Cationic lipid with side chain containing functional group and application thereof

Abstract

The invention discloses a novel cationic lipid and a pegylated derivative thereof, and a cationic liposome, a cationic liposome nucleic acid pharmaceutical composition and a cationic liposome nucleic acid pharmaceutical composition preparation containing the cationic lipid and the pegylated derivative. The invention provides an ionizable lipid compound with a structure as shown in a formula (1), the compound is less ionized or electrically neutral under physiological pH, has a greater degree of ionization under a slightly acidic condition, and has lower systemic circulation toxicity and higher endosomal escape ability; the polar head of the compound is provided with an ionizable tertiary amine group, a side chain can be provided with a functional group, and a tail chain can be provided with an easily degradable connecting bond; the cationic liposome containing the compound has relatively high nucleic acid drug compounding capacity and serum stability, has no obvious cytotoxicity, and also has high transfection efficiency. Compared with the traditional liposome, the cationic liposome provided by the invention has higher nucleic acid delivery and transfection efficiency, and can be used as a novel carrier functional reagent in nucleic acid drugs and vaccines. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of drug delivery, and specifically relates to a pharmaceutical carrier cationic lipid, and in particular to a cationic lipid with a functional group on the side chain, as well as a lipid composition comprising the cationic lipid, a lipid pharmaceutical composition, and a preparation and application thereof. Background Art

[0002] In addition to negatively charged mRNA, LNP also contains four components: ionizable cationic lipids, neutral co-lipids, sterol lipids and PEGylated lipids. Among them, cationic lipids interact with negatively charged mRNA through electrostatics. Neutral co-lipids are generally phospholipids, which prevent lipid oxidation or connect ligands to the surface of liposomes or reduce the aggregation of lipid particles. Sterol lipids have strong membrane fusion properties and promote mRNA intracellular uptake and cytoplasmic entry. PEGylated lipids are located on the surface of lipid nanoparticles, improving their hydrophilicity, avoiding rapid clearance by the immune system, preventing particle aggregation, and increasing stability. Among the four lipids used to prepare LNP, the most critical one is the ionizable cationic lipid, which is not ionized or weakly ionized under physiological conditions and is basically electrically neutral, avoiding excessive interference with the biological membrane in the systemic circulation, thereby reducing toxicity. After being taken up by cells, it can ionize under the acidic conditions of the endosomal cavity and carry partial positive charge, increasing membrane permeability. At the same time, the liposomes are degraded or reorganized, promoting the escape of mRNA from the endosomes and releasing the mRNA into the cytoplasm for further translation, thereby completing the delivery and transfection of the mRNA molecules.

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

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

[0005] The present invention provides a novel cationic lipid, and a lipid composition, a lipid pharmaceutical composition and a preparation thereof comprising the cationic lipid, which are used in the field of drug delivery. The cationic lipid of the present invention has a cationizable nitrogen-branched polar head, and in particular, the polar head has a side chain containing a functional group. The lipid composition prepared by the cationic lipid of the present invention has a high nucleic acid drug complexing ability and good in vivo stability, and can fully exert the efficacy of nucleic acid drugs without causing obvious cytotoxicity, thereby improving the immune or therapeutic effect.

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

[0007] An embodiment of the present invention:

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

[0009]

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

[0011] L 1 , L 2 Each is independently a connecting bond or a divalent connecting group;

[0012] L 3 is a trivalent connecting group, and R 3 The side chain is R 3 The divalent linking group -L 3 (R 3 )-;

[0013] B 1 , B 2 Each independently is a connecting bond or C 1-30 Alkylene;

[0014] R 1 , R 2 Each independently is C 2-30 Aliphatic hydrocarbon or C containing 1-2 O 2-30 Aliphatic hydrocarbon derivative residues;

[0015] R 3 For-OR d 、-NR d R d 、-SR d 、-(C=O)R d 、-(C=O)OR d 、-O(C=O)R d 、-O(C=O)OR d or Among them, R dEach occurrence is independently C 1-12 Alkyl, G 1 is a branched group with a valence of k+1, j is 0 or 1, F 1 Contains functional group R 01 ; When j is 0, G 1 does not exist; when j is 1, G 1 Introduce k F 1 , and k F 1 Each independently has the same or different structure, and k is an integer from 2 to 8;

[0016] R 4 is a carbocyclic group, a heterocyclic group or R'N(R')-R"-N(R')-; wherein the heterocyclic group is a cyclic group whose ring atoms contain 1, 2 or more heteroatoms, and the heteroatoms are B, O, N, Si, P or S; wherein R' is independently H or C each time it appears 1-3 Alkyl, R" is C 2-4 Alkylene;

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

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

[0019] The present invention also provides another embodiment:

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

[0021] The present invention also provides another embodiment:

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

[0023] The present invention also provides another embodiment:

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

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

[0026] The novel cationic lipid compound of the present invention is a cationic lipid containing a functional group in the polar head side chain, which enriches the types of cationic lipids and provides more choices for the construction of lipid nanoparticles. Specifically, it can be applied to the delivery of nucleic acid drugs, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs, etc., thereby improving the therapeutic and / or diagnostic effects of these drugs as preventive and / or therapeutic agents. The number, type and position of the functional groups in the side chains of cationic lipids are all important entry points for regulating the properties of lipid nanoparticles such as cell uptake efficiency, stability under different physiological environments, biomolecule affinity, drug binding force and drug delivery efficiency. In particular, the cationic lipid of the present invention can not only be used in conjunction with conventional PEGylated lipids to make lipid nanoparticles have a suitable size, excellent nucleic acid drug complexing ability and drug encapsulation rate, and have both serum stability and low toxicity, so as to achieve safe and efficient nucleic acid drug delivery and transfection; in addition, the present invention also provides a structurally similar PEGylated lipid derived from the cationic lipid of the present invention, which can be used in conjunction with the cationic lipid of the present invention to further improve the stability of lipid nanoparticles and drug delivery efficiency.

[0027] Implementation

[0028] Terminology

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

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

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

[0032] In the present invention, when at least two items are listed, the "combination" of the listed items refers to the combination of any two or more of the aforementioned listed items; and the number of items is not limited, the number of any item can be one or more than one, and when the number of the same item is greater than 1, it can be the same or different specific forms that satisfy the item. For example, "L is a connecting bond or selected from alkylene, divalent hydrocarbon group containing heteroatoms, -O-, -S-, -SS-, -C(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -NH-, any one or a combination of any one or more", L can be any one of the listed items, or any two or a combination of any two or more, and the combination can be -CH 2 -O-CH 2 -(i.e., the combination of -O- and alkylene in the listed items, wherein the specific form of alkylene is 2 methylene groups), or -CH 2 -NH-CH 2 CH 2 - (i.e., the combination of -NH- and an alkylene group in the listed items, wherein the specific form of the alkylene group is 1 methylene group and 1 ethylene group). In particular, the combination of a linker and any linking group is still the linking group itself, and the "linking group" in the present invention defaults to containing at least one atom.

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

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

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

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

[0037] In the present invention, for polymer molecular weight, "about" or "around" generally refers to a numerical range of ±10%, which can be enlarged to ±15% in some cases, but not more than ±20%. The preset value is used as the base. For example, the deviations between 11kDa, 12kDa and 10kDa are 10% and 20% respectively, and "about 10kDa" includes but is not limited to 11kDa and 12kDa. For another example, when the molecular weight of a PEG component in the general formula is specified to be about 5kDa, the corresponding molecular weight or number average molecular weight is allowed to vary within the range of 5kDa±10%, that is, 4500 to 5500Da.

[0038] In the present invention, for percentages, when the numerical value given (excluding the percentage sign) is accurate to the Nth decimal place, "about" or "approximately" generally means ±(0.5*0.1 N )% of the numerical range. For example, about 1% means 1±(0.5*0.1 0 )% means the range of 0.5%-1.5%, and about 2.2% means 2.2±(0.5*0.1 1 )% is 2.15%-2.25%, about 2.33% refers to 2.2±(0.5*0.1 2 )% that is, the range of 2.335%-2.325%.

[0039] In the present invention, the degree of polymerization of the polyethylene glycol chain is represented by n i Indicates (i is a natural number selected from 1, 2, 3, ...), n with the same value in the same structure i Interchangeable expressions, for example, n 2 ≈n 3 When n 2 Use n 3 It means that n 3 Use n 2 express.

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

[0041] In the present invention, when the structure of a group is represented, To mark the connection key, such as represents a group -G or G-, Represents the group -CH 3 or CH 3 -.

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

[0043] In the present invention, the range of carbon atoms in a group is marked in the subscript form at the subscript position of C, indicating the number of carbon atoms in the group. Unless otherwise specified, the carbon number does not include the contribution of substituents. For example, C 1-12 means "having 1 to 12 carbon atoms". 1-10 Alkylene refers to any alkylene group with carbon atoms in the range indicated by the subscript, i.e., C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 Any of the alkylene groups, including but not limited to straight chain C 1-10 Alkylene (e.g. -(CH 2 ) 6 -) and branched C 1-10 Alkylene (e.g. -(CH 2 ) 3 -CH(CH 3 )-(CH 2 ) 3 -). Another example is, "substituted C 1-12 "Alkyl" refers to C1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 or C 12 The alkyl group is obtained by replacing at least one hydrogen atom of the alkyl group with a substituent, and the number of carbon atoms and heteroatoms in the substituent is not particularly limited.

[0044] In the present invention, when the structure involved has isomers, it can be any one of the isomers unless otherwise specified. For example, for a structure with cis-trans isomers, it can be either a cis structure or a trans structure; for a structure with E / Z isomers, it can be either an E structure or a Z structure; and when optically active, it can be either levorotatory or dextrorotatory.

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

[0046] In the present invention, the molecular weight of polyethylene glycol and its derivatives refers to the average molecular weight by default, and when there is no special provision, the "average molecular weight" generally refers to the number average molecular weight (M n ). For the number average molecular weight, it can be the molecular weight of a polydisperse block or substance, or the molecular weight of a monodisperse block or substance. Unless otherwise specified, the unit of measurement of molecular weight is Dalton (Da). The molecular weight of the polyethylene glycol chain can also be characterized by the "degree of polymerization", which specifically refers to the number of repeating units (ethylene oxide units). Accordingly, it is preferred to use "average degree of polymerization" or "number average degree of polymerization" to characterize the average value or number average of the number of repeating units.

[0047] In the present invention, the "any suitable" in "any suitable linking group", "any suitable reactive group", etc. refers to a structure that conforms to the basic principles of chemical structure and enables the preparation method of the present invention to be smoothly implemented. The chemical structure described in this way can be regarded as having a clear and definite scope.

[0048] In the present invention, "stable existence" and "degradable" of a group are a pair of relative concepts.

[0049] In the present invention, "degradable" refers to the breaking of the chemical bond of the invention, and the breaking is at least two residues independent of each other. If the structure is changed after chemical changes, but the entire linker is still only a complete linker, then the linker still belongs to the category of "can stably exist". The degradable conditions are not particularly limited, and can be physiological conditions in vivo, or simulated physiological environments or other conditions in vitro, preferably physiological conditions in vivo and simulated physiological conditions in vitro. The physiological conditions are not particularly limited, including but not limited to serum, heart, liver, spleen, lung, kidney, bone, muscle, fat, brain, lymph nodes, small intestine, gonads and other parts, can refer to cells, can also refer to extracellular matrix, can refer to normal physiological tissues, can also refer to pathological physiological tissues (such as tumors, inflammation, etc.). The simulated environment in vitro is not particularly limited, including but not limited to physiological saline, buffer, culture medium, etc. The degradable speed is not particularly limited, for example, it can be rapid degradation under the action of enzymes, or it can refer to slow hydrolysis under physiological conditions, etc. The physiological conditions in vivo include physiological conditions during treatment, such as ultraviolet irradiation, hyperthermia, etc. Including but not limited to being degradable under conditions such as light, heat, low temperature, enzyme, redox, acid, alkalinity, physiological conditions, in vitro simulated environment, etc., preferably degradable under conditions such as light, heat, enzyme, redox, acid, alkalinity, etc. The degradable means that degradation occurs under the stimulation of any of the above conditions. The light conditions include but are not limited to light conditions such as visible light, ultraviolet light, infrared light, near infrared light, and mid-infrared light. The thermal conditions refer to temperatures higher than normal physiological temperatures, usually 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 temperatures, preferably lower than 25°C, more preferably ≤10°C, specifically 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 enzymes that can be generated under physiological conditions are all included, such as peptidases, proteases, lyases, etc. The redox conditions are not particularly limited, such as redox transitions and hydrogenation reduction transitions between sulfhydryl groups and disulfide bonds. The acidic and alkaline conditions mainly refer to the pH conditions of normal tissues, diseased tissues, organs or tissues in the treatment period, etc., such as the stomach is acidic, and the tumor site is often acidic. Degradable here means that it can be degraded by metabolic effects in the body (such as physiological effects, such as enzymes, such as oxidation-reduction, etc.), degraded due to microenvironmental stimulation in specific parts of the body (such as acidity, alkalinity), or degraded under clinical treatment stimulation (such as light, heat, and low temperature). It should be noted that some extreme conditions in organic chemistry relative to organisms, such as bond breakage under strong acid, strong base, high temperature (such as above 100°C), are not included in the scope of the degradable conditions of the present invention. For example, although the ether bond can be broken under strong acid conditions such as hydrobromic acid, it is always classified as a linking group that can exist stably in the present invention.

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

[0051] In the present invention, for the same linking group, "can exist stably" is not an absolute concept. For example, an amide bond is much more stable than an ester bond under acidic or alkaline conditions. The "can exist stably" linking group in the present invention includes an amide bond. However, for example, a peptide bond, an amide bond formed by dehydration condensation of an α-carboxyl group of an amino acid molecule and an α-amino group of an amino acid molecule, can be broken when it encounters the action of a specific enzyme, and is therefore also included in the "degradable" linking group. Similarly, carbamate groups, thiocarbamate groups, etc. can be both stable linking groups and degradable linking groups. More generally, carbamate groups, thiocarbamate groups, etc. are more likely to degrade slowly, while non-peptide amide bonds can exist stably during the in vivo circulation process. For example, common ester bonds can be degraded under acidic and alkaline conditions, while ester bonds contained in special structures can also be degraded under ultraviolet light conditions. For example, even if certain chemical bonds can be degraded under the action of specific enzymes, if the circulation pathway does not pass through or basically does not pass through the specific enzyme environment during clinical use (such as in the case of targeted drug administration), the corresponding chemical bonds can still be considered to be stable.

[0052] In the present invention, all compounds of the general formula should be understood to include salts of the compounds of the general formula. The term "salt" used is selected from any one, any two or any combination of two or more of acid addition salts formed with inorganic and / or organic acids and base addition salts formed with inorganic and / or organic bases. When the compound of the general formula contains a basic part (such as but not limited to pyridine or imidazole) and an acidic part (such as but not limited to carboxylic acid), zwitterions ("inner salts") can be formed and included in the term "salt" used. "Salt" can be a pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salt, or it can be other salts. The salt of the compound of the general formula can be formed by reacting the compound of the general formula with a certain amount (such as an equivalent) of an acid or base in a medium such as a salt precipitation or in an aqueous medium and then freeze-dried. Exemplary acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthoate, sulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates, sulfonates, tartrates, thiocyanates, toluenesulfonates, undecanoates, and the like; exemplary base addition salts include ammonium salts, alkali metal salts (such as sodium, lithium, and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts), salts with organic bases (e.g., organic amines), and salts with amino acids (such as arginine or lysine). Basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl and diamyl sulfates), long chain halides (e.g., decyl, lauryl, tetradecyl and stearyl chlorides, bromides and iodides), aryl alkyl halides (e.g., benzyl and phenethyl bromides) and others. Both the acid addition salts and the base addition salts are preferably pharmaceutically acceptable salts and are considered equivalent to the free forms of the corresponding compounds of the general formula for the purposes of this disclosure.

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

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

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

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

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

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

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

[0060] In the present invention, unless otherwise specified, "amino" and "amine" have the same meaning, including monovalent, divalent, trivalent, tetravalent neutral groups or cationic groups, substituted or unsubstituted. 3 -NH 2 -NH 2 It can be called "amino", "amine" or "primary amine". For example, CH 3 -NH-CH 3 The -NH- in the -NH- can be called a "secondary amine" or "secondary amino group", where -NH-CH 3 It can also be understood as an amine group substituted with a methyl group.

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

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

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

[0064] The secondary amine bond and hydrazine bond in the present invention refer to "-NH-" or "-NH-NH-" with both ends capped by hydrocarbon groups, such as -CH 2 -NH-CH 2 -and-CH 2 -NH-NH-CH 2 -; while -C(=O)-NH- is called an amide bond and is not considered to contain a secondary amine bond.

[0065] In the present invention, "functional group" is also referred to as "functional group", preferably a reactive group, a protected reactive group, a precursor of a reactive group, etc. "Polymer" means that the number of functional groups is at least 3, such as polyol refers to a compound containing at least 3 hydroxyl groups, polythiol refers to a compound containing at least 3 thiol groups, etc. It should be noted that it is allowed to contain other types of heterogeneous functional groups, such as tris (hydroxymethyl) aminomethane is a triol containing an amino group, and citric acid is a tricarboxylic acid containing a hydroxyl group.

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

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

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

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

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

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

[0072] In the present invention, "slightly changed form" corresponds to "slightly modified", and refers to a structural form that can form a target reactive group after undergoing simple chemical reaction processes such as protection, deprotection, salt complexation, decomplexation, ionization, protonation, deprotonation or change of leaving group. The change of leaving group, i.e., the transformation of leaving group, is, for example, but not limited to, the transformation of ester form to acyl chloride form.

[0073] In some specific embodiments of the present invention, the reaction process also involves the "protection" and "deprotection" process of related groups. In order to prevent a certain reactive group from affecting the reaction, the reactive group is usually protected. In some specific embodiments of the present invention, when there are more than two reactive groups, only the target reactive group is selectively reacted, so other reactive groups are protected. The protecting group not only remains stable during the target reaction, but can also be removed by conventional technical means in the art as needed.

[0074] In the present invention, "protection" of a reactive group means a strategy of reversibly converting the reactive group to be protected into an inert group (non-reactive group) by a specific reagent. The part of the protected group that is different from the unprotected form is called a "protecting group". For example, -OTBS is a protected form of a hydroxyl group (-OH), wherein TBS is a protecting group of the hydroxyl group.

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

[0076] In the present invention, "hydroxyl protecting group" includes all groups that can be used as a protecting group for a common hydroxyl group. The hydroxyl protecting group is preferably an alkanoyl group (e.g., acetyl, tert-butyryl), an aralkanoyl group (e.g., benzoyl), benzyl, trityl, trimethylsilyl, tert-butyldimethylsilyl, allyl, acetal or ketal. The removal of the acetyl group is generally carried out under alkaline conditions, and the most commonly used is NH 3 / MeOH ammonolysis and methanolysis catalyzed by methanol anion; benzyl can be easily removed by palladium-catalyzed hydrogenolysis in neutral solution at room temperature, and can also be reduced by metal sodium in ethanol or liquid ammonia; trityl is generally removed by catalytic hydrogenolysis; trimethylsilyl is usually removed by reagents containing fluoride ions (such as tetrabutylammonium fluoride / anhydrous THF, etc.); tert-butyldimethylsilyl ether is relatively stable and can withstand the ester hydrolysis conditions of alcoholic potassium hydroxide and mild reduction conditions (such as Zn / CH 3 OH, etc.), fluoride ions (such as Bu 4 N + F - ) in tetrahydrofuran solution, or with aqueous acetic acid at room temperature. The protection of the diol preferably forms dioxolane, dioxane, cyclic carbonate or cyclic borate.

[0077] In the present invention, the "thiol protecting group" includes all groups that can be used as a conventional thiol protecting group. Similar to the hydroxyl group, the thiol group can be protected in the form of a thioether and a thioester. The thiol protecting group is preferably a tert-butyl, benzyl, substituted benzyl, diphenylmethyl, substituted diphenylmethyl, triphenylmethyl, acetyl, benzoyl, tert-butyloxycarbonyl, benzyloxycarbonyl, thioacetal or thioketal group. The deprotection of the thioether can be carried out by using Na / NH under acid catalysis. 3 Reduction or use of heavy metal ions such as Ag + , Hg + Some groups include S-diphenylmethyl, S-triphenylmethyl sulfide, S-2-tetrahydropyranyl, S-isobutoxymethyl semithiol acetal, which can be used (SCN) 2The thioesters are oxidized to disulfides by iodine or thionyl chloride and then reduced to thiols. The formation and deprotection of thioesters are the same as those of carboxylates.

[0078] In the present invention, "carboxyl protecting group" refers to a protecting group that can be converted into a carboxyl group by hydrolysis or deprotection reaction of the carboxyl protecting group. The carboxyl protecting group is preferably an alkyl group (e.g., methyl, ethyl, tert-butyl) or an aralkyl group (e.g., benzyl), more preferably a tert-butyl group (tBu), a methyl group (Me) or an ethyl group (Et). In the present invention, "protected carboxyl" refers to a group formed after a carboxyl group is protected by a suitable carboxyl protecting group, preferably a methoxycarbonyl group, an ethoxycarbonyl group, a tert-butyloxycarbonyl group, or a benzyloxycarbonyl group. The carboxyl protecting group can be removed by hydrolysis under the catalysis of an acid or a base, and occasionally by a thermal decomposition 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, H 2 O, LiOH, NaOH, KOH, MeOH, EtOH and combinations thereof, preferably TFA and H 2 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.

[0079] In the present invention, "amino protecting group" is equivalent to "amine protecting group" and includes all groups that can be used as conventional amino / amine protecting groups, such as aryl C 1-6 Alkyl, C 1-6 Alkoxy C 1-6 Alkyl, C 1-6 Alkoxycarbonyl, aryloxycarbonyl, C 1-6 The amino protecting group is preferably Boc (tert-butyloxycarbonyl), Moz (p-methoxybenzyloxycarbonyl) and Fmoc (9-fluorenylmethyleneoxycarbonyl). The reagent for removing the amino protecting group is selected from TFA, H 2 O, LiOH, MeOH, EtOH and combinations thereof, preferably TFA and H 2 The deprotection agent used for removing the Fmoc protection is a solution of N,N-dimethylformamide (DMF) containing 20% ​​piperidine.

[0080] In the present invention, the "alkynyl protecting group" includes all groups that can be used as a common alkynyl protecting group, preferably trimethylsilyl (TMS), triethylsilyl, tert-butyldimethylsilyl (TBS) or biphenyldimethylsilyl. The TMS-protected alkynyl can be easily deprotected under alkaline conditions, such as K 2 CO 3 / MeOH or KOH / MeOH. The TBS-protected alkynyl group can be deprotected in tetrabutylammonium fluoride in tetrahydrofuran (TBAF / THF).

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

[0082] In the present invention, the repeating unit of the polyethylene glycol component is an oxyethylene unit, i.e., -CH 2 CH 2 O- or -OCH 2 CH 2 -, also recorded as "EO unit", the number of repeating units is also recorded as EO unit number, the average number of repeating units is also recorded as EO unit average number, and preferably the number average number.

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

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

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

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

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

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

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

[0090] In the present invention, lipids include simple esters, complex esters and derived lipids. The simple esters are esters composed of fatty acids and alcohols, which can be divided into three subcategories: fats, oils and waxes. The complex esters are "lipid compounds", also called "lipids" or "lipoids", including phospholipids, sphingolipids, glycolipids, steroids and sterols, and lipoproteins. The derived lipids, including simple lipid derivatives and complex lipid derivatives, have the general properties of lipids.

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

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

[0093] In the present invention, "cation" means that the corresponding structure can be permanently or non-permanently responsive to certain conditions (such as pH) and carry a positive charge. Therefore, cations include both permanent cations and cationizable. Permanent cations refer to the corresponding compounds or groups or atoms that are positively charged at any pH value or hydrogen ion activity of their environment. Typically, the positive charge is generated by the presence of quaternary nitrogen atoms. When a compound carries multiple such positive charges, it can be called a permanent cation. Cationizable refers to a compound or group or atom that is positively charged at a lower pH and uncharged at a higher pH of its environment. In addition, in a non-aqueous environment where the pH value cannot be determined, a cationizable compound, group or atom is positively charged at a high hydrogen ion concentration and uncharged at a low hydrogen ion concentration or activity. It depends on the individual properties of the cationizable or polycationizable compound, in particular the pKa of the corresponding cationizable group or atom, at which pH or hydrogen ion concentration it is charged or uncharged. In a dilute aqueous environment, the fraction of cationizable compounds, groups or atoms with a positive charge can be estimated using the so-called Henderson-Hasselbalch equation, which is well known to those skilled in the art. In some embodiments, the whole or part of the cationizable compound is positively charged at a physiological pH value (e.g., about 7.0-7.4). In some preferred embodiments, the whole or part of the cationizable compound is neutral at a physiological pH value (e.g., about 7.0-7.4), but is positively charged at a lower pH value (e.g., about 5.5 to 6.5). In some embodiments, the pKa of the whole or part of the cationizable compound is preferably in the range of about 5 to about 7.

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

[0095] In the present invention, "PEGylated lipid" refers to a molecule comprising a lipid portion and a polyethylene glycol portion, and can be divided into linear PEGylated lipids and non-linear PEGylated lipids based on their structure. In addition to the PEGylated lipids described in the present invention, the PEGylated lipids also include, but are not limited to, polyethylene glycol-1,2 dimyristin (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) and the like.

[0096] In the present invention, "neutral lipid" refers to any of a number of lipid substances that exist in the form of uncharged or neutral zwitterions at a selected pH, preferably phospholipids. Such lipids include but are not limited to 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (MPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 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-dioleoyl-sn-glycero-3-phosphocholine (DPPC ... Alkanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0DietherPC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dioleoylphosphatidylserine (DOPS), dipalmitoylphosphatidylglycerol (DPPG) , palmitoyloleylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine (LPE) and any one thereof and compositions thereof. Neutral lipids can be synthetic or of natural origin.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0110] In the present invention, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle administered together with the therapeutic agent, and is suitable for contacting the tissues of humans and / or other animals without excessive toxicity, irritation, allergic reaction or other problems or complications corresponding to a reasonable benefit / risk ratio within the scope of reasonable medical judgment. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, sterile liquids, such as water and oils, including those oils of petroleum, animal, plant 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 glucose and glycerol aqueous solutions can also be used as liquid carriers, particularly for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. The composition can also contain a small amount of wetting agent, emulsifier or pH buffer as needed. Oral formulations may contain standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Specifically, for example, excipients include, but are not limited to, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), demulcents, emulsifiers, fillers (diluents), film formers or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending agents or dispersing agents, sweeteners, and hydration water. More specifically, excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dicalcium phosphate, calcium stearate, cross-linked carboxymethylcellulose sodium, cross-linked polyvinyl pyrrolidone, citric acid, cross-linked polyvinyl pyrrolidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, phenylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E (α-tocopherol), vitamin C, and xylitol.

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

[0112] In the present invention, a vaccine is a preventive or therapeutic material that provides at least one antigen or antigenic function that can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0113] In the present invention, treatment refers to the treatment and care of a patient in order to resist a disease, disorder or condition, and is intended to include delaying the progression of the disease, disorder or condition, alleviating or relieving symptoms and complications, and / or curing or eliminating the disease, disorder or condition. The patient to be treated is preferably a mammal, especially a human.

[0114] 1. Cationic lipids

[0115] An embodiment of the present invention:

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

[0117]

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

[0119] L 1 , L 2 Each is independently a connecting bond or a divalent connecting group;

[0120] L 3 is a trivalent connecting group, and R 3 The side chain is R 3 The divalent linking group -L 3 (R 3 )-;

[0121] B 1 , B 2 Each independently is a connecting bond or C 1-30 Alkylene;

[0122] R 1 , R 2 Each independently is C 2-30 Aliphatic hydrocarbon or C containing 1-2 O2-30 Aliphatic hydrocarbon derivative residues;

[0123] R 3 For-OR d 、-NR d R d 、-SR d 、-(C=O)R d 、-(C=O)OR d 、-O(C=O)R d 、-O(C=O)OR d or Among them, R d Each occurrence is independently C 1-12 Alkyl, G 1 is a branched group with a valence of k+1, j is 0 or 1, F 1 Contains functional group R 01 ; When j is 0, G 1 does not exist; when j is 1, G 1 Introduce k F 1 , and k F 1 Each independently has the same or different structure, and k is an integer from 2 to 8;

[0124] R 4 is a carbocyclic group, a heterocyclic group or R'N(R')-R"-N(R')-; wherein the heterocyclic group is a cyclic group whose ring atoms contain 1, 2 or more heteroatoms, and the heteroatoms are B, O, N, Si, P or S; wherein R' is independently H or C each time it appears 1-3 Alkyl, R" is C 2-4 Alkylene;

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

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

[0127] 1.1. Stable and degradable divalent linking groups

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

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

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

[0131] The type of STAG is not particularly limited, and includes but is not limited to alkylene, divalent heteroalkyl, double bond, triple bond, divalent diene, divalent cycloalkyl, divalent cycloalkenyl, divalent cycloalkene, divalent cycloalkenyl, aromatic ring, alicyclic ring, heterophenyl ring, aromatic heterocyclic ring, condensed 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 cycloalkene, substituted divalent cycloalkenyl, substituted divalent cycloalkenyl, substituted divalent cycloalkyne, substituted aromatic ring, substituted Aliphatic heterocycles, substituted heterobenzene rings, substituted aromatic heterocycles, substituted condensed heterocycles, ether bonds, thioether bonds, urea bonds, thiourea bonds, carbamate groups, thiocarbamate groups, -P(=O)-, divalent silicon groups not containing active hydrogen, divalent linking groups containing boron atoms, secondary amino groups, tertiary amino groups, carbonyl groups, thiocarbonyl groups, amide groups, thioamide groups, sulfonamide groups, enamine groups, triazoles, 4,5-dihydroisoxazoles, any divalent linking groups in the skeleton of amino acids and derivatives thereof, and any two or more stable divalent linking groups composed of any two groups.

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

[0627] to

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

[704] of CN104530417A.

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

[0134] There is no particular restriction on the conditions under which DEGG can be degraded. DEGG can be degraded under any condition including but not limited to light, heat, low temperature, enzyme, redox, acidity, alkalinity, physiological conditions, in vitro simulated environment, etc., preferably under any condition including but not limited to light, heat, enzyme, redox, acidity, alkalinity, etc.

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

[0136] The type of DEGG is not particularly limited, and includes but is not limited to disulfide bonds, vinyl ether bonds, ester groups, thioester groups, thioester groups, dithioester groups, carbonate groups, thiocarbonate groups, dithiocarbonate groups, trithiocarbonate groups, carbamate groups, thiocarbamate groups, dithiocarbamate groups, acetals, cyclic acetals, thioacetals, azaacetals, azacyclic acetals, azathiaacetals, dithioacetals, hemiacetals, hemithioacetals, azahemiacetals, ketals, thioketals, azaketals, azacyclic ketals, azathiaketals, imine bonds, hydrazone bonds, acylhydrazone bonds, oxime bonds, thioximate ether groups, semicarbazone bonds, thiosemicarbazone bonds, hydrazine groups, hydrazide groups, thiocarbonylhydrazide groups, azocarbonylhydrazide groups, thioazocarbonylhydrazide groups, carbamate groups, hydrazide groups The invention also includes but is not limited to: thioformate group, carbohydrazide, thiocarbohydrazide, azo group, isourea group, isothiourea group, allophanate group, thioalloyate group, guanidine group, amidine group, aminoguanidine group, aminoamidine group, imidic acid group, imidic acid thioester group, sulfonate group, sulfinic acid group, sulfonylhydrazine group, sulfonylurea group, maleimide, orthoester group, phosphate group, phosphite group, hypophosphite group, phosphonate group, phosphosilane ester group, silane ester group, carbonamide, thioamide, sulfonamide group, polyamide, phosphoramide, phosphoramidite, pyrophosphamide, cyclophosphamide, ifosfamide, thiophosphamide, aconityl group, polypeptide fragment, nucleotide and its derivative backbone, deoxynucleotide and its derivative backbone, any one divalent linking group, any two or more divalent linking groups in combination.

[0137] Here, the carbamate group, thiocarbamate group, carbonamide, phosphoramide, etc. can be used as a stable linking group or a degradable linking group, depending on the environment in which it is used.

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

[705] to

[0725] .

[0139] 1.2.L 1 , L 2

[0140] In a specific embodiment of the present invention, L 1 , L 2 Any of the following situations:

[0141] Case (1): L 1 , L 2 One of them is a connecting bond, and the other is a divalent connecting group;

[0142] Case (2): L 1 , L 2 All are connection keys;

[0143] Case (3): L 1 , L 2 are both divalent linking groups, and L 1 , L 2 be of the same or different structure;

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

[0145] Preferred L 1 , L 2 Each of them is independently -O-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)-, -NHC(=O)-, -C(=O)NH-, -(C=O)O(CH 2 ) y OC(=O)-、-O(C=O)(CH 2 ) y OC(=O)-、-(C=O)O(CH 2 ) y C(=O)O-, -O(C=O)(CH 2 ) y C(=O)O-, wherein y is an integer of 2-8.

[0146] In a specific embodiment of the present invention, L 1 , L 2 Each is independently selected from any one of -O(C=O)-, -(C=O)O- and -O(C=O)O-.

[0147] In a specific embodiment of the present invention, L 1 , L 2 One of them is -(C=O)-, and the other is -O(C=O)-, -(C=O)O-, -O(C=O)O- or -O-.

[0148] In a specific embodiment of the present invention, L 1 , L 2 One of them is -O(C=O)O-, and the other is -O(C=O)- or -(C=O)O-.

[0149] In a specific embodiment of the present invention, L 1 and L 2 They are -O(C=O)- at the same time or -(C=O)O- at the same time.

[0150] In a specific embodiment of the present invention, L 1 and L 2 At the same time, it is -O(C=O)O-.

[0151] In a specific embodiment of the present invention, L 1 and L 2 Also -O-.

[0152] In a specific embodiment of the present invention, L 1 , L 2 Each is independently selected from any one of -NH(C=O)-, -(C=O)NH-, -O(C=O)-, and -(C=O)O-.

[0153] In a specific embodiment of the present invention, L 1 and L 2 At the same time, -(C=O)O(CH 2 ) y OC(=O)-、-O(C=O)(CH 2 ) y OC(=O)-、-(C=O)O(CH 2 ) y C(=O)O-, -O(C=O)(CH 2 ) y C(=O)O-, wherein y is an integer of 2-8.

[0154] 1.3.L 3 , R 3

[0155] In one specific embodiment of the present invention, the divalent linking group -L 3 (R 3 )-Selected from-L 4-、-ZL 4 -、-L 5 -ZL 4 -、-L 4 -ZL 5 -、-ZL 4 -ZL 5 -、-L 5 -ZL 4 -ZL 5 -、-L 4 -ZL 5 -ZL 5 -、-ZL 5 -ZL 4 -and-L 5 -ZL 5 -ZL 4 -, and the right end is connected to the nitrogen branching center; wherein L 4 For -(CH 2 ) x -CR 3 H-(CH 2 ) x -, L 5 For -(CH 2 ) x -, x is independently an integer from 0 to 3 at each occurrence, and Z is independently -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, -NR c -、-NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-; wherein R c Each occurrence is independently H or methyl, preferably H;

[0156] Preferred-L 3 (R 3 )-for-(CH 2 ) x -CR 3 H-(CH 2 ) x -.

[0157] In a specific embodiment of the present invention, the aforementioned -L3 (R 3 )- has the structure where a 1 For R 4 The connecting key, a 2 is a connecting bond connected to the nitrogen branching center in the general formula (1); Preferably any one of the following structures:

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

[0159] In one specific embodiment of the present invention, the divalent linking group -L 3 (R 3 )-by 1 L 4 , 0-4 Z and 0-4 L 5 Combined in a linear manner, and L 4 , Z, L 5 There is no particular restriction on the arrangement of 4 For -(CH 2 ) x -CR 3 H-(CH 2 ) x -, L 5 For -(CH 2 ) x -, x is independently an integer from 0 to 3 at each occurrence, and Z is independently -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -SS-, -C(=O)S-, -SC(=O)-, -NR c -、-NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-; wherein R c Each occurrence is independently H or methyl; preferably -L 3 (R 3 )-is any of the following structures:

[0160]

[0161] 1.4.B 1 , B 2

[0162] In a specific embodiment of the present invention, B 1 , B 2 Each independently is a connecting bond or C 1-20 Alkylene; preferably B 1 , B 2 Any of the following situations:

[0163] Case (1): B 1 , B 2 One of them is the connection key and the other is C 1-20 Alkylene;

[0164] Case (2): B 1 , B 2 All are connection keys;

[0165] Case (3): B 1 , B 2 Each independently is C 1-20 Alkylene;

[0166] The C 1-20 The alkylene group is substituted or unsubstituted and is selected from methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene, eicosylene, 1-CR q H- and 0-19 -CH 2 - a divalent linking group or a divalent linking group composed of 2 -CR q H- and 0-18 -CH 2 - a divalent linking group formed by combination; said R q Each occurrence is independently selected from -OH, -(CH 2 ) tq OH, -(CH 2 ) tq CH 3 、-(CH 2 ) tq OCH 3 ,-SH,-(CH 2 ) tq SH, -(CH 2 ) tq SCH 3 、-NH 2 、-N((CH 2 ) tqCH 3 ) 2 、-(CH 2 ) tq NH 2 and -(CH 2 ) tq N(CH 3 ) 2 Any one of, wherein tq is selected from an integer of 0-4; R q Preferred is -OH.

[0167] In a specific embodiment of the present invention, the aforementioned B 1 , B 2 Each is independently selected from any one of the following: a linker, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, -OH-substituted butylene, -OH-substituted pentylene, -OH-substituted hexylene, -OH-substituted heptylene, -OH-substituted octylene;

[0168] Preferred B 1 , B 2 All are ethylene, or B 1 , B 2 All are butylene, or B 1 , B 2 All are hexamethylene, or B 1 , B 2 All are heptylene, or B 1 For butylene, B 2 is heptylene, or B 1 For pentylene, B 2 For hexamethylene, or B 1 For pentylene, B 2 is heptylene, or B 1 is the connecting key, B 2 For hexamethylene, or B 1 For pentylene, B 2 is a hexamethylene group substituted with -OH, or B 1 , B 2 are all hexamethylene substituted with -OH, or B 1 For hexamethylene, B 2 It is a hexamethylene group substituted with -OH.

[0169] In a specific embodiment of the present invention, B 1 , B 2 Each is independently any one of butylene, pentylene, hexylene and heptylene.

[0170] In a specific embodiment of the present invention, B 1 , B 2One of them is a connecting bond, and the other is selected from any one of pentylene, hexylene and heptylene.

[0171] In a specific embodiment of the present invention, B 1 , B 2 wherein one is selected from pentylene, hexylene, heptylene, and the other is

[0172] In a specific embodiment of the present invention, B 1 , B 2 are all ethylene, or are all propylene, or are all butylene, or are all pentylene, or are all hexylene, or are all heptylene, or are all

[0173] In a specific embodiment of the present invention, B 1 For pentylene, B 2 It is heptylene.

[0174] 1.5.R 1 , R 2

[0175] In a specific embodiment of the present invention, R 1 , R 2 Each independently selected from R L , R B , Rr; the R L , R B , Rr each independently contain 0-4 R m Substituent; said R m Each occurrence is independently selected from the linear C 1-12 Hydrocarbon or branched C 1-3 Alkyl, preferably methyl;

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

[0177] Among them, t L An integer selected from 0-20, t L1 ,t L2 Each independently selected from an integer from 2 to 10;

[0178] The R B The structure is Where t is 0, 1 or 2; t 01 ,t 02 ,t 03 ,t 04Each independently is 0 or 1; R e , R f Each independently is C 1-12 Alkyl, C 3-12 Alkenyl and C 3-12 Any of the alkynyl groups; preferably R B Any of the following structures:

[0179]

[0180] The Rr is a C containing a ring structure 3-30 Aliphatic hydrocarbon group; the ring structure is C 3-20 Carbon ring, preferably benzene ring or C 3-20 The residue of a cycloalkane; preferably Rr is any one of the following structures:

[0181] or Among them, t a Each occurrence is independently selected from an integer between 0 and 12, t b An integer selected from 1 to 12, t L An integer selected from 0 to 20, R g Each occurrence is independently a substituent derived from any position on the ring, the substituent being selected from H, C 1-12 Alkyl, C 2-12 Alkenyl, C 2-12 Any of alkynyl;

[0182] Rr is further preferably any one of the following structures:

[0183]

[0184]

[0185] In a specific embodiment of the present invention, R 1 , R 2 Each independently contains 0-8 carbon-carbon double bonds and / or 0-8 carbon-carbon triple bonds, preferably R 1 , R 2 Each is independently any one of a straight-chain alkyl group, a branched-chain alkyl group, a straight-chain alkenyl group, a branched-chain alkenyl group, a straight-chain alkynyl group and a branched-chain alkynyl group. The alkenyl group preferably contains 1 to 4 carbon-carbon double bonds, and the alkynyl group preferably contains 1 to 4 carbon-carbon triple bonds.

[0186] 1.6.R 3 , G 1 、F 1 , R 01

[0187] In a specific embodiment of the present invention, R 3 Selected from -O(CH2 ) r CH 3 、-S(CH 2 ) r CH 3 , -C(=O)(CH 2 ) r CH 3 、-C(=O)O(CH 2 ) r CH 3 、-OC(=O)(CH 2 ) r CH 3 、-OC(=O)O(CH 2 ) r CH 3 and -(CH 2 ) r N(CH 3 ) 2 Any one of the foregoing, wherein r is an integer from 0 to 3, preferably 0 or 1.

[0188] In a specific embodiment of the present invention, R 3 for F 1 The structure is -(CH 2 ) h -R 01 or -(CH 2 ) f -Z 3 -(CH 2 ) g -R 01 ; wherein h is an integer from 0 to 6, f is 0, 1 or 2, and g is 2, 3 or 4; wherein Z 3 Selected from -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c -、-NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-; wherein R c Each occurrence is independently H or methyl, preferably H;

[0189] Preferred F 1For-R 01 、-CH 2 -R 01 、-(CH 2 ) 2 -R 01 、-O-(CH 2 ) 2 -R 01 、-(C=O)O-(CH 2 ) 2 -R 01 、-O(C=O)-(CH 2 ) 2 -R 01 、-O(C=O)O-(CH 2 ) 2 -R 01 Any one of.

[0190] In a specific embodiment of the present invention, j is 1, R 3 for G 1 The structure is -(CH 2 ) f -(Z 0 ) q -(CH 2 ) f -G 01 ; where q is 0 or 1; f is independently 0, 1 or 2 each time it appears; G 01 is a trivalent branched core or a tetravalent branched core selected from -OCH<, -N< or And the left end and -(CH 2 ) f - connected; Z 0 Z 1 , Z 2 or -(Z 2 ) q -(CH 2 ) g -(Z 1 ) q -, where Z 1 , Z 2 Each independently represents -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c -、-NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NRc C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-; R c Each occurrence is independently H or methyl, preferably H; g is 2, 3 or 4;

[0191] Preferred G 1 For -(CH 2 ) f O(CH 2 ) f CH<, more preferably -OCH 2 CH<.

[0192] In a specific embodiment of the present invention, R 01 Any one selected from alkoxy, alcoholic hydroxyl, protected alcoholic hydroxyl, sulfhydryl, protected sulfhydryl, carboxyl, protected carboxyl, amino, protected amino, aldehyde, protected aldehyde, ester, carbonate, carbamate, succinimide, maleimide, protected maleimide, dimethylamino, alkenyl, alkenoate, azido, alkynyl, folic acid, rhodamine and biotinyl.

[0193] In a specific embodiment of the present invention, R 01 Selected from: any one of a reactive group, a variation of a reactive group, a functional group with therapeutic targeting, and a fluorescent functional group; wherein the variation is selected from any one of a precursor of a reactive group, an active form with a reactive group as a precursor, an active form in which a reactive group is substituted, and an inactive form in which a reactive group is protected; wherein the precursor of the reactive group refers to a structure that can be converted into the reactive group through at least one process of oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, and deprotonation.

[0194] In a specific embodiment of the present invention, the aforementioned R 01 Any functional group or its variation selected from the following classes A to I:

[0195] Category A: active ester group, active ester group-like structures; wherein the active ester group is selected from any one of succinimide active ester group, p-nitrobenzene active ester group, o-nitrobenzene active ester group, 1,3,5-trifluorobenzene active ester group, 1,3,5-trichlorobenzene active ester group, 1,3,5-tribromobenzene active ester group, 1,3,5-triiodobenzene active ester group, pentafluorobenzene active ester group, imidazole active ester group, benzotriazole active ester group, thiazolidine-2-thione active ester group, tetrahydropyrrole-2-thione active ester group, 2-mercaptobenzothiazole active ester group, 1-oxo-3-thioxoisoindoline active ester group; wherein the active ester group-like structure is active carboxylic acid ester group or active acyl group;

[0196] Class B: carboxyl, protected carboxyl, sulfonic acid, sulfonate, sulfinic acid, sulfinic acid, sulfenic acid, ester, thioester, dithioester, carbonate, thiocarbonate, dithiocarbonate, trithiocarbonate, xanthate, tetrathiodiester, sulfone, sulfoxide, methacryloyl, hydroxamic acid, thiohydroxamic acid, sulfonyl halide, thiocarboxyl;

[0197] Class C: aldehyde group, hydrated aldehyde group, thialdehyde group, acyl halide group, ketone group, hydrated ketone group, thiol group, thiol hydrate group, glyoxal group, acetal group, monothioacetal group, bisthioacetal group, ketal group, monothioketal group, bisthioketal group, hemiacetal group, hemithioacetal group, hemiketal group, ortho acid group, protected ortho acid group, ortho ester group, cyanate group, thiocyanate group, isocyanate group, isothiocyanate group, oxazoline group, isoxazoline group;

[0198] Class D: primary amino group, secondary amino group, protected amino group, hydroxylamine group, sulfhydryl group, disulfide group, halogen atom, haloacetamide group, ammonium salt, hydrazine group, tetramethylpiperidinyloxy group, dioxapiperidinyloxy group, O-carbonylhydroxylamine group, amide group, imide group, hydrazide group, sulfonylhydrazide group, hydrazone group, imine group, enamine group, alkynylamine group, carbamate group, monothiocarbamate group, dithiocarbamate group;

[0199] Class E: urea group, thiourea group, guanidine group and its protonated form, amidine group and its protonated form, anhydride group, squaryl group, squarate group, semisquaryl group, semisquarate group, imidazole-1-carboxamide group, imidate group, nitrone group, aldoxime group, ketoxime group;

[0200] Class F: maleimide, furan protected maleimide, acrylate, N-acrylamide, N-methylacrylamide, methacrylate, maleamic acid, 1,2,4-triazine-3,5-dione, linear azo compound, cyclic azo compound;

[0201] Class G: alkenyl, alkenyl alkyl, cycloalkene, alkynyl, alkynyl alkyl, protected alkynyl, cycloalkynyl, linear conjugated diene, cyclic conjugated diene, cyclic conjugated diene containing heteroatoms, epoxy, 1,2,4,5-tetrazine, azido, nitrile oxide, cyano, isocyano, diazo, diazonium ion, azoxy, nitrile imine, N-oxidized aldimine, tetrazolyl, 4-acetyl-2-methoxy-5-nitrophenoxy and its diazotized form, imidazolyl, indolyl; wherein the cycloalkene is selected from any one of cyclooctenyl, norbornene, norbornadienyl, oxa-norbornene, and oxa-norbornadienyl;

[0202] Class H: hydroxyl, protected hydroxyl, protected dihydroxyl, siloxy, trihydroxysilyl, protected trihydroxysilyl; wherein the hydroxyl is selected from any one of alcoholic hydroxyl, phenolic hydroxyl, enol hydroxyl, and hemiacetal hydroxyl;

[0203] Class I: monosaccharide selected from allose, altrose, arabinose, cladinose, erythrose, erythrulose, fructose, fucose, fucosamine, fucose, fucose, galactosamine, galactosamine alcohol, N-acetyl-galactosamine, galactose, glucosamine, N-acetyl-glucosamine, glucosamine alcohol, glucose, glucose-6-phosphate, gulose glyceraldehyde, L-glycero-D-mannoheptose, glycerol, glyceraldehyde, dihydroxyacetone, gulose, arachid ... The residue of any one of dulose, lyxose, mannosamine, mannose, mannose-6-phosphate, mannoheptulose, psicose, quinose, quinosamine, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, deoxyribose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, threose, xylose, and xylulose, or a functional derivative thereof; the monosaccharide group is in D-configuration or L-configuration, is a cyclic or chain structure, and is substituted or unsubstituted;

[0204] Among them, the protected hydroxyl group is preferably any one of ether, silyl ether, ester, carbonate, and sulfonate; the protected amino group is preferably any one of carbamate, amide, imide, N-alkylamine, N-arylamine, imine, enamine, imidazole, pyrrole, and indole; the protected thiol group is preferably any one of sulfide, disulfide, silyl sulfide, and thioester; the protected carboxyl group is preferably a form in which the carboxyl group is protected by any one of methyl, ethyl, tert-butyl, and benzyl; the protected alkynyl group is preferably a form in which the alkynyl group is protected by a silyl group; the protected dihydroxyl group is preferably an acetal structure in which its protecting group and two oxygen atoms form a five-membered ring or a six-membered ring; the protecting group of the dihydroxyl group is preferably a methylene or substituted methylene, and more preferably any one of methylene, 1-methylmethylene, 1,1-dimethylmethylene, 1,1-cyclopentylene, 1,1-cyclohexylene, 1-phenylmethylene, and 3,4-dimethylphenylmethylene.

[0205] In a specific embodiment of the present invention, R 01 Any functional group selected from the following classes A to I or their variations:

[0206] Class A:

[0207]

[0208] Class B:

[0209]

[0210] Class C:

[0211]

[0212] Class D:

[0213]

[0214] Class E:

[0215]

[0216] Class F:

[0217]

[0218]

[0219] Class G:

[0220]

[0221] Class H:

[0222]

[0223] Class I:

[0224]

[0225] Wherein, X is a halogen atom selected from any one of a fluorine atom, a chlorine atom, a bromine atom and an iodine atom;

[0226] Among them, Y 1 Selected from C 1-5 Any one of alkyl, vinyl, phenyl, benzyl, p-methylphenyl, 4-(trifluoromethoxy)phenyl, trifluoromethyl and 2,2,2-trifluoroethyl;

[0227] Among them, R d2 is an organic group, preferably each occurrence is independently selected from C 1-5 Alkyl, C 2-5 Alkenyl, C 2-5Any of alkynyl and phenyl, wherein the alkyl, alkenyl, alkynyl and phenyl are each independently substituted or unsubstituted;

[0228] Wherein, W is a leaving group selected from any one of -F, -Cl, -Br, -I and -SPh;

[0229] Among them, M 5 M is a ring-forming atom selected from any one of a carbon atom, a nitrogen atom, a phosphorus atom and a silicon atom; 5 The cyclic structure is a 3- to 30-membered ring, preferably a 3- to 20-membered ring, more preferably a 3- to 16-membered ring, and more preferably a 5- to 16-membered ring; the cyclic structure is preferably any one of the following groups, any substituted form, or any hybridized form: cyclohexane, furanose ring, pyranose ring, benzene, tetrahydrofuran, pyrrolidine, thiazolidine, cyclohexene, tetrahydropyran, piperidine, 1,4-dioxane, pyridine, pyridazine, pyrimidine, pyrazine , 1,3,5-triazine, 1,4,7-triazacyclononane, cyclotripeptide, indene, indane, indole, isoindole, purine, naphthalene, dihydroanthracene, xanthene, thioxanthene, dihydrophenanthrene, 10,11-dihydro-5H-dibenzo[a,d]cycloheptane, dibenzocycloheptene, 5-dibenzocycloheptenone, quinoline, isoquinoline, fluorene, carbazole, iminodibenzyl, naphthyl ring, dibenzocyclooctyne, azadibenzocyclooctyne;

[0230] in, The ring structure is a ring structure containing an acetal group, a disulfide bond, an amine group, an imide group, an anhydride group, an azo group, a carbon-carbon double bond, a carbon-carbon triple bond, or a conjugated diene on the ring skeleton, and the ring structure is selected from a carbocycle, a heterocycle, a benzoheterocycle, a substituted carbocycle, a substituted heterocycle, or a substituted benzoheterocycle;

[0231] Wherein, Q is an atom or substituent that contributes to the induction and conjugation effect of unsaturated bond electrons; when Q is on a ring, the number is one or more; when the number is multiple, they are the same structure, or a combination of two or more different structures; when it is a substituent, Q has a straight chain structure, a branched structure containing a side group, or a cyclic structure;

[0232] The changed form is selected from any one of a precursor of a reactive group, an active form with a reactive group as a precursor, an active form in which a reactive group is substituted, and an inactive form in which a reactive group is protected; wherein the precursor of the reactive group refers to a structure that can be converted into the reactive group through at least one process of oxidation, reduction, hydration, dehydration, electron rearrangement, structural rearrangement, salt complexation and decomplexation, ionization, protonation, and deprotonation.

[0233] In a specific embodiment of the present invention, R 01Selected from any one of hydroxyl, carboxyl, aldehyde, amino, thiol or their protected forms, or selected from any one of halogen, ester, sulfonate, active ester; preferably -OH, -COOH, -C(=O)OCH 3 Any one of.

[0234] 1.7.R 4

[0235] In a specific embodiment of the present invention, R 4 is R'N(R')-R"-N(R')-, selected from Any one, preferably

[0236] In a specific embodiment of the present invention, R 4 is a carbocyclic or heterocyclic group and contains 0-5 substituents, each of which is independently -F, -Cl, -Br, -I, -R d '、-OR d ',-NR d 'R d '、-SR d ', -(C=O)R d ', -(C=O)OR d ', -O(C=O)R d 'or-O(C=O)OR d ', where R d ' is C 1-3 alkyl;

[0237] Preferred R 4 Any of the following structures:

[0238]

[0239] More preferably, it is any one of the following structures:

[0240]

[0241] In a specific embodiment of the present invention, R 4phenyl, benzyl, naphthyl, phenanthrenyl, anthracenyl, biphenyl, oxacycloalkyl, epoxyalkyl, furanyl, dihydrofuranyl, tetrahydrofuranyl, pyranyl, dihydropyranyl, tetrahydropyranyl, thienyl, dihydrothienyl, tetrahydrothienyl, thiopyranyl, dihydrothiopyranyl, tetrahydrothiopyranyl, aziridinyl, 2-aziridine, azetidinyl, pyrrolidinyl, pyrrolidonyl, piperidin ... any one of oxazinyl, homopiperazinyl, morpholinyl, pyridinyl, dihydropyridinyl, tetrahydropyridinyl, pyrrolyl, pyrrolinyl, hydantoinyl, imidazolyl, triazolyl, tetrazolyl, pyrimidinyl, dihydropyrimidinyl, tetrahydropyrimidinyl, purinyl, dihydropurinyl, tetrahydropurinyl, thiazolyl, lactamyl, succinimidyl, indolyl and isoindolyl, or a benzo derivative of any one of them; R 4 is unsubstituted or contains R n Substituent, R n One or more; R n When the number is multiple, they are of the same structure, or a combination of two or more different structures; each R n Independently selected from C 1-10 Alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, phenyl, =O, acetal, -NH 2 、-OR j 、-NR j R j 、-SR j 、-(C=O)R j 、-(C=O)OR j 、-O(C=O)R j 、-O(C=O)OR j , piperidinyl, piperazinyl and morpholinyl, wherein R j C 1-10 Alkyl, the alkyl, alkenyl, alkynyl is a linear structure or a cyclic structure; preferably, each R n Independently for C 3-8 Cycloalkyl, C 3-8 Cycloalkenyl, cyclooctynyl.

[0242] 1.8. Specific structural examples

[0243] In a specific embodiment of the present invention, the structure of the cationic lipid is selected from any one of the following:

[0244]

[0245]

[0246]

[0247] 2. PEGylated lipids

[0248] An embodiment of the present invention:

[0249] A PEGylated lipid derived from the aforementioned cationic lipid, whose structure is shown in general formula (2):

[0250]

[0251] Wherein, N is a nitrogen branching center;

[0252] L 1 , L 2 Each is independently a connecting bond or a divalent connecting group;

[0253] L 3 Is a trivalent linking group, and -L A (A 1 ) n1 OR 3 The side chain is -L A (A 1 ) n1 OR 3 The divalent linking group -L 3 [L A (A 1 ) n1 OR 3 ]-;

[0254] B 1 , B 2 Each independently is a connecting bond or C 1-30 Alkylene;

[0255] R 1 , R 2 Each independently is C 2-30 Aliphatic hydrocarbon or C containing 1-2 O 2-30 Aliphatic hydrocarbon derivative residues;

[0256] R 3 H, -R d 、-(CH 2 ) h NR d R d 、-(CH 2 ) h SR d 、-(C=O)R d 、-(C=O)OR d or

[0257] Among them, R d Each occurrence is independently C 1-12 Alkyl; h is an integer of 0-6; G1 is a branched group with a valence of k+1, j is 0 or 1, F 1 Contains functional group R 01 ; When j is 0, G 1 does not exist; when j is 1, G 1 Introduce k F 1 , and k F 1 Each independently has the same or different structure, and k is an integer from 2 to 8;

[0258] R 5 C 3-14 Alkyl, carbocyclic group, heterocyclic group or R'N(R')-R"-N(R')-; wherein the heterocyclic group is a cyclic group whose ring atoms contain 1, 2 or more heteroatoms, and the heteroatoms are B, O, N, Si, P or S; wherein R' is independently H or C each time it appears 1-3 Alkyl, R" is C 2-4 Alkylene;

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

[0260] A 1 OCH 2 CH 2 - and the left end is L A Connected;

[0261] n 1 A1 The number of repetitions is an integer selected from 20-250;

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

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

[0264]

[0265]

[0266] 3. Preparation of lipids

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

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

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

[0270] In the present invention, the PEGylated lipid is obtained through a preparation process including a coupling reaction and / or a polymerization reaction. The polymerization reaction undergoes at least the following two steps: deprotonation of a small molecule initiator and polymerization of ethylene oxide; the small molecule initiator can be a directly obtained raw material or an intermediate in the preparation process. During the preparation process, when the coupling reaction and the polymerization reaction exist at the same time, there is no particular restriction on the order of the coupling reaction and the polymerization reaction.

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

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

[0273] The intermediates and final products prepared in the present invention can be purified by purification methods including but not limited to extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, thin film dialysis or supercritical extraction. The characterization and confirmation of the structure and molecular weight of the final product can be carried out by characterization methods including but not limited to nuclear magnetic resonance, electrophoresis, UV-visible spectrophotometer, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism, etc.

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

[0275] 3.1. Preparation of cationic lipids

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

[0277] General Reaction Scheme I:

[0278]

[0279] Among them, R 3 'With R 3 The structures of can be the same or different; when R 3 'With R 3 When the specific structure of R is different, 3 ' can be transformed into R after slight modification before or after any reaction step 3 The micro-modification is selected from any one, any two or more of the following chemical reactions: deprotection, salt complexation and decomplexation, ionization, protonation, deprotonation, and changing the leaving group; preferably R 3 For R 3 ' deprotected form, for example, -OTBS is converted to -OH after deprotection reaction;

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

[0281] Among them, L 1 , L 2 , L 3 , B 1 , B 2 , R 3 , R 4 , R 1 and R 2 The definition of is consistent with that in general formula (1), and will not be repeated here;

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

[0283]

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

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

[0286] General Reaction Scheme II:

[0287]

[0288] Among them, R 3 'With R 3 The transformation relationship and F N Refer to General Reaction Scheme I for the definition of 1 'Get B 1 Contains a hydroxyl substituent, consisting of B 2 'Get B 2 Contains a hydroxyl substituent;

[0289] For example, A-1 is C-2 is The structure of IM-1 obtained by the reaction is: Among them B 1 ' is butylene, B 1 for

[0290] Among them, L 1 , L 2 , L 3 , B 1 , B 2 , R 3 , R 4 , R 1 and R 2 The definition of is consistent with that in the general formula (1).

[0291] General Reaction Scheme III:

[0292]

[0293] Among them, B 1 , B 2 All are ethylene;

[0294] Among them, R 3 'With R 3 The transformation relationship and F N The definition of refers to general reaction scheme I;

[0295] Among them, L 1 , L 2 All are -(C=O)O(CH 2 ) y OC(=O)-, y is an integer from 2 to 8;

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

[0297] Among them, L 1 , L 2 , L 3 , B 1 , B 2 , R 3 , R 4 , R 1 and R 2 The definition of is consistent with that in the general formula (1).

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

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

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

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

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

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

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

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

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

[0307] 3.2.2. “Protection” and “deprotection” of related groups involved in the reaction process

[0308] In the present invention, the reaction process also involves the "protection" and "deprotection" processes of related groups. In order to prevent the functional group from affecting the reaction, the functional group is usually protected. In addition, when there are more than two functional groups, only the target functional group is selectively reacted, so the other functional groups are protected. The protecting group not only stably protects the functional group as the object, 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.

[0309] In the present invention, "carboxyl protecting group" refers to a protecting group that can be converted into a carboxyl group by hydrolysis or deprotection reaction of the carboxyl protecting group. The carboxyl protecting group is preferably an alkyl group (e.g., methyl, ethyl, tert-butyl) or an aralkyl group (e.g., benzyl), more preferably a tert-butyl group (tBu), a methyl group (Me) or an ethyl group (Et). In the present invention, "protected carboxyl" refers to a group formed after a carboxyl group is protected by a suitable carboxyl protecting group, preferably a methoxycarbonyl group, an ethoxycarbonyl group, a tert-butyloxycarbonyl group, or a benzyloxycarbonyl group. The carboxyl protecting group can be removed by hydrolysis under the catalysis of an acid or a base, and occasionally by a thermal decomposition 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, H 2 O, LiOH, NaOH, KOH, MeOH, EtOH and combinations thereof, preferably TFA and H2 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.

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

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

[0312] A: Deprotection of benzyl group

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

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

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

[0316] B: Deprotection of acetals and ketals

[0317] The acetal or ketal compound used for this type of hydroxyl protection is preferably ethyl vinyl ether, tetrahydropyran, acetone, 2,2-dimethoxypropane, benzaldehyde, etc. The deprotection of this type of acetal and ketal is achieved under acidic conditions, and the solution pH is preferably 0 to 4. The acid is not particularly limited, but acetic acid, phosphoric acid, sulfuric acid, hydrochloric acid, 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.

[0318] C: Deprotection of silyl ether

[0319] Compounds used for this type of hydroxyl protection include trimethylsilyl ether, triethylsilyl ether, dimethyl tert-butylsilyl ether, tert-butyldiphenylsilyl ether, etc. The deprotection of this type of silyl ether is carried out by a fluorine-containing compound, preferably tetrabutylammonium fluoride, tetraethylammonium fluoride, hydrofluoric acid, potassium fluoride, and more preferably tetrabutylammonium fluoride and potassium fluoride. The amount of the fluorine-containing reagent is 5 to 20 times the molar equivalent of the protected hydroxyl group, preferably 8 to 15 times the initiator. If the amount of fluorine-containing reagent is less than 5 times the molar equivalent of the protected hydroxyl group, incomplete deprotection will result; when the amount of the deprotection reagent is greater than 20 times the molar equivalent of the protected hydroxyl group, the excess reagent or compound will cause trouble for purification and may be mixed into the subsequent steps, thereby causing side reactions. There is no particular restriction on the reaction solvent, as long as it can dissolve the reactants and products, preferably a non-protonic solvent, more preferably tetrahydrofuran and dichloromethane. 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.

[0320] D: Deprotection of tert-butyl group

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

[0322] In the terminal functionalization method, preferably q = 0, q 1 =1, Z 1 When q is not 0, A and R 01 When there are connecting groups such as amino acids and succinyl groups, Z can be generated by using the present technical field. 2 or Z 1 The prior art (including but not limited to alkylation, condensation, click reaction, etc.) is used, and the preparation is carried out with reference to the following linear functionalization reaction.

[0323] 3.2.3. Alkylation reaction

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

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

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

[0327] The obtained product is a mixture of an ether intermediate and excess sulfonate and halide, which can be purified by anion exchange resin, permeation, ultrafiltration and the like. 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, preferably an ion exchange resin with a tertiary amine or quaternary ammonium salt having a skeleton of dextran, agarose, polypropionate, polystyrene, polystyrene and the like. The solvent for permeation and ultrafiltration is not limited, generally water or an organic solvent can be used, wherein the organic solvent is not particularly limited, as long as the product can be dissolved therein, preferably dichloromethane, chloroform and the like.

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

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

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

[0331] After the substrate amine and the aldehyde derivative react to obtain the imine intermediate, the intermediate is obtained under the action of a reducing agent. Wherein, the molar equivalent of the aldehyde derivative is 1 to 20 times that of the substrate amine, preferably 1 to 2 times, and more preferably 1 to 1.5 times. When the molar equivalent of the aldehyde derivative is greater than 20 times that of the substrate amine, the excess reagent brings trouble to the purification and may be mixed into the subsequent steps, increasing the difficulty of purification. When the molar equivalent of the aldehyde derivative is less than 1 times that of the substrate amine, the reaction is incomplete, increasing the difficulty of purification. Wherein, the product after the reaction can be purified by means of cation exchange resin, permeation, ultrafiltration, etc. to obtain an intermediate. The cation exchange resin is not particularly limited, as long as it can exchange with quaternary ammonium cations to achieve a separation effect. The solvent for permeation and ultrafiltration is not limited, and generally can be water or an organic solvent, wherein the organic solvent is not particularly limited, as long as the product can be dissolved therein, preferably dichloromethane, chloroform, etc.

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

[0333] The reducing agent is not particularly limited, as long as it can reduce the imine to an amine, preferably sodium borohydride, lithium aluminum hydride, sodium cyanoborohydride, Zn / AcOH, etc., more preferably sodium cyanoborohydride. The amount of the reducing agent is generally 0.5 to 50 times the amount of the aldehyde derivative substance, more preferably 1-10 times.

[0334] 3.2.4.R 3 Linear functionalization of side chain ends

[0335] R 3 The method of terminal linear functionalization of the side chain is not particularly limited and is related to the type of the final functional group or its protected form. 3 Still belongs to the present invention R 3 within the range.

[0336] (1) Functionalization of terminal hydroxyl groups

[0337] From F 1 Starting from the terminal hydroxyl group of 2 ) h -R 01 To-(CH 2 ) f -Z 3 -(CH 2 ) g -R 01wherein h is an integer from 0 to 6, f is 0, 1 or 2, and g is 2, 3 or 4; wherein Z 3 Selected from -(C=O)-, -O(C=O)-, -(C=O)O-, -O(C=O)O-, -O-, -S-, -C(=O)S-, -SC(=O)-, -NR c -、-NR c C(=O)-, -C(=O)NR c -、-NR c C(=O)NR c -、-OC(=O)NR c -、-NR c C(=O)O-, -SC(=O)NR c -and-NR c C(=O)S-; wherein R c Each occurrence is independently H or methyl, preferably H; the specific preparation method includes but is not limited to that described in paragraphs

[0960] to

[1205] of document CN104530417A.

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

[0339] Method 1: Direct modification, based on direct modification of reactive groups, to obtain the target functional group or its protected form. For example, the conversion of carboxyl groups to acyl halides, hydrazides, esters, thioesters, dithioesters, the conversion of hydroxyl groups, sulfhydryl groups, alkynyl groups, amino groups, carboxyl groups, etc. to corresponding protected structures, etc. Another example is the modification of hydroxyl groups, amino groups, etc. by acid anhydrides.

[0340] Method 2: Coupling reaction between two reactive groups, using a heterofunctionalized reagent containing one reactive group and the target functional group or its protected form as raw material, through one of the reactive groups and F 1The reaction between the reactive groups at the ends introduces the target functional group or its protected form. The reaction mode and method between the two reactive groups are not particularly limited, and the reaction conditions are related to the type of divalent linking group generated by the reaction, and the existing public technology 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 sulfhydryl or amino group, corresponding to the formation of thioether bond, secondary amino group or tertiary amino group in turn. Among them, the condensation reaction includes but is not limited to the condensation reaction of generating ester group, thioester group, amide group, imine bond, hydrazone bond, carbamate group, etc. For another example, a heterofunctionalizing reagent containing azide, alkynyl, alkenyl, trithioester group, sulfhydryl, diene group, furanyl, 12,4,5-tetrazine group, cyanate group and the target functional group or its protected form is used as a raw material to introduce the target functional group or its protected form through a click reaction. The reaction between 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, and the like.

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

[0342] 4.1. Lipid composition

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

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

[0345] An embodiment of the present invention:

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

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

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

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

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

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

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

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

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

[0355] It is preferred that the lipids include phospholipids, steroid lipids and PEGylated lipids.

[0356] In a specific embodiment of the present invention, the phospholipids in the aforementioned lipid composition are selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, Phosphocholine, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-Diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dioleoyl Any one of acylphosphatidylserine, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine, and combinations thereof.

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

[0358] In a specific embodiment of the present invention, the PEGylated lipid in the aforementioned lipid composition is selected from polyethylene glycol-1,2-dimyristyl glyceride, 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.

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

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

[0361]

[0362]

[0363]

[0364] Where n 1 is an integer selected from 20-250.

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

[0366] The molar percentage of PEGylated lipids to total lipids is 0.5-5%, preferably 1-3%, more preferably 1.5%, 1.6%, 1.7%, 1.8%, 1.9%;

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

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

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

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

[0371] 4.2. Lipid pharmaceutical compositions and their preparations

[0372] An embodiment of the present invention:

[0373] A lipid pharmaceutical composition comprises any of the aforementioned lipid compositions and a drug, wherein the drug is selected from any of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs.

[0374] In a specific embodiment of the present invention, the drug in the aforementioned lipid pharmaceutical composition is a nucleic acid drug, selected from any one of DNA, RNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir and ribozyme, and the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA and siRNA; preferably any one of DNA, mRNA, miRNA and siRNA.

[0375] In a specific embodiment of the present invention, the aforementioned lipid pharmaceutical composition is used as a drug, and the drug is selected from any one of the following: drugs for treating cancer, anti-infective agents, antibiotics, antiviral agents, antifungal agents, and vaccines.

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

[0377] In a specific embodiment of the present invention, the drugs in the aforementioned lipid pharmaceutical composition include but are not limited to doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, streptozotocin, actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracycline, nitrogen mustard, thiotepa, chlorambucil, razithromycin, melphalan, carmustine, lomustine, busulfan, dibromomannitol, mitomycin C, cis-dichlorodiamine platinum (II), methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil dacarbazine, dibucaine, chlorpromazine, propranolol, dimerol, labetalol, clonidine, hydralazine, imipramine, amitriptyline , doxepin, phenytoin, diphenhydramine, chlorpheniramine, promethazine, gentamicin, ciprofloxacin, cefoxitin, miconazole, terconazole, econazole, isoconazole, butoconazole, clotrimazole, itraconazole, nystatin, 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, dihydroxyanthraquinone, mithramycin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, puromycin, maytansine.

[0378] In a specific embodiment of the present invention, the drug in the aforementioned lipid pharmaceutical composition is a nucleic acid drug, and the N / P ratio is (0.1-100):1, preferably (0.2-30):1, and more preferably (0.5-20):1.

[0379] An embodiment of the present invention:

[0380] A lipid pharmaceutical composition preparation, comprising any of the aforementioned lipid pharmaceutical compositions and a pharmaceutically acceptable diluent or excipient, wherein the diluent or excipient is preferably any of deionized water, ultrapure water, phosphate buffer and physiological saline, more preferably phosphate buffer or physiological saline, and most preferably physiological saline.

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

[0382] Preparation method

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

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

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

[0386] Among them, the equilibration time is not particularly limited, preferably 0.1 to 12 hours, more preferably 0.2 to 6 hours, and most preferably 0.5 to 3 hours; the recombination time is not particularly limited, preferably 0.1 to 12 hours, more preferably 0.2 to 5 hours, and most preferably 0.5 to 2 hours.

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

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

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

[0390] (3) mixing the organic phase solution and the aqueous phase solution to obtain an LNP-nucleic acid pharmaceutical composition, washing by ultrafiltration to remove the organic solvent and free molecules, and finally passing through a sterile filter for standby use;

[0391] Among them, the organic solvent is preferably any one of methanol, ethanol, propanol, tert-butanol, acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone, or a mixed solvent of any one or more thereof; the buffer is preferably a citrate buffer, and further, preferably, its concentration is 5-80mM, pH is 2-6, more preferably the concentration is 10-50mM, and pH is 3-5; the volume ratio of the organic phase solution to the aqueous phase solution is preferably 1:1-10, more preferably 1:2.

[0392] In a specific embodiment of the present invention, ultrasound, extrusion or microfluidics is used to control the particle size of lipid nanoparticles, and the particle size is 1 to 1000 nm, preferably 20 to 500 nm, more preferably 60 to 200 nm, and most preferably 60 to 150 nm. DETAILED DESCRIPTION

[0393] The preparation of cationic lipids, lipid compositions, lipid pharmaceutical compositions, and the biological activity test of LNP-nucleic acid pharmaceutical compositions are further described below in conjunction with some specific examples. The specific examples are for further illustrating the present invention in detail, but do not limit the scope of protection of the present invention.

[0394] 5.1. Preparation of lipid compounds

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

[0396]

[0397] Corresponding to the general formula (1), in E1-1, R 1 , R 2 Both B 1 , B 2 All are ethylene, L 1 , L 2 All are -(C=O)O-(CH 2 ) 4 -OC(=O)-,-L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 938 Da.

[0398] The preparation process is as follows:

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

[0400]

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

[0402]

[0403] Corresponding to the general formula (1), in E2-1, R1 is undecyl, R 2 for B 1 For pentylene, B 2 For heptylene, L 1 , L 2 All are ester groups (-C(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 838 Da.

[0404] The preparation process is as follows:

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

[0406] Step b: S2-4 (3.31 g, 7.2 mmol, obtained by condensation reaction of 8-bromooctanoic acid and 9-heptadecanol) was dissolved in 50 mL of DMF, and S2-3 (1.82 g, 6.0 mmol) and K 2 CO 3 (1.19 g, 8.6 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S2-5 (3.24 g).

[0407] Step c: The above compound S2-5 (2.74 g, 4.0 mmol) was dissolved in dry THF (50 mL), and NaH (60%, 1.59 g, 40.0 mmol) was slowly added under ice bath, and the mixture was reacted under ice bath for 1 hour. After the reaction was completed, S2-6 (1.67 g, 4.8 mmol, obtained by condensation reaction of 6-bromohexanoic acid and 1-undecanol) was added, and the mixture was stirred under ice bath for 1 hour, and the reaction solution was slowly restored to room temperature and reacted overnight. After the reaction was completed, the reaction was placed under ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (50 mL) was added and stirred to mix, and then the mixture was extracted twice with dichloromethane (25 mL*2), the organic phases were collected and combined, and backwashed once with saturated sodium chloride (50 mL). After the organic phase was concentrated under reduced pressure, 20 mL of tetrahydrofuran was added, and then 20 mL of TBAF tetrahydrofuran solution (1 M) was added, and the mixture was reacted overnight to remove TBS protection. After the reaction is completed, the organic phases are concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain a crude product of compound E2-1. The cationic lipid E2-1 (2.41 g) is obtained by column chromatography purification, concentration, and oil pump drying. The main data of the H NMR spectrum of E2-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:4.83-4.77(m,1H),4.06(t,2H),3.83-3.77(m,1H),2.66-2.29(m,16H),2.26-2.20(m,9H),1.69-1.21(m,64H),0.86(t,9H). MS(ESI):m / z=838.83([M+H] + ).

[0408]

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

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

[0411]

[0412] Corresponding to the general formula (1), in E3-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 , L 2 All are ester groups (-OC(=O)-), -L 3 (R 3 )-for Where R 3 is -OH, R4 for The total molecular weight is approximately 863 Da.

[0413] The preparation process is as follows:

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

[0415]

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

[0417]

[0418] Corresponding to the general formula (1), in E3-2, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 , L 2 All are ester groups (-OC(=O)-), -L 3 (R 3 )-for Where R 3is -OH, R 4 for The total molecular weight is approximately 835 Da.

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

[0420]

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

[0422]

[0423] Corresponding to the general formula (1), in E4-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 , L 2 All are ester groups (-C(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 820 Da.

[0424] The preparation process is as follows:

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

[0426] Step b: S4-3 (2.76 g, 6.6 mmol) was dissolved in 50 mL of DMF, and 2-amino-1-cyclohexylethanol (S4-4, 0.77 g, 3.0 mmol) containing TBS-protected hydroxyl groups and K 2 CO 3 (1.09g, 7.9mmol), stirred overnight at room temperature. After the reaction is completed, the reaction mixture is concentrated under reduced pressure and poured into 50mL of dichloromethane. After washing with 10% citric acid (25mL*2) and brine (25mL*2) in sequence, the organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 25mL of tetrahydrofuran is added, followed by 25mL of TBAF tetrahydrofuran solution (1M), and the reaction is allowed to proceed overnight to remove the TBS protection. After the reaction is completed, the organic phases are concentrated, extracted, combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product is purified by silica gel column chromatography to obtain E4-1 (1.99g). The main data of the H NMR spectrum of E4-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:4.89-4.82(m,2H),3.35-3.28(m,1H),2.60-2.20(m,10H),1.90(d,1H),1.77-1.39(m,20H),1.32-0.96(m,54H),0.86(t,12H). MS(ESI):m / z=820.58([M+H] + ).

[0427]

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

[0429]

[0430] Corresponding to the general formula (1), in E5-1, R 1 , R 2 Both B 1 , B 2 All are heptylene, L 1 , L 2 All are ester groups (-C(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 912 Da.

[0431] The preparation process is as follows:

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

[0433] Step b: Under argon atmosphere, DCC (2.78 g, 13.5 mmol) was added to a round-bottom flask containing 8-bromooctanoic acid (S5-4, 2.00 g, 9.0 mmol), S5-3 (2.57 g, 9.9 mmol) and DMAP (0.27 g, 2.3 mmol) dissolved in dichloromethane (50 mL) under argon atmosphere, 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 obtained residue was purified by silica gel column chromatography to obtain S5-5 (3.49 g).

[0434] Step c: S5-5 (3.06 g, 6.6 mmol) was dissolved in 50 mL of DMF, and 2-amino-1-cyclohexylethanol (S4-4, 0.77 g, 3.0 mmol) containing TBS-protected hydroxyl groups and K 2 CO 3 (1.09g, 7.9mmol), stirred overnight at room temperature. After the reaction is completed, the reaction mixture is concentrated under reduced pressure and poured into 50mL of dichloromethane. After washing with 10% citric acid (25mL*2) and brine (25mL*2) in sequence, the organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 25mL of tetrahydrofuran is added, followed by 25mL of TBAF tetrahydrofuran solution (1M), and the reaction is allowed to proceed overnight to remove the TBS protection. After the reaction is completed, the organic phases are concentrated, extracted, combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product is purified by silica gel column chromatography to obtain E5-1 (2.19g). The main data of the H NMR spectrum of E5-1 are as follows: 1 H NMR (400 MHz, CDCl 3)δ:4.97-4.92(m,2H),3.59-3.27(m,17H),2.60-2.19(m,10H),1.90(d,1H),1.79-1.40(m,20H),1.33-0.95(m,42H),0.86(t,12H). MS(ESI):m / z=912.84([M+H] + ).

[0435]

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

[0437]

[0438] Corresponding to the general formula (1), in E6-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 , L 2 All of them are carbonate groups (-OC(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 882 Da.

[0439] The preparation process is as follows:

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

[0441] Step b: S6-2 (2.86 g, 6.6 mmol) was dissolved in 50 mL of DMF, and S1-2 (0.86 g, 3.0 mmol) and K were added. 2 CO 3(1.09g, 7.9mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50mL of dichloromethane. After washing with 10% citric acid (25mL*2) and brine (25mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 25mL of tetrahydrofuran was added, followed by 25mL of TBAF tetrahydrofuran solution (1M), and the reaction was allowed to proceed overnight to remove the TBS protection. After the reaction was completed, the organic phases were concentrated, extracted, combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain E6-1 (2.20g). The main data of the H NMR spectrum of E6-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:4.15-4.04(m,6H),3.38-2.92(m,12H),2.88-2.37(m,8H),1.75-1.20(m,64H),0.88(t,12H). MS(ESI):m / z=882.86([M+H] + ).

[0442]

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

[0444]

[0445] Corresponding to the general formula (1), in E7-1, R 1 is undecyl, R 2 for B 1 For pentylene, B 2 For heptylene, L 1 , L 2 All are ester groups (-C(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 787 Da.

[0446] The preparation process is as follows:

[0447] Step a: S2-4 (4.42 g, 9.6 mmol) was dissolved in 60 mL of DMF, and 2-amino-1-(4-pyridyl)ethanol (S7-3, 2.02 g, 8.0 mmol) and K were added. 2 CO 3(1.59 g, 11.5 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 60 mL of dichloromethane. After washing with 10% citric acid (30 mL*2) and brine (30 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S7-4 (4.15 g).

[0448] Step b: The above compound S7-4 (3.80 g, 6.0 mmol) was dissolved in dry THF (60 mL), and NaH (60%, 2.40 g, 60.0 mmol) was slowly added under ice bath, and the mixture was reacted under ice bath for 1 hour. After the reaction was completed, S2-6 (2.51 g, 7.2 mmol) was added, and the mixture was stirred under ice bath for 1 hour, and the reaction solution was slowly returned to room temperature and reacted overnight. After the reaction was completed, the reaction was placed under ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (60 mL) was added and stirred to mix, and then the mixture was extracted twice with dichloromethane (30 mL*2), the organic phases were collected and combined, and backwashed once with saturated sodium chloride (60 mL). After the organic phase was concentrated under reduced pressure, 30 mL of tetrahydrofuran was added, and then 30 mL of TBAF tetrahydrofuran solution (1 M) was added, and the reaction was continued overnight to remove TBS protection. After the reaction is completed, the organic phases are concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain a crude product of compound E7-1. The cationic lipid E7-1 (3.64 g) is obtained by column chromatography purification, concentration, and oil pump drying. The main data of the H NMR spectrum of E7-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:8.44(d,2H),7.24(d,2H),4.89-4.83(m,1H),4.72-4.68(m,1H),4.07(t,2H),2.56-2.24(m,10H),1.78-1.22(m,62H),0.88(t,9H). MS(ESI):m / z=787.70([M+H] + ).

[0449]

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

[0451]

[0452] Corresponding to the general formula (1), in E8-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1is an ester group (-OC(=O)-), L 2 is a carbonate group (-OC(=O)O-), -L 3 (R 3 )-for Where R 3 -O(CH 2 ) 2 OH, R 4 for The total molecular weight is approximately 910 Da.

[0453] The preparation process is as follows:

[0454] Step a: S6-2 (3.12 g, 7.2 mmol) was dissolved in 50 mL of DMF, and S8-1 (1.73 g, 6.0 mmol, prepared in Reference Example 42) and K were added. 2 CO 3 (1.19 g, 8.6 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S8-2 (3.09 g).

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

[0456]

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

[0458]

[0459] Corresponding to the general formula (1), in E9-1, R 1 is undecyl, R 2 for B 1 For pentylene, B 2 For heptylene, L 1 is an ester group (-C(=O)O-), L 2 is a carbonate group (-OC(=O)O-), -L 3 (R 3 )-for Where R 3 -(C=O)O(CH 2 ) 2 OH, R 4 for The total molecular weight is approximately 875 Da.

[0460] The preparation process is as follows:

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

[0462] Step b: S9-4 (3.43 g, 7.2 mmol, obtained by reaction of 7-bromoheptyl-4-nitrophenyl carbonate and 9-heptadecanol) was dissolved in 50 mL of DMF, and S9-3 (1.69 g, 6.0 mmol) and K were added. 2 CO 3 (1.19 g, 8.6 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S9-5 (3.59 g).

[0463] Step c: The above compound S9-5 (2.72 g, 4.0 mmol) was dissolved in dry THF (50 mL), and NaH (60%, 1.60 g, 40.0 mmol) was slowly added under ice bath, and the mixture was reacted under ice bath for 1 hour. After the reaction was completed, compound S2-6 (1.67 g, 4.8 mmol) was added, and the mixture was stirred under ice bath for 1 hour, and the reaction solution was slowly returned to room temperature and reacted overnight. After the reaction was completed, the reaction was placed under ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (50 mL) was added and stirred to mix, and then extracted twice with dichloromethane (25 mL*2), the organic phases were collected and combined, and backwashed once with saturated sodium chloride (50 mL). After the organic phase was concentrated under reduced pressure, it was dissolved with methanol, and 1 M hydrochloric acid was added to pH = 3.5. After reacting for 4 hours, it was concentrated, precipitated, filtered, recrystallized, and dried to obtain the crude product of E9-1 with exposed hydroxyl groups. The cationic lipid E9-1 (2.70 g) was obtained by column chromatography purification, concentration, and oil pump drying. The main data of the H-NMR spectrum of E9-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:8.48-7.14(m,4H),4.21-4.05(m,7H),3.86-3.80(m,2H),3.68-3.63(m, 1H),3.08-3.01(m,2H),2.69-2.24(m,6H),1.72-1.23(m,62H),0.89(t,9H). MS(ESI):m / z=875.74([M+H] + ).

[0464]

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

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

[0467]

[0468] Corresponding to the general formula (1), in E10-1, R 1 , R 2 All are 8,11-heptadecadienylidene, B 1 , B 2 All are butylene, L 1 , L 2 All are ester groups (-OC(=O)-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 829 Da.

[0469] The preparation process is as follows:

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

[0471] Step b: S10-3 (2.73 g, 6.6 mmol) was dissolved in 50 mL of DMF, and 1-amino-3-(4-morpholinyl)-2-propanol (S10-4, 0.82 g, 3.0 mmol) and K were added to the mixture. 2 CO 3 (1.09g, 7.9mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50mL of dichloromethane. After washing with 10% citric acid (25mL*2) and brine (25mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 25mL of tetrahydrofuran was added, followed by 25mL of TBAF tetrahydrofuran solution (1M), reacted overnight, and TBS protection was removed. After the reaction was completed, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product of compound E10-1. The crude product was purified by silica gel column chromatography to obtain E10-1 (1.96g). The main data of the nuclear magnetic hydrogen spectrum of E10-1 are as follows: 1 H NMR (400 MHz, CDCl 3)δ:5.37-5.33(m,8H),4.03(t,4H),3.92-3.86(m,1H),3.72-3.67(m,4H),2.76(t,4H),2.7 2-2.42(m,10H),2.39-2.23(m,4H,2H),2.06-2.00(m,8H),1.69-1.21(m,40H),0.87(t,6H). MS(ESI):m / z=829.76([M+H] + ).

[0472]

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

[0474]

[0475] Corresponding to the general formula (1), in E10-2, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 , L 2 All are ester groups (-OC(=O)-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 837 Da.

[0476] Referring to the preparation of E10-1, S10-3 was replaced by S3-3, and the remaining steps were the same to obtain E10-2, the main data of which are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:4.03(t,4H),3.94-3.87(m,1H),3.71-3.66(m,4H),2.72-2.42(m,10H),2.37-2.24 (m,2H,2H),1.62-1.50(m,10H),1.43-1.32(m,10H),1.26-1.19(m,44H),0.85(t,12H). MS(ESI):m / z=837.80([M+H] + ).

[0477]

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

[0479]

[0480] Corresponding to the general formula (1), in E11-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 is an ester group (-C(=O)O-), L 2 is a carbonate group (-OC(=O)O-), -L 3 (R 3 )-for Where R 3 for R 4 for The total molecular weight is approximately 905 Da.

[0481] The preparation process is as follows:

[0482] Step a: Add 200 mL of tetrahydrofuran and an excess of potassium diphenylmethyl (14.42 g, 70.0 mmol) to a closed reaction kettle without water or oxygen, and then add 2-amino-1-(4-pyridyl)ethanol (S11-1, 4.00 g, 16.8 mmol) containing a Boc-protected amino group and compound S11-2 (6.55 g, 16.8 mmol), wherein S11-2 is glycerol with one hydroxyl group replaced by p-toluenesulfonate (-OTs) and the other two hydroxyl groups protected by EE. After reacting at 30°C for 12 hours, open the reaction kettle, concentrate, wash, dissolve with dichloromethane, add TFA to 0.1 M, react for 4 hours, adjust the pH to neutral, concentrate the reaction solution, add purified water, extract with dichloromethane, concentrate the extract, precipitate, filter, and dry to obtain the intermediate S11-3 (3.51 g) with exposed amino groups.

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

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

[0485]

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

[0487]

[0488] Corresponding to the general formula (1), in E12-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 , L 2 All are ester groups (-OC(=O)-), -L 3 (R 3 )-for Where R 3 -(C=O)O(CH 2 )2 OH, R 4 for The total molecular weight is approximately 1111 Da.

[0489] The preparation process is as follows:

[0490] Step a: Under argon atmosphere, add DCC (3.09 g, 15.0 mmol) to a round-bottom flask containing 3-(3-pyridyl)-L-alanine (S12-1, 2.66 g, 10.0 mmol) containing a Boc-protected amino group, ethylene glycol (S9-2, 1.47 g, 11.0 mmol) containing an EE-protected hydroxyl group, and DMAP (0.31 g, 2.5 mmol) in dichloromethane (50 mL), and react at room temperature for 16 h. After the reaction is completed, remove the precipitate by filtration. Concentrate the filtrate, dissolve it in dichloromethane, add TFA to 0.1 M, react for 4 hours, adjust the pH to neutral, concentrate the reaction solution, add purified water, extract with dichloromethane, dry the extract with anhydrous magnesium sulfate, filter, concentrate the filtrate, and recrystallize to obtain the intermediate S12-2 (1.79 g) with exposed amino group.

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

[0492]

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

[0494]

[0495] Corresponding to the general formula (1), in E13-1, R 1 is undecyl, R 2 for B 1 For pentylene, B 2 For heptylene, L 1 , L 2 All are ester groups (-C(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 792 Da.

[0496] The preparation process is as follows:

[0497] Step a: S2-4 (4.42 g, 9.6 mmol) was dissolved in 60 mL of DMF, and S4-4 (2.06 g, 8.0 mmol) and K were added. 2 CO 3 (1.59 g, 11.5 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 60 mL of dichloromethane. After washing with 10% citric acid (30 mL*2) and brine (30 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S13-1 (4.44 g).

[0498] Step b: The above compound S13-1 (3.83 g, 6.0 mmol) was dissolved in dry THF (60 mL), and NaH (60%, 2.40 g, 60.0 mmol) was slowly added under ice bath, and the mixture was reacted under ice bath for 1 hour. After the reaction was completed, S2-6 (2.51 g, 7.2 mmol) was added, and the mixture was stirred under ice bath for 1 hour, and the reaction solution was slowly returned to room temperature and reacted overnight. After the reaction was completed, the reaction was placed under ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (60 mL) was added and stirred to mix, and then the mixture was extracted twice with dichloromethane (30 mL*2), the organic phases were collected and combined, and backwashed once with saturated sodium chloride (60 mL). After the organic phase was concentrated under reduced pressure, 30 mL of tetrahydrofuran was added, and then 30 mL of TBAF tetrahydrofuran solution (1 M) was added, and the reaction was continued overnight to remove TBS protection. After the reaction is completed, the organic phases are concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain a crude product of compound E13-1. The cationic lipid E13-1 (3.37 g) is obtained by column chromatography purification, concentration, and oil pump drying. The main data of the H NMR spectrum of E13-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:4.87-4.81(m,1H),4.09(t,2H),3.35-3.27(m,1H),2.63-2.24(m,10H),1.90(d,1H),1.78-0.97(m,72H),0.88(t,9H). MS(ESI):m / z=792.79([M+H] + ).

[0499]

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

[0501]

[0502] Corresponding to the general formula (1), in E14-1, R 1 is undecyl, R 2 for B 1 For pentylene, B 2 For heptylene, L 1 , L 2 All are ester groups (-C(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 796 Da.

[0503] The preparation process is as follows:

[0504] Step a: S5-5 (4.45 g, 9.6 mmol) was dissolved in 60 mL of DMF, and S4-4 (2.06 g, 8.0 mmol) and K were added. 2 CO 3 (1.59 g, 11.5 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 60 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S14-1 (4.37 g).

[0505] Step b: The above compound S14-1 (3.85 g, 6.0 mmol) was dissolved in dry THF (60 mL), and NaH (60%, 2.40 g, 60.0 mmol) was slowly added under ice bath, and the mixture was reacted under ice bath for 1 hour. After the reaction was completed, S2-6 (2.51 g, 7.2 mmol) was added, and the mixture was stirred under ice bath for 1 hour, and the reaction solution was slowly returned to room temperature and reacted overnight. After the reaction was completed, the reaction was placed under ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (60 mL) was added and stirred to mix, and then the mixture was extracted twice with dichloromethane (30 mL*2), the organic phases were collected and combined, and backwashed once with saturated sodium chloride (60 mL). After the organic phase was concentrated under reduced pressure, 30 mL of tetrahydrofuran was added, and then 30 mL of TBAF tetrahydrofuran solution (1 M) was added, and the reaction was continued overnight to remove TBS protection. After the reaction is completed, the organic phases are concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain a crude product of compound E14-1. The cationic lipid E14-1 (3.63 g) is obtained by column chromatography purification, concentration, and oil pump drying. The main data of the H NMR spectrum of E14-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:4.94-4.89(m,1H),4.11(t,2H),3.58-3.29(m,9H),2.65-2.23(m,10H),1.92(d,1H),1.79-0.99(m,50H),0.87(t,9H). MS(ESI):m / z=796.77([M+H] + ).

[0506]

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

[0508]

[0509] Corresponding to the general formula (1), in E15-1, R 1 is undecyl, R 2 for B 1 For pentylene, B 2 For heptylene, L 1 , L 2 All are ester groups (-C(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 822 Da.

[0510] The preparation process is as follows:

[0511] Step a: S2-4 (4.42 g, 9.6 mmol) was dissolved in 60 mL of DMF, and S1-2 (2.30 g, 8.0 mmol) and K were added. 2 CO 3 (1.59 g, 11.5 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 60 mL of dichloromethane. After washing with 10% citric acid (30 mL*2) and brine (30 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S15-1 (4.36 g).

[0512] Step b: The above compound S15-1 (4.01 g, 6.0 mmol) was dissolved in dry THF (60 mL), and NaH (60%, 2.40 g, 60.0 mmol) was slowly added under ice bath, and the mixture was reacted under ice bath for 1 hour. After the reaction was completed, S2-6 (2.51 g, 7.2 mmol) was added, and the mixture was stirred under ice bath for 1 hour, and the reaction solution was slowly returned to room temperature and reacted overnight. After the reaction was completed, the reaction was placed under ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (60 mL) was added and stirred to mix, and then the mixture was extracted twice with dichloromethane (30 mL*2), the organic phases were collected and combined, and backwashed once with saturated sodium chloride (60 mL). After the organic phase was concentrated under reduced pressure, 30 mL of tetrahydrofuran was added, and then 30 mL of TBAF tetrahydrofuran solution (1 M) was added, and the reaction was continued overnight to remove TBS protection. After the reaction is completed, the organic phases are concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain a crude product of compound E15-1. The cationic lipid E15-1 (3.92 g) is obtained by column chromatography purification, concentration, and oil pump drying. The main data of the H NMR spectrum of E15-1 are as follows: 1 H NMR (400 MHz, CDCl 3)δ:4.88-4.80(m,1H),4.08(t,2H),3.84-3.73(m,1H),2.77-2.19(m,23H),1.72-1.18(m,62H),0.87(t,9H). MS(ESI):m / z=822.81([M+H] + ).

[0513]

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

[0515]

[0516] Corresponding to the general formula (1), in E16-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 , L 2 All are ester groups (-OC(=O)-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is about 850Da.

[0517] The preparation process is as follows:

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

[0519]

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

[0521]

[0522] Corresponding to the general formula (1), in E17-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 , L 2 All of them are carbonate groups (-OC(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 1022 Da.

[0523] The preparation process is as follows:

[0524] Step a: Under the protection of nitrogen, 6-bromohexyl-4-nitrophenyl carbonate (S6-1, 10.35 g, 30.0 mmol) was dissolved in DCM (400 mL), and 2-octyl dodecanol (S17-1, 35.76 g, 120.0 mmol) was added dropwise under stirring at room temperature, followed by slow dropwise addition of pyridine (3.02 mL, 37.5 mmol) over 10 min, and then DMAP (0.73 g, 6.0 mmol) was added at once. The reaction was stirred at room temperature for 16 h, and after the reaction was completed, it was extracted twice with DCM and water, the organic phases were combined and washed with brine, and then dried over anhydrous magnesium sulfate, filtered and concentrated, separated and purified by silica gel column, and the target eluent was collected and concentrated to obtain S17-2 (3.63 g).

[0525] Step b: S17-2 (3.33 g, 6.6 mmol) was dissolved in 50 mL of DMF, and S1-2 (0.86 g, 3.0 mmol) and K were added. 2 CO 3(1.09g, 7.9mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50mL of dichloromethane. After washing with 10% citric acid (25mL*2) and brine (25mL*2) in turn, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 25mL of tetrahydrofuran was added, followed by 25mL of TBAF tetrahydrofuran solution (1M), reacted overnight, and TBS protection was removed. After the reaction was completed, the organic phases were concentrated, extracted, combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain E17-1 (2.42g). The main data of the H NMR spectrum of E17-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:4.13-3.99(m,8H),3.33-2.94(m,12H),2.92-2.57(m,6H),2.55-2.41(m,2H),1.76-1.38(m,16H),1.34-1.22(m,66H),0.88(t,12H). MS(ESI):m / z=1022.95([M+H] + ).

[0526]

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

[0528]

[0529] Corresponding to the general formula (1), in E18-1, R 1 is undecyl, R 2 Cyclohexadecyl, B 1 For pentylene, B 2 For hexamethylene, L 1 Ester group (-C(=O)O-), L 2 is a carbonate group (-OC(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 824 Da.

[0530] The preparation process is as follows:

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

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

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

[0534]

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

[0536]

[0537] Corresponding to the general formula (1), in E19-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 , L 2 All are ester groups (-C(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 814 Da.

[0538] The preparation process is as follows:

[0539] Step a: Under argon atmosphere, DCC (3.09 g, 15.0 mmol) was added to a round-bottom flask containing 7-bromoheptanoic acid (S4-1, 2.08 g, 10.0 mmol), 8-pentadecanol (S19-1, 2.51 g, 11.0 mmol) and DMAP (0.31 g, 2.5 mmol) dissolved in dichloromethane (50 mL) under argon atmosphere, 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 obtained residue was purified by silica gel column chromatography to obtain S19-2 (3.68 g).

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

[0541]

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

[0543]

[0544] Corresponding to the general formula (1), in E20-1, R 1 is undecyl, R 2 for B 1 For pentylene, B 2 is a hexamethylene group substituted with -OH, L 1 , L 2 All are ester groups (-C(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 840 Da.

[0545] The preparation process is as follows:

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

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

[0548] Step c: The above compound S20-4 (3.67 g, 9.6 mmol) was dissolved in 50 mL of ethanol, and S2-3 (2.42 g, 8.0 mmol) containing TBS to protect the hydroxyl group was added, and the mixture was stirred at 50°C for 4 hours. After the reaction was completed, 50 mL of dichloromethane was added for dilution, and the mixture was backwashed with 100 mL of saturated sodium chloride solution, and the organic phase was separated and dried with anhydrous sodium sulfate. The mixture was filtered and concentrated, and purified by silica gel column chromatography to obtain S20-5 (3.62 g).

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

[0550] Step e: S20-6 (3.20 g, 4.0 mmol) was dissolved in dry THF (40 mL), and NaH (60%, 1.59 g, 40.0 mmol) was slowly added under ice bath, and the mixture was reacted under ice bath for 1 hour. After the reaction was completed, S2-6 (1.67 g, 4.8 mmol) was added, and the mixture was stirred under ice bath for 1 hour, and the reaction solution was slowly returned to room temperature and reacted overnight. After the reaction was completed, the reaction was placed under ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (40 mL) was added and stirred to mix, and then the mixture was extracted twice with dichloromethane (20 mL*2), the organic phases were collected and combined, and backwashed once with saturated sodium chloride (40 mL), and the organic phase was concentrated under reduced pressure and 20 mL of tetrahydrofuran was added, and then 20 mL of TBAF tetrahydrofuran solution (1 M) was added, and the reaction was continued overnight to remove TBS protection. After the reaction is completed, the organic phases are concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain a crude product of compound E20-1. The cationic lipid E20-1 (2.72 g) is obtained by column chromatography purification, concentration, and oil pump drying. The main data of the H NMR spectrum of E20-1 are as follows: 1 HNMR (400MHz, CDCl 3 )δ:4.85-4.80(m,1H),4.07(t,2H),3.83-3.78(m,1H),3.63-3.59(m,1H),2.67-2.21(m,25H),1.73-1.23(m,60H),0.87(t,9H). MS(ESI):m / z=840.82([M+H] + ).

[0551]

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

[0553]

[0554] Corresponding to the general formula (1), in E21-1, R 1 is undecyl, R 2 for B 1 , B 2 All are -OH substituted hexamethylene, L 1 is an amide group (-NHC(=O)-), L 2 is an ester group (-C(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 841 Da.

[0555] The preparation process is as follows:

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

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

[0558] Step c: The above compound S21-4 (2.58 g, 9.6 mmol) was dissolved in 50 mL of ethanol, and S2-3 (2.42 g, 8.0 mmol) containing TBS to protect the hydroxyl group was added, and the mixture was stirred at 50°C for 4 hours. After the reaction was completed, 50 mL of dichloromethane was added for dilution, and the mixture was backwashed with 100 mL of saturated sodium chloride solution, and the organic phase was separated and dried with anhydrous sodium sulfate. The mixture was filtered and concentrated, and purified by silica gel column chromatography to obtain S21-5 (3.12 g).

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

[0560] Step e: The above compound S21-6 (2.75 g, 4.0 mmol) was dissolved in 50 mL of ethanol, S20-4 (1.67 g, 4.8 mmol) was added, and the mixture was stirred at 50 ° C for 8 hours. After dilution with 50 mL of dichloromethane, the organic phase was separated after backwashing with 100 mL of saturated sodium chloride solution, and 30 mL of tetrahydrofuran was added after concentration under reduced pressure, and then 30 mL of TBAF tetrahydrofuran solution (1 M) was added, and the reaction was allowed to proceed overnight to remove the TBS protection. After the reaction was completed, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by column chromatography, concentrated, and dried by an oil pump to obtain cationic lipid E21-1 (2.39 g). The main data of the nuclear magnetic hydrogen spectrum of E21-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:4.86-4.81(m,1H),3.84-3.78(m,1H),3.66-3.60(m,2H),3.20-3.15(m,2 H),2.67-2.19(m,23H),2.13-2.08(m,2H),1.71-1.22(m,56H),0.86(t,9H). MS(ESI):m / z=841.81([M+H] + ).

[0561]

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

[0563]

[0564] Corresponding to the general formula (1), in E22-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 is an ester group (-OC(=O)-), L 2 is a carbonate group (-OC(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 866 Da.

[0565] The preparation process is as follows:

[0566] Step a: S6-2 (3.12 g, 7.2 mmol) was dissolved in 50 mL of DMF, and S1-2 (1.72 g, 6.0 mmol) and K were added. 2CO 3 (1.19 g, 8.6 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S22-1 (3.02 g).

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

[0568]

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

[0570]

[0571] Corresponding to the general formula (1), in E23-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 is an ester group (-OC(=O)-), L2 is a carbonate group (-OC(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 853 Da.

[0572] The preparation process is as follows:

[0573] Step a: S6-2 (3.12 g, 7.2 mmol) was dissolved in 50 mL of DMF, and 1-amino-3-(4-morpholinyl)-2-propanol (S10-4, 1.64 g, 6.0 mmol) and K were added to the mixture. 2 CO 3 (1.19 g, 8.6 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S23-1 (2.94 g).

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

[0575]

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

[0577]

[0578] Corresponding to the general formula (1), in E24-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 , L 2 All are -O-, -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 865 Da.

[0579] The preparation process is as follows:

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

[0581] Step b: S24-3 (2.85 g, 6.6 mmol) was dissolved in 50 mL of DMF, and S10-4 (0.82 g, 3.0 mmol) containing TBS-protected hydroxyl groups and K 2 CO 3(1.09g, 7.9mmol), stirred overnight at room temperature. After the reaction is completed, the reaction mixture is concentrated under reduced pressure and poured into 50mL of dichloromethane. After washing with 10% citric acid (25mL*2) and brine (25mL*2) in sequence, the organic phase is dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 25mL of tetrahydrofuran is added, followed by 25mL of TBAF tetrahydrofuran solution (1M), and the reaction is allowed to proceed overnight to remove the TBS protection. After the reaction is completed, the organic phases are concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain the crude product of compound E24-1. The crude product is purified by silica gel column chromatography to obtain E24-1 (2.26g). The main data of the nuclear magnetic hydrogen spectrum of E24-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:3.80-3.41(m,13H),2.72-2.30(m,12H),1.82-1.22(m,74H),0.86(t,12H). MS(ESI):m / z=865.91([M+H] + ).

[0582]

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

[0584]

[0585] Corresponding to the general formula (1), in E25-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 , L 2 All are ester groups (-OC(=O)-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 818 Da.

[0586] The preparation process is as follows:

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

[0588]

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

[0590]

[0591] Corresponding to the general formula (1), in E26-1, R 1 , R 2 Both B 1 , B 2 All are heptylene, L 1 , L 2 All are ester groups (-C(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 902 Da.

[0592] The preparation process is as follows:

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

[0594] Step b: S26-2 (3.04 g, 6.6 mmol) was dissolved in 50 mL of DMF, and 2-amino-1-[2-(1-methylimidazolyl)]ethanol (S26-3, 0.77 g, 3.0 mmol) and K were added. 2 CO 3 (1.09g, 7.9mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50mL of dichloromethane. After washing with 10% citric acid (25mL*2) and brine (25mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 25mL of tetrahydrofuran was added, followed by 25mL of TBAF tetrahydrofuran solution (1M), reacted overnight, and TBS protection was removed. After the reaction was completed, the organic phases were concentrated, extracted, combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain E26-1 (2.19g). The main data of the H NMR spectrum of E26-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:6.94-6.91(m,1H),6.88-6.84(m,1H),4.90-4.84(m,2H),4.82-4.78(m,1H),3. 77(s,3H),2.90-2.75(m,2H),2.53-2.22(m,8H),1.75-1.22(m,76H),0.87(t,12H). MS(ESI):m / z=902.82([M+H] + ).

[0595]

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

[0597]

[0598] Corresponding to the general formula (1), in E27-1, R 1 , R 2 Both B1 , B 2 All are hexamethylene, L 1 is an ester group (-C(=O)O-), L 2 is a carbonate group (-OC(=O)O-), L 3 (R 3 )for Where R 3 is -(C=O)OH, R 4 for The total molecular weight is about 850Da.

[0599] The preparation process is as follows:

[0600] Step a: S6-2 (4.69 g, 10.8 mmol) was dissolved in 60 mL of DMF, and 3-(2-tetrazolyl)-L-alanine (S27-1, 1.41 g, 9.0 mmol) and K 2 CO 3 (1.79 g, 13.0 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 60 mL of dichloromethane. After washing with 10% citric acid (30 mL*2) and brine (30 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S27-2 (3.59 g).

[0601] Step b: The above compound S27-2 (3.07 g, 6.0 mmol) was dissolved in dry THF (50 mL), and NaH (60%, 2.40 g, 60.0 mmol) was slowly added under ice bath, and reacted under ice bath for 1 hour. After the reaction was completed, compound S4-3 (3.01 g, 7.2 mmol) was added, and the reaction was stirred under ice bath for 1 hour, and the reaction solution was slowly restored to room temperature and reacted overnight. After the reaction was completed, the reaction was placed under ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (50 mL) was added and stirred to mix, and then extracted twice with dichloromethane (25 mL*2), the organic phases were collected and combined, backwashed once with saturated sodium chloride (50 mL), the organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product of compound E27-1. Purification by column chromatography, concentration, and oil pumping to dryness obtained cationic lipid E27-1 (4.13 g). The main data of the H-NMR spectrum of E27-1 are as follows: 1 H NMR (400 MHz, CDCl 3)δ:8.40(s,1H),4.98-4.75(m,3H),4.21-4.09(m,3H),3.50-3.45(m,1H) ,2.51-2.42(m,4H),2.38-2.32(t,2H),1.72-1.24(m,64H),0.89(t,12H). MS(ESI):m / z=850.84([M+H] + ).

[0602]

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

[0604]

[0605] Corresponding to the general formula (1), in E28-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 , L 2 All of them are carbonate groups (-OC(=O)O-), -L 3 (R 3 )-for Where R 3 is -(C=O)OH, R 4 for The total molecular weight is approximately 864 Da.

[0606] The preparation process is as follows:

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

[0608]

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

[0610]

[0611] Corresponding to the general formula (1), in E29-1, R 1 is undecyl, R 2 for B 1 For pentylene, B 2 For heptylene, L 1 , L 2 All are ester groups (-C(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 779 Da.

[0612] The preparation process is as follows:

[0613] Step a: Under argon atmosphere, DCC (3.71 g, 18.0 mmol) was added to a round-bottom flask containing S5-4 (2.66 g, 12.0 mmol), S19-1 (3.01 g, 13.2 mmol) and DMAP (0.37 g, 3.0 mmol) dissolved in dichloromethane (50 mL) under argon atmosphere, 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 obtained residue was purified by silica gel column chromatography to obtain S29-1 (4.28 g).

[0614] Step b: S29-1 (4.15 g, 9.6 mmol) was dissolved in 60 mL of DMF, and 1-(3-amino-2-hydroxypropyl)pyrrolidin-2-one (S29-2, 2.18 g, 8.0 mmol) containing TBS-protected hydroxyl group and K 2 CO 3(1.59 g, 11.5 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 60 mL of dichloromethane. After washing with 10% citric acid (30 mL*2) and brine (30 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S29-3 (4.15 g).

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

[0616]

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

[0618]

[0619] Corresponding to the general formula (1), in E30-1, R 1 is undecyl, R 2 for B 1 For pentylene, B2 For heptylene, L 1 is an ester group (-C(=O)O-), L 2 is a carbonate group (-OC(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 856 Da.

[0620] The preparation process is as follows:

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

[0622] Step b: S30-2 (3.43 g, 7.2 mmol) was dissolved in 50 mL of DMF, and 1-amino-3-(1-benzimidazolyl)-2-propanol (S30-3, 1.83 g, 6.0 mmol) and K were added to the mixture. 2 CO 3 (1.19 g, 8.6 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S30-4 (3.30 g).

[0623] Step c: The above compound S30-4 (2.81 g, 4.0 mmol) was dissolved in dry THF (50 mL), and NaH (60%, 1.60 g, 40.0 mmol) was slowly added under ice bath, and the mixture was reacted under ice bath for 1 hour. After the reaction was completed, compound S2-6 (1.67 g, 4.8 mmol) was added, and the mixture was stirred under ice bath for 1 hour, and the reaction solution was slowly returned to room temperature and reacted overnight. After the reaction was completed, the reaction was placed under ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (50 mL) was added and stirred to mix, and then the mixture was extracted twice with dichloromethane (25 mL*2), the organic phases were collected and combined, and backwashed once with saturated sodium chloride (50 mL). After the organic phase was concentrated under reduced pressure, 25 mL of tetrahydrofuran was added, and then 25 mL of TBAF tetrahydrofuran solution (1 M) was added, and the mixture was reacted overnight to remove TBS protection. After the reaction is completed, the organic phases are concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain a crude product of compound E30-1. The cationic lipid E30-1 (2.57 g) is obtained by column chromatography purification, concentration, and oil pump drying. The main data of the H NMR spectrum of E30-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:8.20(s,1H),7.65(d,1H),7.60(d,1H),7.23-7.20(m,2H),4.45-4.39(m,1H),4.36-3.32 (m,2H),4.21-4.08(m,3H),4.07(t,2H),2.63-2.23(m,8H),1.70-1.21(m,62H),0.87(t,9H). MS(ESI):m / z=856.83([M+H] + ).

[0624]

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

[0626]

[0627] Corresponding to the general formula (1), in E31-1, R 1 is 8,11-heptadecadienylidene, R 2 for B 1 For butylene, B 2 For heptylene, L 1 is an ester group (-OC(=O)-), L 2 is an ester group (-C(=O)O-), -L 3 (R 3 )-for Where R3 is -OH, R 4 for The total molecular weight is approximately 853 Da.

[0628] The preparation process is as follows:

[0629] Step a: S2-4 (4.42 g, 9.6 mmol) was dissolved in 60 mL of DMF, and S19-3 (2.01 g, 8.0 mmol) containing TBS-protected hydroxyl groups and K 2 CO 3 (1.59 g, 11.5 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 60 mL of dichloromethane. After washing with 10% citric acid (30 mL*2) and brine (30 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S31-1 (4.41 g).

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

[0631]

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

[0633]

[0634] Corresponding to the general formula (1), in E32-1, R 1 , R 2 All are 7-phenylheptyl, B 1 , B 2 All are butylene, L 1 , L 2 All are ester groups (-OC(=O)-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 709 Da.

[0635] The preparation process is as follows:

[0636] Step a: Under argon atmosphere, DCC (3.09 g, 15.0 mmol) was added to a round-bottom flask containing 8-phenyloctanoic acid (S32-1, 2.20 g, 10.0 mmol), 4-bromobutanol (S10-2, 1.67 g, 11.0 mmol) and DMAP (0.31 g, 2.5 mmol) dissolved in dichloromethane (40 mL) under argon atmosphere, 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 obtained residue was purified by silica gel column chromatography to give S32-2 (3.08 g).

[0637] Step b: S32-2 (2.34 g, 6.6 mmol) was dissolved in 40 mL of DMF, and S10-4 (0.82 g, 3.0 mmol) containing TBS-protected hydroxyl groups and K 2 CO 3 (1.09g, 7.9mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 40mL of dichloromethane. After washing with 10% citric acid (20mL*2) and brine (20mL*2) in turn, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and 20mL of tetrahydrofuran was added, followed by 20mL of TBAF tetrahydrofuran solution (1M), and the reaction was allowed to proceed overnight to remove the TBS protection. After the reaction was completed, the organic phases were concentrated, extracted, combined, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain E32-1 (1.75g). The main data of the H NMR spectrum of E32-1 are as follows: 1H NMR (400 MHz, CDCl 3 )δ:7.24-7.19(m,10H),3.90-3.85(m,1H),3.74-3.68(m,4H),4.05(t,4H),2.68-2.23(m,20H),1.69-1.24(m,28H). MS(ESI):m / z=709.66Da([M+H] + ).

[0638]

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

[0640]

[0641] Corresponding to the general formula (1), in E33-1, R 1 is undecyl, R 2 for B 1 For pentylene, B 2 For heptylene, L 1 , L 2 All of them are carbonate groups (-OC(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 985 Da.

[0642] The preparation process is as follows:

[0643] Step a: S8-3 (2.62 g, 7.2 mmol, obtained by reaction of 5-bromopentyl-4-nitrophenyl carbonate and 1-undecanol) was dissolved in 50 mL of DMF, and 2-(3-amino-2-hydroxypropyl)-2,3-dihydro-1H-isoindole-1,3-dione (S33-1, 2.00 g, 6.0 mmol) and K were added. 2 CO 3 (1.19 g, 8.6 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S33-2 (2.89 g).

[0644] Step b: The above compound S33-2 (2.47 g, 4.0 mmol) was dissolved in dry THF (40 mL), and NaH (60%, 1.60 g, 40.0 mmol) was slowly added under ice bath, and the mixture was reacted under ice bath for 1 hour. After the reaction was completed, compound S33-3 (2.69 g, 4.8 mmol, obtained by the reaction of 7-bromoheptyl-4-nitrophenyl carbonate and 3-decyl tridecanol) was added, and the mixture was stirred under ice bath for 1 hour, and the reaction solution was slowly returned to room temperature and reacted overnight. After the reaction was completed, the reaction mixture was placed under ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (40 mL) was added and stirred to mix, and then the mixture was extracted twice with dichloromethane (20 mL*2), the organic phases were collected and combined, and backwashed once with saturated sodium chloride (40 mL). After the organic phase was concentrated under reduced pressure, 20 mL of tetrahydrofuran was added, and then 20 mL of TBAF tetrahydrofuran solution (1 M) was added, and the reaction was allowed to proceed overnight to remove the TBS protection. After the reaction was completed, the organic phases were concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product of compound E33-1. The cationic lipid E33-1 (2.99 g) was obtained by column chromatography purification, concentration, and oil pump drying. The main data of the H NMR spectrum of E33-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:7.90-7.65(m,4H),4.20-3.98(m,9H),3.66-3.59(m,2H),2.61-2.44(m,6H),1.78-1.24(m,73H),0.90(t,9H). MS(ESI):m / z=985.79([M+H] + ).

[0645]

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

[0647]

[0648] Corresponding to the general formula (1), in E34-1, R 1 is tridecyl, R 2 for B 1 For pentylene, B 2 For heptylene, L 1 is an ester group (-C(=O)O-), L 2 is a carbonate group (-OC(=O)O-), -L 3 (R 3 )-for Where R 3 is -(C=O)OH, R 4 for The total molecular weight is approximately 859 Da.

[0649] The preparation process is as follows:

[0650] Step a: S30-2 (3.43 g, 7.2 mmol) was dissolved in 50 mL of DMF, and 3-(3-pyridyl)-L-alanine (S34-1, 1.00 g, 6.0 mmol) and K were added. 2 CO 3 (1.19 g, 8.6 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S34-2 (2.66 g).

[0651] Step b: The above compound S34-2 (2.25 g, 4.0 mmol) was dissolved in dry THF (40 mL), and NaH (60%, 1.60 g, 40.0 mmol) was slowly added under ice bath, and the mixture was reacted under ice bath for 1 hour. After the reaction was completed, compound 6-bromohexanoic acid tridecyl ester (S34-3, 1.80 g, 7.2 mmol, obtained by condensation reaction of 6-bromohexanoic acid and 1-tridecanol) was added, and the mixture was stirred under ice bath for 1 hour, and the reaction solution was slowly returned to room temperature to react overnight. After the reaction was completed, the reaction mixture was placed under ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (400 mL) was added and stirred to mix, and then the mixture was extracted twice with dichloromethane (20 mL*2), the organic phases were collected and combined, and backwashed once with saturated sodium chloride (40 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product of compound E34-1. The cationic lipid E34-1 (2.56 g) was obtained by column chromatography purification, concentration, and oil pump drying. The main data of the H-NMR spectrum of E34-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:8.48-7.14(m,4H),4.22-4.06(m,3H),4.09(t,2H),3.73-3.69(m,1H) ,3.08-3.03(m,2H),2.54-2.23(m,6H),1.70-1.20(m,66H),0.87(t,9H). MS(ESI):m / z=859.75([M+H] + ).

[0652]

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

[0654]

[0655] Corresponding to the general formula (1), in E35-1, R 1 is heptadecyl, R 2 for B 1 is the connecting key, B 2 For hexamethylene, L 1 is a carbonyl group (-C(=O)-), L 2 is an ester group (-OC(=O)-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 771 Da.

[0656] The preparation process is as follows:

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

[0658] Step b: S35-2 (5.35 g, 12.0 mmol) was dissolved in 60 mL of DMF, and 2-amino-1-(2-pyridyl)ethanol (S35-3, 2.52 g, 10.0 mmol) and K were added. 2 CO 3 (1.99 g, 14.4 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 60 mL of dichloromethane. After washing with 10% citric acid (30 mL*2) and brine (30 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S35-4 (4.85 g).

[0659] Step c: Compound S35-4 (4.64 g, 7.5 mmol), stearic acid (S35-5, 1.42 g, 5.0 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.44 g, 7.5 mmol), 1-hydroxybenzotriazole (HOBt, 0.88 g, 6.5 mmol), triethanol (TEA, 1.01 g, 10.0 mmol) were dissolved in dichloromethane (60 mL) in sequence, and stirred at room temperature overnight. After the reaction was completed, the reaction solution was backwashed twice with 0.1 mol / L HCl aqueous solution (10% NaCl) (60 mL*2), and then backwashed once with saturated NaCl (60 mL). After the organic phase was concentrated under reduced pressure, 30 mL of tetrahydrofuran was added, and then 30 mL of TBAF tetrahydrofuran solution (1 M) was added, and the reaction was allowed to proceed overnight to remove the TBS protection. After the reaction is completed, the organic phases are concentrated, extracted, and combined, dried over anhydrous magnesium sulfate, filtered, and the filtrate is concentrated to obtain a crude product. The target compound E35-1 (2.97 g) is obtained by column chromatography purification. The main data of the H NMR spectrum of E35-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:8.56-8.52(m,1H),7.73-7.65(m,1H),7.55-7.51(m,1H),7.20-7.15(m,1H),4.79-7.76(m,1H),4.10-4. 05(t,2H),3.71-3.59(m,2H),3.16(t,2H),2.35-2.28(m,1H),2.23(t,2H),1.70-1.19(m,66H),0.88(t,9H). MS(ESI):m / z=771.74([M+H] + ).

[0660]

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

[0662]

[0663] Corresponding to the general formula (1), in E36-1, R 1 is undecyl, R 2 for B 1 For pentylene, B 2 For heptylene, L 1 is a carbonate group (-OC(=O)O-), L 2 is an ester group (-C(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 825 Da.

[0664] The preparation process is as follows:

[0665] Step a: S8-3 (2.62 g, 7.2 mmol) was dissolved in 50 mL of DMF, and S10-4 (1.64 g, 6.0 mmol) containing TBS-protected hydroxyl groups and K 2 CO 3 (1.19 g, 8.6 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S36-1 (2.73 g).

[0666] Step b: The above compound S36-1 (2.23 g, 4.0 mmol) was dissolved in dry THF (30 mL), and NaH (60%, 1.60 g, 40.0 mmol) was slowly added under ice bath, and the mixture was reacted under ice bath for 1 hour. After the reaction was completed, compound S2-4 (2.21 g, 4.8 mmol) was added, and the mixture was stirred under ice bath for 1 hour, and the reaction solution was slowly returned to room temperature and reacted overnight. After the reaction was completed, the reaction was placed under ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (30 mL) was added and stirred to mix, and then the mixture was extracted twice with dichloromethane (15 mL*2), the organic phases were collected and combined, and backwashed once with saturated sodium chloride (30 mL). After the organic phase was concentrated under reduced pressure, 15 mL of tetrahydrofuran was added, and then 15 mL of TBAF tetrahydrofuran solution (1 M) was added, and the mixture was reacted overnight to remove TBS protection. After the reaction is completed, the organic phases are concentrated, extracted, and combined, dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated to obtain a crude product of compound E36-1. The cationic lipid E36-1 (2.61 g) is obtained by column chromatography purification, concentration, and oil pump drying. The main data of the H NMR spectrum of E36-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:4.89-4.85(m,1H),4.22-4.03(m,4H),3.93-3.85(m,1H),3.73-3.67(m,4H),2.69-2.22(m,14H),1.76-1.22(m,62H),0.87(t,9H). MS(ESI):m / z=825.78([M+H] + ).

[0667]

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

[0669]

[0670] Corresponding to the general formula (1), in E37-1, R 1 is heptadecyl, R 2 for B 1 is the connecting key, B 2 For hexamethylene, L 1 is a carbonyl group (-C(=O)-), L 2 is an ester group (-OC(=O)-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 793 Da.

[0671] The preparation process is as follows:

[0672] Step a: S35-2 (5.35 g, 12.0 mmol) was dissolved in 60 mL of DMF, and S10-4 (2.74 g, 10.0 mmol) containing TBS-protected hydroxyl groups and K 2 CO 3 (1.99 g, 14.4 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 60 mL of dichloromethane. After washing with 10% citric acid (30 mL*2) and brine (30 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S37-1 (5.06 g).

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

[0674]

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

[0676]

[0677] Corresponding to the general formula (1), in E38-1, R 1 is heptadecyl, R 2 for B 1 is the connecting key, B 2 For hexamethylene, L 1 is a carbonyl group (-C(=O)-), L 2 -O-, -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 757 Da.

[0678] The preparation process is as follows:

[0679] Step a: S24-3 (3.11 g, 7.2 mmol) was dissolved in 50 mL of DMF, and S7-3 (1.51 g, 6.0 mmol) containing TBS-protected hydroxyl groups and K 2 CO 3 (1.19 g, 8.6 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S38-1 (2.90 g).

[0680] Step b: Compound S38-1 (2.72 g, 4.5 mmol), S35-5 (0.85 g, 3.0 mmol), EDCI (0.86 g, 4.5 mmol), HOBt (0.53 g, 3.9 mmol), TEA (0.61 g, 6.0 mmol) were dissolved in dichloromethane (50 mL) in turn, and stirred at room temperature for overnight reaction. After the reaction was completed, the reaction solution was backwashed twice with 0.1 mol / L HCl aqueous solution (10% NaCl) (50 mL*2), and then backwashed once with saturated NaCl (50 mL). After the organic phase was concentrated under reduced pressure, 25 mL of tetrahydrofuran was added, and then 25 mL of TBAF tetrahydrofuran solution (1 M) was added, and the reaction was continued overnight to remove the TBS protection. After the reaction was completed, the organic phase was concentrated, extracted, and combined, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated to obtain a crude product. Purify by column chromatography, concentrate, and pump dry to obtain the target compound E38-1 (1.75 g). The main data of the H NMR spectrum of E38-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:8.44(d,2H),7.24(d,2H),4.74-4.69(m,1H),3.71-3.41(m,6H),3.18(t,2H),2.24(t,2H),1.55-1.21(m,67H),0.88(t,9H). MS(ESI):m / z=757.77([M+H] + ).

[0681]

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

[0683]

[0684] Corresponding to the general formula (1), in E39-1, R1 is heptadecyl, R 2 for B 1 is the connecting key, B 2 For hexamethylene, L 1 is a carbonyl group (-C(=O)-), L 2 -O-, -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 763 Da.

[0685] The preparation process is as follows:

[0686] Step a: S24-3 (3.11 g, 7.2 mmol) was dissolved in 50 mL of DMF, and 1-amino-3-(1-pyrrolidinyl)-2-propanol (S39-1, 1.55 g, 6.0 mmol) and K 2 CO 3 (1.19 g, 8.6 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S39-2 (3.01 g).

[0687] Step b: Compound S39-2 (2.75 g, 4.5 mmol), S35-5 (0.85 g, 3.0 mmol), EDCI (0.86 g, 4.5 mmol), HOBt (0.53 g, 3.9 mmol), TEA (0.61 g, 6.0 mmol) were dissolved in dichloromethane (50 mL) in turn, and stirred at room temperature overnight. After the reaction was completed, the reaction solution was backwashed twice with 0.1 mol / L HCl aqueous solution (10% NaCl) (50 mL*2), and then backwashed once with saturated NaCl (50 mL). The organic phase was retained, concentrated under reduced pressure, and then 25 mL of tetrahydrofuran was added, and then 25 mL of TBAF tetrahydrofuran solution (1 M) was added, and the reaction was continued overnight to remove the TBS protection. After the reaction was completed, the organic phase was concentrated, extracted, and combined, dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain the crude product of E39-1. Purify by column chromatography, concentrate, and pump dry to obtain the target compound E39-1 (1.79 g). The main data of the H NMR spectrum of E39-1 are as follows: 1 H NMR (400 MHz, CDCl 3)δ:3.82-3.21(m,7H),3.19(t,2H),2.45-2.38(m,2H),2.25-2.19(m,6H),1.63-1.20(m,71H),0.87(t,9H). MS(ESI):m / z=763.81([M+H] + ).

[0688]

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

[0690]

[0691] Corresponding to the general formula (1), in E40-1, R 1 is heptadecyl, R 2 for B 1 is the connecting key, B 2 For hexamethylene, L 1 is a carbonyl group (-C(=O)-), L 2 is a carbonate group (-OC(=O)O-), -L 3 (R 3 )-for Where R 3 is -OH, R 4 for The total molecular weight is approximately 794 Da.

[0692] The preparation process is as follows:

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

[0694]

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

[0696]

[0697] Corresponding to the general formula (1), in E41-1, R 1 for R 2 for B 1 For hexamethylene, B 2 is a hexamethylene group substituted with -OH, L 1 is an ester group (-OC(=O)-), L 2 is an ester group (-C(=O)O-), -L 3 (R 3 )-for Where R 3 for G 1 OCH 2 CH<, k is 2, two F 1 -OH and -C(=O)OCH 3 , R 4 for The total molecular weight is approximately 1012 Da.

[0698] The preparation process is as follows:

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

[0700] Step b: Add 150 mL of tetrahydrofuran and an excess of diphenylmethyl potassium (10.30 g, 50.0 mmol) to a closed reaction kettle without water or oxygen, and then add compound S41-1 (3.76 g, 13.0 mmol) and compound S41-2 (5.04 g, 13.0 mmol), wherein S41-2 is methyl 2,3-dihydroxypropionate in which one hydroxyl group is replaced by p-toluenesulfonate and the other hydroxyl group is protected by TBS. After reacting at 30°C for 12 hours, the reaction kettle is opened, concentrated, washed, dissolved with dichloromethane, trifluoroacetic acid (TFA) is added to 0.1 M, reacted for 4 hours, the pH is adjusted to neutral, the reaction solution is concentrated, purified water is added, extracted with dichloromethane, the extract is dried over anhydrous magnesium sulfate, filtered, the filtrate is concentrated, and recrystallized to obtain the intermediate S41-3 (3.56 g) with exposed amino groups.

[0701] Step c: S20-4 (3.21 g, 8.4 mmol) was dissolved in 50 mL of ethanol, S41-3 (2.84 g, 7.0 mmol) was added, and the mixture was stirred at 50°C for 4 hours. After the reaction was completed, 50 mL of dichloromethane was added for dilution, and the mixture was backwashed with 100 mL of saturated sodium chloride solution, and the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered and concentrated, and purified by silica gel column chromatography to obtain S41-4 (3.64 g).

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

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

[0704]

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

[0706]

[0707] Corresponding to the general formula (1), in E42-1, R 1 , R 2 Both B 1 , B 2 All are hexamethylene, L 1 , L 2 All are ester groups (-OC(=O)-), -L 3 (R 3 )-for Where R 3 -O(CH 2 ) 2 OH, R4 for The total molecular weight is approximately 958 Da.

[0708] The preparation process is as follows:

[0709] Step a: Add 150 mL of tetrahydrofuran and an excess of diphenylmethyl potassium (10.30 g, 50.0 mmol) to a closed reaction kettle without water or oxygen, and then add compound S42-1 (2.73 g, 10.0 mmol) and compound S42-2 (2.88 g, 10.0 mmol), wherein S42-2 is ethylene glycol in which one hydroxyl group is replaced by p-toluenesulfonate (-OTs) and the other hydroxyl group is protected by EE. After reacting at 30°C for 12 hours, the reaction kettle is opened, and compound S8-1 (3.27 g) having two protected hydroxyl groups and one protected amino group is obtained after concentration, washing, and column chromatography.

[0710] Step b: Add S8-1 (2.33 g, 6.0 mmol) to a dry and clean container, dissolve it with dichloromethane, add TFA to 0.1 M, react for 4 hours, adjust the pH to neutral, concentrate the reaction solution, add purified water, extract with dichloromethane, concentrate the extract, precipitate, filter, and dry to obtain the amino-exposed intermediate S42-3 (1.42 g).

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

[0712]

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

[0714]

[0715] Corresponding to the general formula (1), in E43-1, R 1 is undecyl, R 2 for B 1 For pentylene, B 2 For heptylene, L 1 , L 2 All are ester groups (-C(=O)O-), -L 3 (R 3 )-for Where R 3 for R 4 for The total molecular weight is approximately 861 Da.

[0716] The preparation process is as follows:

[0717] Step a: S2-4 (3.31 g, 7.2 mmol) was dissolved in 50 mL of DMF, and S11-3 (2.14 g, 6.0 mmol) and K were added. 2 CO 3 (1.19 g, 8.6 mmol), stirred overnight at room temperature. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and poured into 50 mL of dichloromethane. After washing with 10% citric acid (25 mL*2) and brine (25 mL*2) in sequence, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain S43-5 (3.63 g).

[0718] Step b: The above compound S43-5 (2.95 g, 4.0 mmol) was dissolved in dry THF (50 mL), and NaH (60%, 1.60 g, 40.0 mmol) was slowly added under ice bath, and the mixture was reacted under ice bath for 1 hour. After the reaction was completed, S2-6 (1.67 g, 4.8 mmol) was added, and the mixture was stirred under ice bath for 1 hour, and the reaction solution was slowly returned to room temperature and reacted overnight. After the reaction was completed, the reaction was placed under ice bath, and 2 mL of water was slowly added to quench the reaction. After stirring for 30 minutes, water (50 mL) was added and stirred to mix, and then extracted twice with dichloromethane (25 mL*2), the organic phases were collected and combined, and backwashed once with saturated sodium chloride (50 mL). After the organic phase was concentrated under reduced pressure, it was dissolved with methanol, 1 M hydrochloric acid was added to pH = 3.5, and the mixture was reacted for 4 hours, concentrated, precipitated, filtered, recrystallized, and dried to obtain the crude product of E43-1 with exposed hydroxyl groups. Cationic lipid E43-1 (2.65 g) was obtained by column chromatography purification. The main data of the H-NMR spectrum of E43-1 are as follows: 1 H NMR (400 MHz, CDCl 3 )δ:8.55(d,2H),7.20(d,2H),4.84-4.77(m,1H),4.29-4.25(m,1H),4.07(t,2H),3.77-3.72(m,1H),3.63 -3.56(m,2H),3.42-3.36(m,2H),3.01-2.94(m,2H),2.57-2.24(m,8H),1.75-1.23(m,62H),0.89(t,9H). MS(ESI):m / z=861.76([M+H] + ).

[0719]

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

[0721]

[0722] Corresponding to the general formula (2), in E44-1, R 1 , R 2 All are tetradecyl, B 1 , B 2 All are connecting keys, L 1 , L 2 All are connecting keys, L 3 for L A For -(CH 2 ) h - and h is 0, A 1 OCH 2 CH2 -, n 1 ≈45, R 3 is methyl, R 5 It is hexyl and has a total molecular weight of approximately 2.5 kDa.

[0723] The preparation process is as follows:

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

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

[0726]

[0727] 5.2. Lipid Pharmaceutical Compositions

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

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

[0730]

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

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

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

[0734] Step (3): The ethanol phase solution and the aqueous phase solution were mixed (1:3 v / v) to prepare LNP / Fluc-mRNA, and washed by DPBS ultrafiltration multiple times to remove ethanol and free molecules, and finally passed through a 0.2 μm sterile filter for use.

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

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

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

[0738] Nucleic acid complexing ability determination: The nucleic acid complexing ability of LNP / Fluc-mRNA was investigated by gel permeation electrophoresis experiment. Weigh 0.8g of agarose and dissolve it in 40mL of TAE solution, heat it in a microwave oven to completely dissolve the agarose particles, cool it, add 5μL of nucleic acid dye GelGreen to the cooled agarose gel, add the gel to the gel tank, and dry it naturally. Add the mixture of LNP / Fluc-mRNA and 2μL of LoadingBuffer to the agarose gel hole, set the electrophoresis voltage to 90V for electrophoresis experiment, and electrophoresed for 10min at room temperature. There is basically no free Fluc-mRNA in the LNP / Fluc-mRNA experimental group (L-1~L-43, L-3-2-d, PL-0, PL-3-2, PL-4, PL-10-2, PL-16, PL-17) and the LNP / Fluc-mRNA control group D-0, indicating that the LNP of the present invention has a good nucleic acid drug complexing ability.

[0739] Encapsulation efficiency determination: LNP / Fluc-mRNA was ultracentrifuged (4°C, 60000rpm, 1h) using an ultracentrifuge, and the concentration of unencapsulated Fluc-mRNA in the supernatant was detected using a nucleic acid quantifier, and the encapsulation efficiency of liposomes for Fluc-mRNA was calculated. The results are summarized in Table 1, showing that the liposomes of the present invention have a high encapsulation efficiency for nucleic acid drugs, wherein the encapsulation efficiency of the experimental group is above 80%. In particular, compared with E16-1, the polar head of d-1 does not contain a side chain hydroxyl group, d-2 does not contain a polar head with a branched structure, and the polar head of d-3 does not contain a nitrogen heterocycle, and accordingly, the encapsulation efficiency of the experimental group L-16 (95%) is greater than that of the control groups D-1, D-2 and D-3 (81%, 91%, 83%). E1-1 and d-4 also contain multiple ester bonds, and accordingly, the encapsulation efficiency of L-1 (87%) is greater than that of D-4 (81%). Compared with L3-2, L3-2-d contains the same types of lipids and has a higher encapsulation rate by adjusting the ratio of each lipid to a certain extent.

[0740] Particle size determination: According to the literature (Hassett et al., J. Controlled Release 2021, 335, 237-246), the particle size of the LNP preparation containing nucleic acid drugs can exert better efficacy at 60 to 150 nm. In this embodiment, the particle size of LNP / Fluc-mRNA was measured by dynamic light scattering (DLS). The measured LNP / Fluc-mRNA has a high size uniformity, and its PDI is less than 0.3. The experimental results show that the particle size of LNP / Fluc-mRNA prepared using the cationic lipids of the present invention is in the range of about 70 nm to about 113 nm, which are particle sizes that can achieve better efficacy.

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

[0742]

[0743]

[0744] (2) Serum stability evaluation

[0745] The above-mentioned LNP / Fluc-mRNA (control group D-0, D-1, D-2, D-3, D-4; experimental group L-1, L-3-2, L-4, L-10-2, L-16, L-17, PL-0, PL-3-2, PL-4, PL-10-2, PL-16, PL-17) was added to a culture medium containing 10% fetal bovine serum (FBS), stirred at 37°C, and samples were taken at regular intervals to determine the particle size change of LNP / Fluc-mRNA. The serum stability of the nucleic acid drug preparation was analyzed by testing the particle size change. The experimental results show (Table 2) that within 7 days, the particle size of PL-3-2, PL-4, PL-16, and PL-10-2 containing both the cationic lipid of the present invention and the PEGylated lipid of the present invention changed the least (0-3%), and the particle size of the remaining experimental groups and the control group changed by 5-16%, among which the cationic lipids containing carbonate groups or multiple ester groups, D-4, L-1, L-17, and PL-17, had slightly larger particle size changes. The results show that the cationic lipid of the present invention is used for the preparation of the lipid pharmaceutical composition, and the obtained lipid nanoparticles can have sufficient stability under physiological conditions, and the PEGylated lipid of the present invention has a better effect.

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

[0747]

[0748]

[0749] (3) Cytotoxicity evaluation

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

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

[0752] Hela press 1×10 4The cells were seeded in 96-well plates at a ratio of 100 μL per well and divided into control group (blank control group), L2K / Fluc-mRNA group (positive control group) and LNP / Fluc-mRNA group (experimental groups in Table 1) and incubated at 37°C and 5% CO 2 After 24 hours of incubation, 3.3 μg / mL of L2K / Fluc-mRNA and 3.3 μg / mL of LNP / Fluc-mRNA were added to the positive control group and the experimental group, respectively, and the cells were incubated at 37°C and 5% CO. 2 After 24 hours of incubation, remove the 96-well plate, remove the culture medium, and add 120 μL of the diluted CCK-8 solution to each well. 2 Incubate for 1-4 hours.

[0753] Take out and measure the absorbance of each well at a wavelength of 450 nm using an enzyme marker.

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

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

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

[0757] Table 3: Cell transfection test results

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

[0759] In order to investigate the mRNA transfection rate of each group of LNP / Fluc-mRNA compositions prepared in Example 45 of the present invention at the cellular level, Luciferase bioluminescence was used for testing. The LNP / Fluc-mRNA composition preparation was dissolved in the culture medium to prepare the required dose, and Hela cells were used as a cell model. The cell suspension 100 μL / well was inoculated into a 96-well plate with a black-rimmed transparent bottom at an inoculation density of 6000 cells / well. After inoculation, the cells were incubated in a cell culture incubator for 24 hours, and then administered at a dose of 0.2ug mRNA per well. The blank control group was added with a corresponding dose of free Fluc-mRNA. After transfection for 24 hours, the old culture medium was removed and replaced with a new culture medium containing D-fluorescein sodium (1.5mg / mL) substrate, and after incubation for 5 minutes, the bioluminescence was detected using an ELISA instrument. The stronger the fluorescence, the more Fluc-mRNA transported into the cytoplasm and translated into the corresponding fluorescent protein. The experimental results are shown in Table 3, wherein the relative value of fluorescence intensity is the ratio of the fluorescence intensity value of each group to the fluorescence intensity of the blank control group. The results show that the LNP / Fluc-mRNA compositions prepared by the present invention have excellent in vitro transfection effects, that is, the LNPs of the control group and the experimental group are effective delivery vectors, and the transfection effects of most experimental groups are better than those of the control group. When the pegylated lipid in the LNP / Fluc-mRNA composition is DMG, the transfection effects of L-1, L-3-2, L-10-2 and L-16 containing nitrogen heterocycles at the polar head of the cationic lipid are better; among them, the transfection effect of L-1 is the best, which may be due to the fact that the multiple ester bonds it contains are more conducive to the degradation of cationic lipids in cells and promote the release of nucleic acid drugs Fluc-mRNA into the cytoplasm. When the pegylated lipid in the LNP / Fluc-mRNA composition is E44-1 of the present invention, the transfection efficiency will not be significantly reduced (such as the transfection efficiency of L-0 and PL-0 is equivalent), and when the cationic lipid of the present invention is matched, the transfection efficiency is improved to a certain extent (such as PL-1 and PL-3-2).

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

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

Claims

1. A cationic lipid, It is characterized in that The structure is shown in the general formula (1): Wherein, N is a nitrogen branching center; L 1 , L 2 Each independently represents -OC(=O)-, -C(=O)O-, -OC(=O)O-, -C(=O)O(CH 2 ) y OC(=O)-、-OC(=O)(CH 2 ) y OC(=O)-、-C(=O)O(CH 2 ) y C(=O)O-, -OC(=O)(CH 2 ) y Any one of C(=O)O-, wherein y is an integer from 2 to 8; L 3 is a trivalent connecting group, and R 3 The side chain is R 3 The divalent linking group -L 3 (R 3 )-; B 1 , B 2 Each independently is C 1-30 Alkylene; R 1 , R 2 Each independently is C 2-30 Aliphatic hydrocarbon or C containing 1-2 O 2-30 Aliphatic hydrocarbon derivative residues; R 3 For -(CH 2 ) h -R 01 or -(CH 2 ) f -Z 3 -(CH 2 ) g -R 01 ; wherein h is an integer from 0 to 6, f is 0, 1 or 2, g is 2, 3 or 4, Z 3 For-O-, R 01 is -OH; R 4 is a carbocyclic group or R'N(R')-R"-N(R')-; wherein R' is independently H or C 1-3 Alkyl, R" is C 2-4 Alkylene; The-L 3 (R 3 )- has the structure The alkylene group, aliphatic hydrocarbon group, aliphatic hydrocarbon derivative residue, and carbocyclic group are each independently unsubstituted; or a salt, tautomer, stereoisomer or solvate thereof.

2. The cationic lipid according to claim 1, It is characterized in that The-L 3 (R 3 )-for 3. The cationic lipid according to claim 1, It is characterized in that B 1 , B 2 Each independently is C 1-20 The alkylene groups are each independently selected from any one of methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, hexadecylene, heptadecylene, octadecylene, nonadecylene and eicosylene.

4. according to the cationic lipid of claim 3, described B 1 , B 2 Each is independently selected from any one of the following: ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene; Preferred B 1 , B 2 All are ethylene, or B 1 , B 2 All are butylene, or B 1 , B 2 All are hexamethylene, or B 1 , B 2 All are heptylene, or B 1 For butylene, B 2 is heptylene, or B 1 For pentylene, B 2 For hexamethylene, or B 1 For pentylene, B 2 It is heptylene.

5. The cationic lipid according to claim 1, It is characterized in that The R 1 , R 2 Each independently selected from R L , R B , Rr; The R L For straight chain C 2-30 Aliphatic hydrocarbon group, containing 0-4 carbon-carbon double bonds; preferably any one of the following structures or any cis-trans isomers thereof: Among them, t L An integer selected from 0-20; The R B The structure is Where t is 0, 1 or 2; t 01 ,t 02 ,t 03 ,t 04 Each independently is 0 or 1; R e , R f Each independently is C 1-12 Alkyl, C 3-12 Alkenyl and C 3-12 Any of the alkynyl groups; preferably R B Any of the following structures: The Rr is a C containing a ring structure 3-30 Aliphatic hydrocarbon group; the ring structure is C 3-20 Carbocyclic ring, preferably C 3-20 The residue of cycloalkane; the Rr is preferably any one of the following structures:

6. The cationic lipid according to claim 1, It is characterized in that The R 4 is R'N(R')-R"-N(R')-, selected from Any one, preferably 7. The cationic lipid according to claim 1, It is characterized in that The R 4 is a carbocyclic group selected from any one of the following structures: More preferably 8. The cationic lipid according to claim 1, It is characterized in that Its structure is selected from any of the following:

9. A lipid composition, It is characterized in that Contains the cationic lipid according to any one of claims 1 to 8.

10. The lipid composition according to claim 9, It is characterized in that It also contains one or more of phospholipids, steroid lipids and pegylated lipids, selected from any one of the following situations: Case (1): also contains phospholipids; Case (2): also contains steroid lipids; Case (3): also contains PEGylated lipids; Case (4): also contains phospholipids and steroid lipids; Case (5): also contains phospholipids and PEGylated lipids; Case (6): also contains steroid lipids and PEGylated lipids; Case (7): also contains phospholipids, steroid lipids and PEGylated lipids; It is preferred that the lipids include phospholipids, steroid lipids and PEGylated lipids.

11. The lipid composition according to claim 10, It is characterized in that The phospholipids are selected from 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-O-octadecanoyl-sn-glycero-3-phosphocholine, sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn- Glycerol-3-phosphoethanolamine, 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycerol-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycerol-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycerol-3-phospho-rac-(1-glycerol) sodium salt, dioleoylphosphatidyl silk Any one of amino acid, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine, and combinations thereof.

12. The lipid composition according to claim 10, It is characterized in that The steroid lipid is selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol and a mixture thereof.

13. The lipid composition according to claim 10, It is characterized in that The PEGylated lipid is selected from polyethylene glycol-1,2 dimyristin, polyethylene glycol-distearoyl phosphatidylethanolamine, PEG-cholesterol, polyethylene glycol-diacylglycerol, polyethylene glycol-dialkoxypropyl, specifically including any one of polyethylene glycol 500-dipalmitoyl phosphatidylcholine, polyethylene glycol 2000-dipalmitoyl phosphatidylcholine, polyethylene glycol 500-stearoyl phosphatidylethanolamine, polyethylene glycol 2000-distearoyl phosphatidylethanolamine, polyethylene glycol 500-1,2-oleoyl phosphatidylethanolamine, polyethylene glycol 2000-1,2-oleoyl phosphatidylethanolamine and polyethylene glycol 2000-2,3-dimyristoyl glycerol.

14. The lipid composition according to claim 10, It is characterized in that The PEGylated lipid is selected from any one of the following: where n 1 An integer selected from 20-250.

15. The lipid composition according to claim 10, It is characterized in that The structure of the PEGylated lipid is selected from any one of the following: where n 1 is an integer selected from 20-250.

16. The lipid composition according to claim 10, It is characterized in that The molar percentage of PEGylated lipids to total lipids is 0.5-5%, preferably 1-3%, more preferably 1.5%, 1.6%, 1.7%, 1.8%, 1.9%; The molar percentage of cationic lipids to total lipids is 30-65%, preferably 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 55%; The molar percentage of phospholipids to total lipids is 7.5-13%, preferably 8%, 9%, 10%, 11%, 12%; The molar percentage of steroid lipids in total lipids is 35-50%, preferably 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%.

17. A lipid pharmaceutical composition, It is characterized in that A lipid composition and a drug according to any one of claims 9 to 16, wherein the drug is selected from any one of nucleic acid drugs, gene vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs or protein drugs.

18. The lipid pharmaceutical composition according to claim 17, It is characterized in that The drug is a nucleic acid drug, selected from any one of DNA, RNA, antisense nucleic acid, plasmid, interfering nucleic acid, aptamer, antagomir and ribozyme, and the RNA is selected from any one of mRNA, saRNA, circRNA, miRNA and siRNA; preferably any one of DNA, mRNA, miRNA and siRNA.

19. The lipid pharmaceutical composition according to claim 17, It is characterized in that The lipid pharmaceutical composition is used as a medicine, and the medicine is selected from any one of the following: a drug for treating cancer, an anti-infective agent, an antibiotic, an antiviral agent, an antifungal agent, and a vaccine.

20. The lipid pharmaceutical composition according to claim 17, It is characterized in that The lipid pharmaceutical composition is an LNP-pharmaceutical composition, preferably an LNP-nucleic acid pharmaceutical composition, and more preferably an LNP-mRNA composition.

21. A lipid pharmaceutical composition preparation, It is characterized in that A lipid pharmaceutical composition comprising the lipid pharmaceutical composition of any one of claims 17 to 20 and a pharmaceutically acceptable diluent or excipient, wherein the diluent or excipient is preferably any one of deionized water, ultrapure water, phosphate buffer and normal saline, more preferably phosphate buffer or normal saline, and most preferably normal saline.

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