 1.0 mmol) in acetonitrile (20 mL). While stirring slowly, sequentially add S10-3 (0.58 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E25-1 (0.68 g). 1 H NMR (400 MHz, CDCl3) δ: 4.09 (t, 2H), 4.06 (t, 2H), 3.51 (t, 2H), 2.55 - 2.37 (m, 12H), 2.30 (t, 2H), 2.26 (tt, 1H), 1.68 - 1.25 (m, 69H), 0.89 (t, 9H). MS (ESI): m / z = 821.73 ([M + H] + )。

[0563]

[0564] Example 26: Cationic Lipid (E26-1)

[0565]

[0566] Corresponding to general formula (1), in E26-1, R1 is undecyl, R2 is B1 is pentamethylene, B2 is heptamethylene, L1 is an ester group (-OC(=O)-), L2 is a carbonate group (-OC(=O)O-), X is N, L3 is ethylene, and N ring is R3 is -CH2CH2OH, and the total molecular weight is about 837 Da.

[0567] The preparation process is as follows:

[0568] Under nitrogen protection, dissolve S25-1 (0.44 g, 1.0 mmol) in acetonitrile (20 mL). Under slow stirring, sequentially add S11-2 (0.60 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E26-1 (0.70 g). 1 HNMR(400MHz,CDCl3)δ:4.71-4.68(m,1H),4.20(t,2H),4.08(t,2H),3.50(t,2H),2.52-2.39(m,12H),2.30(t,2H),1.73-1.25(m,69H),0.88(t,9H). MS(ESI):m / z=837.76([M+H] + )。

[0569]

[0570] Example 27: Cationic Lipid (E27-1)

[0571]

[0572] Corresponding to the general formula (1), in E27-1, R1 is undecyl, R2 is B1 is pentamethylene, B2 is heptamethylene, L1 is carbonate group (-OC(=O)O-), L2 is ester group (-OC(=O)-), X is N, L3 is ethylene, N ring is R3 is -CH2CH2OH, and the total molecular weight is about 837 Da.

[0573] The preparation process is as follows:

[0574] Step a: Under nitrogen protection, dissolve S18-1 (0.34 g, 2.0 mmol) in acetonitrile (50 mL). Under slow stirring, sequentially add S12-3 (0.91 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound S27-1 (0.75 g).

[0575] Step b: Under nitrogen protection, dissolve S27-1 (0.46 g, 1.0 mmol) in acetonitrile (20 mL). Under slow stirring, sequentially add S8-3 (0.58 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E27-1 (0.69 g). 1 1H NMR (400 MHz, CDCl3) δ: 4.88 (t, 1H), 4.16 (t, 4H), 3.50 (t, 2H), 2.52 - 2.33 (m, 12H), 2.30 (t, 2H), 1.68 - 1.25 (m, 69H), 0.90 (t, 9H). MS (ESI): m / z = 837.73 ([M + H] + )。

[0576]

[0577] Example 28: Cationic Lipid (E28-1)

[0578]

[0579] Corresponding to general formula (1), in E28-1, R1 is undecyl, R2 is B1 is pentamethylene, B2 is heptamethylene, L1 and L2 are both carbonate groups (-OC(=O)O-), X is N, L3 is ethylene, and the N ring is R3 is -CH2CH2OH, and the total molecular weight is about 853 Da.

[0580] The preparation process is as follows:[[]]END]]

[0581] Under nitrogen protection, dissolve S27-1 (0.46 g, 1.0 mmol) in acetonitrile (20 mL). Under slow stirring, sequentially add S11-2 (0.60 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E28-1 (0.71 g). 11H NMR (400 MHz, CDCl3) δ: 4.71 - 4.68 (m, 1H), 4.23 (t, 2H), 4.16 (t, 4H), 3.51 (t, 2H), 2.53 - 2.36 (m, 12H), 1.73 - 1.25 (m, 69H), 0.88 (t, 9H). MS (ESI): m / z = 853.78 ([M+H] + )。

[0582]

[0583] Example 29: Cationic Lipid (E29-1)

[0584]

[0585] Corresponding to the general formula (1), in E29-1, R1 is undecyl, R2 is B1 is pentamethylene, B2 is heptamethylene, L1, L2 are ester groups (-OC(=O)-), X is N, L3 is ethylene, and N ring is R3 is -CH2OH, and the total molecular weight is about 809 Da.

[0586] The preparation process is as follows:

[0587] Step a: Under nitrogen protection, dissolve 4-(2-aminoethyl)-2-(hydroxymethyl)morpholine (S29-1, 0.32 g, 2.0 mmol) in acetonitrile (50 mL). Under slow stirring, successively add S8-5 (0.87 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound S29-1 (0.71 g).

[0588] Step b: Under nitrogen protection, dissolve S29-1 (0.43 g, 1.0 mmol) in acetonitrile (20 mL). Under slow stirring, successively add S8-3 (0.58 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol), and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it in dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E29-1 (0.66 g). 11H NMR (400 MHz, CDCl3) δ: 4.87 (t, 1H), 4.07 (t, 2H), 3.85 - 3.50 (m, 5H), 2.72 - 2.20 (m, 16H), 1.68 - 1.25 (m, 62H), 0.89 (t, 9H). MS (ESI): m / z = 809.73 ([M+H] + )。

[0589]

[0590] Example 30: Cationic Lipid (E30-1)

[0591]

[0592] Corresponding to the general formula (1), in E30-1, R1 is undecyl, R2 is B1 is pentamethylene, B2 is heptamethylene, L1 is an ester group (-OC(=O)-), L2 is a carbonate group (-OC(=O)O-), X is N, L3 is ethylene, N ring is R3 is -CH2OH, and the total molecular weight is about 825 Da.

[0593] The preparation process is as follows:

[0594] Under nitrogen protection, dissolve S29-1 (0.43 g, 1.0 mmol) in acetonitrile (20 mL), and successively add S11-2 (0.60 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) under slow stirring. Stir and react at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it successively with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain the cationic lipid E30-1 (0.69 g). 1 1H NMR (400 MHz, CDCl3) δ: 4.72 - 4.67 (m, 1H), 4.21 (t, 2H), 4.05 (t, 2H), 3.84 - 3.50 (m, 5H), 2.73 - 2.20 (m, 14H), 1.73 - 1.25 (m, 62H), 0.89 (t, 9H). MS (ESI): m / z = 825.73 ([M+H] + )。

[0595]

[0596] Example 31: Cationic Lipid (E31-1)

[0597]

[0598] Corresponding to General Formula (1), in E31-1, R1 is undecyl, and R2 is B1 is pentamethylene, B2 is heptamethylene, L1 is carbonate group (-OC(=O)O-), L2 is ester group (-OC(=O)-), X is N, L3 is ethylene, and N ring is R3 is -CH2OH, and the total molecular weight is about 825 Da.

[0599] The preparation process is as follows:

[0600] Step a: Under nitrogen protection, dissolve S29-1 (0.32 g, 2.0 mmol) in acetonitrile (50 mL). While stirring slowly, add S12-3 (0.91 g, 2.5 mmol) and DIPEA (0.18 g, 2.0 mmol) in sequence, and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain compound S31-1 (0.74 g).

[0601] Step b: Under nitrogen protection, dissolve S31-1 (0.45 g, 1.0 mmol) in acetonitrile (20 mL). While stirring slowly, add S8-3 (0.58 g, 1.3 mmol) and DIPEA (0.09 g, 1.0 mmol) in sequence, and stir the reaction at room temperature for about 20 h. After the reaction is completed, concentrate the reaction solution, dissolve it with dichloromethane, extract it with 0.6 M hydrochloric acid / 10% sodium chloride solution and saturated sodium bicarbonate solution in sequence, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify by column chromatography to obtain cationic lipid E31-1 (0.69 g). 1 H NMR (400 MHz, CDCl3) δ: 4.86 (t, 1H), 4.16 (t, 4H), 3.85 - 3.50 (m, 5H), 2.73 - 2.20 (m, 14H), 1.73 - 1.25 (m, 62H), 0.88 (t, 9H). MS (ESI): m / z = 825.69 ([M + H] + )。

[0602]

[0603] Example 32: Preparation of Lipid Drug Composition (LNP-mRNA Composition) and Testing of Its Physicochemical Properties

[0604] Example 32.1: Preparation of LNP-mRNA Composition

[0605] In this example, multiple groups of LNP-mRNA compositions were prepared for comparison. The cationic lipids in each group were different, but the phospholipids were all DSPC, the sterol lipids were all cholesterol, and the polyethylene glycolated lipids were all PEG2k-DMG. Among them, the cationic lipids in the experimental group series (L-1 to L-28) were the cationic lipids prepared in the examples of this application; another positive control group (L-CT1 and L-CT2) was set: the cationic lipids were MC3 and (prepared according to the method disclosed in CN114206827A); specifically, as shown in Table 1.

[0606] The preparation method of the LNP-mRNA composition is as follows:

[0607] Dissolve the ionizable cationic lipid, DSPC, cholesterol, and polyethylene glycolated lipid in ethanol according to a certain molar ratio (50:10:38:1.5 or 48:9:42:1.5) to obtain an ethanol-phase solution; add Fluc-mRNA to 50 mM citrate buffer (pH = 4) according to an N / P ratio of 6:1 to obtain an aqueous-phase solution; mix the ethanol-phase lipid solution and the mRNA aqueous-phase solution with a volume ratio of 1:3 using a microfluidic device, and wash by ultrafiltration with DPBS multiple times to remove ethanol and free molecules, and finally filter through a 0.2 μm sterile filter to obtain the LNP-mRNA composition. The results are shown in Table 1 below.

[0608] Example 32.2: Physicochemical property test of LNP-mRNA composition

[0609] Encapsulation efficiency determination: In this example, the Quant-it Ribogreen RNA quantification assay kit was used to determine the encapsulation efficiency of the LNP-mRNA composition. The results showed that the lipid composition of the present invention had a high encapsulation efficiency for nucleic acid drugs (mRNA), all within the range of 80%-95%, and most of the encapsulation efficiencies were within the range of 90%-95%. The results are shown in Table 1 below. The results indicate that the lipid compositions prepared from the cationic lipids of each experimental group can well encapsulate mRNA, showing an encapsulation efficiency equivalent to or better than that of existing cationic lipids, and there are also differences in the encapsulation efficiencies of different cationic lipids, but the differences are not significant.

[0610] Particle size determination: In this example, the particle size of LNP-mRNA was determined by dynamic light scattering (DLS). The results are shown in Table 1 below. The measured LNP-mRNA had a high size uniformity, and its PDI was less than 0.3. The particle size of the LNP-mRNA prepared from the lipid composition of this application was within the range of 90-150 nm.

[0611] Example 33: Biological activity test of LNP-mRNA composition

[0612] (1) Study on cytotoxicity (biocompatibility)

[0613] The MTT staining method was used to test the cytotoxicity of the LNP-mRNA composition preparation of the present invention. The LNP-mRNA composition preparation was dissolved in the culture medium to prepare the required concentration. When necessary, an appropriate amount of cosolvent could be added. Hela cells were used as the cell model, and the seeding density was 1×10 4 cells / well. The cell suspension was inoculated into a 96-well plate at 100 μL / well. After inoculation, it was incubated in a cell culture incubator at 37 °C and 4% CO2 for 24 h. Then, the old culture medium was aspirated, and 100 μL of the culture medium containing 3.3 μg / mL of LNP-mRNA (lipid nucleic acid drug composition preparation, prepared in Example 32.1) was added to each well. 100 μL of fresh culture medium was added to the blank control group. Each concentration in each group had 6 replicates. After the LNP-mRNA composition preparation was co-incubated with Hela cells for 24 h, 20 μL of PBS buffer containing 5 mg / mL of MTT was added to each well. After MTT was incubated with Hela cells for 4 h, the mixed solution of the culture medium and MTT buffer was aspirated, and 150 μL / well of DMSO was added to dissolve the purple crystal formazan of living cells. After sufficient oscillation, the absorbance at 490 nm was detected with an enzyme-linked immunosorbent assay (ELISA) reader. According to the measured absorbance values, the calculation results showed that compared with the blank control group, the cell survival rate of the LNP-mRNA composition prepared with the cationic lipid of the present invention was greater than 95%, indicating that the cationic lipid of the present invention had good biocompatibility and biosafety.

[0614] Table 1 - Summary table of the formulations of each lipid composition and the particle size and encapsulation efficiency of the LNP-mRNA prepared therefrom

[0615]

[0616]

[0617] (2) Serum stability evaluation

[0618] The LNP-mRNA composition was added to the culture medium containing 10% fetal bovine serum (FBS), and stirred at 37 °C. Samples were taken at regular intervals to measure the particle size change of LNP-mRNA, and the serum stability of the LNP-mRNA composition preparation was analyzed by testing its particle size change. The experimental results showed that within 7 days, the particle size change of both the control group and the experimental group was less than 10%. In particular, the particle size change of the experimental groups L-1, L-2, L-8, L-9, L-10, L-14, and L-15 was less than 5%, indicating that the LNP-mRNA composition preparation prepared with the cationic lipid of the present invention had good serum stability.

[0619] Table 2: Results of cell transfection test

[0620] Serial number Relative fluorescence value Serial number Relative fluorescence value Serial number Relative fluorescence value Blank 1 L-9 3.3 L-20 3.2 L-CT1 2.0 L-10 3.2 L-21 2.8 L-CT2 1.2 L-11 2.7 L-22 2.3 L-1 4.1 L-12 2.8 L-23 2.9 L-2 3.1 L-13 2.6 L-24 2.7 L-3 2.7 L-14 3.1 L-25 2.3 L-4 2.5 L-15 3.2 L-26 2.2 L-5 3.0 L-16 3.5 L-27 2.9 L-6 3.2 L-17 2.6 L-28 2.8 L-7 3.1 L-18 3.7 L-8 3.3 L-19 2.7

[0621] (3) Study on cell transfection activity

[0622] To investigate the mRNA transfection efficiency of each group of LNP-mRNA compositions prepared in Example 31 of the present invention at the cellular level, Luciferase bioluminescence was used for testing. The LNP / Fluc-mRNA composition preparation was dissolved in the culture medium to prepare the required dose. Hela cells were used as the cell model, and the cell suspension was inoculated into a 96-well plate with a black-edge transparent bottom at a seeding density of 6000 cells / well at 100 μL / well. After inoculation, the cells were incubated in a cell culture incubator for 24 h, 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. After 24 h of transfection, the old culture medium was removed and replaced with a new culture medium containing the substrate of sodium D-luciferin (1.5 mg / mL). After incubating for 5 minutes, the bioluminescence was detected using a microplate reader. The stronger the fluorescence, the more Fluc-mRNA was transported into the cytoplasm and translated into the corresponding fluorescent protein. The experimental results are shown in Table 2. Among them, the relative fluorescence intensity is the ratio of the fluorescence intensity value of each group to the fluorescence intensity of the blank control group. The results show that the LNP-mRNA compositions prepared by the present invention all have excellent in vitro transfection effects, that is, the LNPs in the experimental groups are all effective delivery vectors. Among them, in the experimental groups L-1 and L-18, the relative fluorescence value of L-1 is higher; in the experimental groups L-8 and L-23, the relative fluorescence value of L-8 is higher. It shows that when the nitrogen-containing heterocycle in the polar head of the cationic lipid is a piperazine ring or a piperidine ring, the cationic lipid containing a piperazine ring has a better transfection effect, probably because the piperazine ring contains two ionizable tertiary amines; the hydrophobic ends of the cationic lipids used in L-1, L-16 and L-18 with relatively high relative fluorescence values are all double-branched. It is preliminarily speculated that the cationic lipids with double-branched hydrophobic ends have better delivery effects.

[0623] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, shall be equally included in the patent protection scope of the present invention.

[0624] For those skilled in the art, the present invention can be implemented within a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experiments. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including changes made by using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. A cationic lipid, the structure of which is selected from any one of the following structures:

2. A lipid composition, characterized in that, Comprising the cationic lipid described in Claim 1.

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

4. The lipid composition according to claim 2, characterized in that, It also contains three lipids, namely phospholipids, steroid lipids, and polyethylene glycolated lipids, simultaneously.

5. The lipid composition according to claim 3, characterized in that, The phospholipids are selected from any one of 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-dielaidoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecenoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-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-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dioleoyl phosphatidylserine, dipalmitoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylethanolamine, distearoyl-phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphoethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine and their combinations; Or the steroid lipids are selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and their combinations; Alternatively, the polyethylene glycolated lipid is selected from polyethylene glycol-1,2-dimyristoyl glycerol, polyethylene glycol-distearoyl phosphatidylethanolamine, PEG-cholesterol, polyethylene glycol-diacylglycerol, polyethylene glycol-dialkoxypropyl, specifically including any one of polyethylene glycol 500-dipalmitoyl phosphatidylcholine, polyethylene glycol 2000-dipalmitoyl phosphatidylcholine, polyethylene glycol 500-stearoyl phosphatidylethanolamine, polyethylene glycol 2000-distearoyl phosphatidylethanolamine, polyethylene glycol 500-1,2-oleoyl phosphatidylethanolamine, polyethylene glycol 2000-1,2-oleoyl phosphatidylethanolamine, and polyethylene glycol 2000-2,3-dimyristoyl glycerol and its combinations.

6. The lipid composition according to any one of claims 3-5, characterized in that, It contains 20-80% of the cationic lipid described in claim 1, and also contains any one, any two, or any three of 5-15% of phospholipids, 25-65% of steroid lipids, and 0.5-10% of polyethylene glycolated lipid, and the percentages are the molar percentages of each lipid in the total lipids in the solution containing the solvent.

7. The lipid composition according to any one of claims 3-5, characterized in that, The molar percentage of the cationic lipid in the total lipids in the solution containing the solvent is 30-65%.

8. The lipid composition according to claim 7, wherein The molar percentage of the cationic lipid in the total lipids in the solution containing the solvent is 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 55%.

9. The lipid composition according to any one of claims 3 to 5, characterized in that, The molar percentage of the phospholipid in the total lipids in the solution containing the solvent is 7.5-13%.

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

11. The lipid composition according to any one of claims 3-5, characterized in that, The molar percentage of the steroid lipid in the total lipids in the solution containing the solvent is 35-50%.

12. The lipid composition according to claim 11, characterized in that, The molar percentage of the steroid lipid in the total lipids in the solution containing the solvent is 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.

13. The lipid composition according to any one of claims 3-5, characterized in that, The molar percentage of the polyethylene glycolated lipid in the total lipids in the solution containing the solvent is 0.5-5%.

14. The lipid composition according to claim 13, wherein, The molar percentage of the polyethylene glycolated lipid in the total lipids in the solution containing the solvent is 1-3%.

15. The lipid composition according to claim 13, characterized in that, The molar percentage of the polyethylene glycolated lipid in the total lipids in the solution containing the solvent is 1.5%, 1.6%, 1.7%, 1.8%, 1.9%.

16. A lipid pharmaceutical composition, characterized in that, It contains the lipid composition described in any one of claims 3-15 and a drug, and the drug is selected from any one of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs, or protein drugs.

17. The lipid pharmaceutical composition according to claim 16, wherein, The nucleic acid drug is selected from any one of RNA, DNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir, and ribozyme, and the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA, and siRNA.

18. The lipid pharmaceutical composition according to claim 17, characterized in that, The nucleic acid drug is any one of DNA, mRNA, miRNA, and siRNA.

19. The lipid pharmaceutical composition according to claim 16, wherein The lipid drug composition is used for the preparation of drugs, and is selected from any one of the following drugs: anti-tumor agents, antiviral agents, anti-fungal agents, and vaccines.

20. A lipid pharmaceutical composition preparation, characterized in that, Comprising the lipid pharmaceutical composition according to any one of claims 16-19 and a pharmaceutically acceptable diluent or excipient.

21. The lipid pharmaceutical composition preparation according to claim 20, characterized in that, The diluent or excipient is any one of deionized water, ultrapure water, phosphate buffer solution, and physiological saline.

22. The lipid pharmaceutical composition preparation according to claim 20, characterized in that, The diluent or excipient is phosphate buffer solution or physiological saline.

23. The lipid pharmaceutical composition preparation according to claim 20, characterized in that, The diluent or excipient is physiological saline.

24. A liposome or lipid nanoparticle, characterized in that, Comprising the lipid pharmaceutical composition according to any one of claims 16-19.

25. The liposome or lipid nanoparticle according to claim 24, wherein, The lipid nanoparticles are LNP-drug composition, LPP-drug composition, or PNP-drug composition.

26. The liposome or lipid nanoparticle according to claim 24, wherein The lipid nanoparticles are LNP-drug composition.

27. The liposome or lipid nanoparticle according to claim 24, wherein The lipid nanoparticles are LNP-nucleic acid drug composition.

28. The liposome or lipid nanoparticle according to claim 24, characterized in that, The lipid nanoparticles are LNP-mRNA composition.

Images