Cationic lipid compound, preparation method thereof, composition containing cationic lipid compound and application

Abstract

The invention provides a cationic lipid compound, a preparation method thereof, a composition containing the cationic lipid compound and application, and belongs to the technical field of biological medicine. The cationic lipid compound has a structure as shown in a formula (I): # imgabs0 #, one or more of R1, R2 and R3 are independently selected from secondary amine or tertiary amine, and the rest are independently selected from C3-C30 straight chain or branched chain saturated or unsaturated alkyl; l1 and L2 are each independently selected from a single bond or C1-C20 saturated or unsaturated alkylene, and q methylene groups of L1 and / or L2 are each independently replaced by-O-,-C (O)-or-C (O) O-; l3 is selected from saturated or unsaturated alkylene groups with 1 to 10 carbon atoms; x1, X2 and X4 are respectively and independently selected from-C (O)-,-C (O)-NH-,-C (O)-S-,-C (O)-O-,-NH-,-S-and-O-, and X3 is selected from-NH-,-S-or-O-.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and in particular to a cationic lipid compound, a preparation method thereof, a composition containing the same, and an application thereof. Background Art

[0002] In order to effectively deliver drugs such as nucleic acid drugs and gene vaccines, a delivery system centered on cationic lipid molecules has been widely used clinically. Most of the polar heads of cationic lipids contain amine groups (except for one lipid containing an amidino group), which play a role in the binding of lipids to nucleic acid molecules, and lipid nanoparticle-nucleic acid complexes to cell membranes or other components within cells.

[0003] In related technologies, quaternary ammonium salts in which the amino group of the polar head is substituted by methyl or hydroxyethyl are representative of permanently charged cationic lipids. For example, there are: monovalent cationic lipids such as (2,3-dioleyloxypropyl) trimethylammonium chloride (DOTAP), dioleoylpropyl trimethylammonium bromide (DOTMA), dimethyl-2-hydroxyethyl-2,3-bis(tetradecyloxy)propylammonium bromide (DMRIE), and 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM); multivalent cationic lipids such as N-(2-sperminecarbonyl)-N',N'-dioctadecylglycinamide (DOGS) and dimethyl-2,3-dioleenyloxypropyl-2-(2-sperminecarbonylamino)ethylammonium trifluoroacetate (DOSPA).

[0004] The defects of the above-mentioned charged cationic lipids are that they have relatively high toxicity or are prone to inducing immune responses. Therefore, ionizable cationic lipids have been further developed, with protonatable amino groups as the head groups and unsaturated alkyl chains to improve transfection efficiency. For example, the first-generation ionizable cationic lipid 1,2-dihydroxy-3-dimethylaminopropane (DLin-DMA) with a pKa of 6.5; and again, based on DLin-DMA, many available cationic lipids have been obtained by modifying the length and degree of unsaturation, such as dioleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA). Based on ionizable cationic lipids, lipid nanoparticle delivery systems have been successfully applied to a variety of nucleic acid drugs and vaccines. However, the rapid development of this field still requires the development of highly efficient nucleic acid delivery carriers. Summary of the Invention

[0005] In view of this, the main object of the present invention is to provide a cationic lipid compound, a preparation method thereof, a composition containing the same, and an application thereof. By constructing a lipid nanoparticle delivery system with a cationic lipid compound having high transfection efficiency and low cytotoxicity, it is beneficial to improve the nucleic acid delivery efficiency.

[0006] To achieve the above object, in one aspect of the present invention, there is provided a cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof, wherein the cationic lipid compound has the structure shown in formula (I):

[0007]

[0008] Wherein, one or more of R1, R2, and R3 are each independently selected from secondary amines or tertiary amines, and the rest are each independently selected from C3-C 30 linear or branched saturated or unsaturated hydrocarbon groups, and R1, R2, and R3 are not all secondary amines or tertiary amines; L1 and L2 are each independently selected from a single bond, or a substituted or unsubstituted C1-C 20 saturated or unsaturated alkylene group, q methylene groups of L1 and / or L2 are each independently replaced by -O-, -C(O)- or -C(O)O-, and when the substituted or unsubstituted C1-C 20 saturated or unsaturated alkylene group has a substituent, the substituent is selected from one or more combinations of hydroxyl group, mercapto group, C1-C 20 alkyl groups; L3 is selected from a substituted or unsubstituted C1-C 10 alkylene group, and when the substituted or unsubstituted C1-C 10 alkylene group has a substituent, the substituent is selected from one or more combinations of C1-C 20 alkyl groups, C3-C8 cycloalkyl groups; X1, X2, and X4 are each independently selected from -C(O)-, -C(O)-NH-, -C(O)-S-, -C(O)-O-, -NH-, -S-, -O-, X3 is selected from -NH-, -S- or -O-; p is 1 or 2; q is an integer from 0 to 10.

[0009] In another aspect of the present invention, there is provided a composition, which comprises the above-mentioned cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof.

[0010] In yet another aspect of the present invention, there is provided an application of the above-mentioned cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof or the above-mentioned composition in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides or protein drugs.

[0011] The above-mentioned cationic lipid compound shown in formula (I) provided by the present invention has a large difference in structure compared with representative cationic lipids in the related art, such as DLin-DMA, DLin-MC3-DMA, etc., and has the characteristics of high transfection efficiency and low cytotoxicity. The lipid nanoparticle delivery system constructed by it can exhibit good nucleic acid delivery efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the particle size distribution diagram of the lipid nanoparticles obtained by the dynamic light scattering method (DLS) in Example 60 of the present invention. Detailed implementation manners

[0013] The terms used herein are merely for describing specific embodiments and are not intended to limit the present invention. The terms "including", "comprising", etc. used herein indicate the presence of features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components. All terms used herein (including technical and scientific terms) have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein should be interpreted as having a meaning consistent with the context of this specification and should not be interpreted in an idealized or overly rigid manner.

[0014] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meanings understood by those skilled in the art to which the present invention pertains. If descriptions such as "first", "second", etc. are involved throughout the text, such "first", "second", etc. descriptions are only used to distinguish similar objects and should not be construed as indicating or implying their relative importance, sequence, or implicitly indicating the quantity of the technical features indicated. It should be understood that the data described by "first", "second", etc. can be interchanged under appropriate circumstances.

[0015] In the present invention, the term "pharmaceutically acceptable" refers to a compound, substance, composition, and / or dosage form that is applicable to contact with human and animal tissues within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic reaction, or other problems or complications and is commensurate with a reasonable benefit / risk ratio.

[0016] In the present invention, the term "solvate" refers to a complex formed by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof and a solvent (such as ethanol or water). The term "hydrate" refers to the case where the solvent in the above-mentioned term "solvate" is water. Further, the compound of formula (I) or a pharmaceutically acceptable salt thereof can be separated in the form of a solvate, and any solvate is included within the scope of protection of the present invention.

[0017] In the present invention, the term "pharmaceutically acceptable salt" refers to relatively non-toxic organic or inorganic acid addition salts of the compounds of formula (I). Among them, inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, etc., and organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, cinnamic acid, picric acid, trifluoromethanesulfonic acid, dodecyl sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid, itaconic acid, naphthalenedisulfonic acid, malic acid, adipic acid, alginic acid, maleic acid, D-gluconic acid, aspartic acid, etc.

[0018] In the present invention, the term "stereoisomer" includes geometric isomers, diastereoisomers and enantiomers. Therefore, the compounds claimed in the present invention also include racemic mixtures, single stereoisomers and optically active mixtures. It is understood that one stereoisomer may have better efficacy and / or lower side effects than other stereoisomers. Single stereoisomers and optically active mixtures can be obtained by chiral source synthesis, chiral catalysis, chiral resolution and other methods. Racemates can be resolved by chromatographic resolution or chemical resolution.

[0019] In the present invention, the group of "substituted or unsubstituted" can be substituted with one substituent or multiple substituents. When there are multiple substituents, they can be selected from different substituents. When the same expression is involved in the present invention, it has the same meaning, and the selection range of substituents is as shown above and will not be elaborated one by one.

[0020] In the present invention, "independently of each other" means that when its subject has multiple, they can be the same or different from each other.

[0021] In the present invention, for the expression of chemical elements, unless otherwise specified, it usually includes the concept of isotopes with the same chemical properties. For example, the expression of "hydrogen (H)" also includes 1 H (protium or H), 2 H (deuterium or D); carbon (C) includes 12 C, 13 C, etc., and will not be elaborated.

[0022] In the present invention, C a ~C b The expression represents that the number of carbon atoms of the group is a to b. Unless otherwise specified, generally the number of carbon atoms does not include the carbon atoms of the substituents.

[0023] In the present invention, the term "alkyl" may include branched or straight-chain saturated aliphatic monovalent hydrocarbon groups having a specified number of carbon atoms. For example, as C1~C 20Examples of alkyl groups include: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, etc.

[0024] In the present invention, the term "linear or branched saturated or unsaturated hydrocarbon group" may include a linear or branched saturated aliphatic monovalent hydrocarbon group or an unsaturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. For example, as C3-C 30 Examples of linear or branched saturated hydrocarbon groups include: propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, heptyl, octyl, isooctyl, nonyl, decyl, undecyl, dodecyl, etc. As C3-C 30 The linear or branched unsaturated hydrocarbon group may be one or more single bonds in the linear or branched saturated hydrocarbon group replaced by double bonds, etc. 30 The linear or branched saturated hydrocarbon group may have one or more single bonds replaced by double bonds, etc.

[0025] In the present invention, the term "saturated or unsaturated alkylene group" may include a saturated aliphatic divalent hydrocarbon group or an unsaturated aliphatic divalent hydrocarbon group having a specified number of carbon atoms. For example, as C1-C 20 Examples of linear or branched saturated alkylene groups include: methylene, ethylene, propylene, pentylene, etc. As C1-C 20 The unsaturated alkylene group may be one or more single bonds in the saturated alkylene group replaced by double bonds or triple bonds, etc. 20 The unsaturated alkylene group may be one or more single bonds in the saturated alkylene group replaced by double bonds or triple bonds, etc.

[0026] In the process of implementing the present invention, it was found that the cationic lipid compound of the present invention can be used for delivering nucleic acids, etc., and has a different structure compared with some representative compounds in the related art such as DLin-DMA, DLin-MC3-DMA, etc., showing higher transfection efficiency and lower cytotoxicity. The lipid nanoparticle delivery system constructed therefrom is beneficial to improving the delivery efficiency of nucleic acids, etc.

[0027] According to an embodiment of one aspect of the present invention, there is provided a cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof, and the cationic lipid compound has a structure shown in formula (I):

[0028]

[0029] Wherein, one or more of R1, R2, and R3 are each independently selected from secondary amines or tertiary amines, preferably tertiary amines, and the rest are each independently selected from C3-C 30 linear or branched saturated or unsaturated hydrocarbon groups, preferably C3-C 30A linear or branched saturated hydrocarbon group, and R1, R2, and R3 are not all secondary amines or tertiary amines; L1 and L2 are each independently selected from a single bond, or a substituted or unsubstituted C1-C 20 Saturated or unsaturated alkylene group, and q methylene groups of L1 and / or L2 are each independently replaced by -O-, -C(O)- or -C(O)O-, and the substituted or unsubstituted C1-C 20 When the saturated or unsaturated alkylene group has a substituent, the substituent is selected from one or more combinations of hydroxyl group, mercapto group, C1-C 20 One or more combinations of alkyl groups; L3 is selected from substituted or unsubstituted C1-C 10 Alkylene group, and when the substituted or unsubstituted C1-C 10 Alkylene group has a substituent, the substituent is selected from one or more combinations of C1-C 20 Alkyl group, C3-C8 cycloalkyl group; X1, X2 and X4 are each independently selected from -C(O)-, -C(O)-NH-, -C(O)-S-, -C(O)-O-, -NH-, -S-, -O-, X3 is selected from -NH-, -S- or -O-; P is 1 or 2; q is an integer from 0 to 10. It should be noted that the above "secondary amine" or "tertiary amine" refers to a group containing -NH- or -NR- (R is not H).

[0030] According to an embodiment of the present invention, by introducing at least one protonatable polar head containing a secondary amine or a tertiary amine, at least one hydrophobic tail containing a specified number of carbons, the polar head and the hydrophobic tail are connected by a specific linking chain, having high transfection efficiency and low cytotoxicity.

[0031] According to an embodiment of the present invention, the cationic lipid compound of the present invention has the structure shown in formula (II-A):

[0032]

[0033] Wherein, R1 and R3 are selected from C3-C 30 Linear or branched saturated or unsaturated hydrocarbon group; L2 is selected from substituted or unsubstituted C1-C 20 Saturated or unsaturated alkylene group, q methylene groups in L2 are replaced by -O-, -C(O)- or -C(O)O-, and when the substituted or unsubstituted C1-C 20 Saturated or unsaturated alkylene group has a substituent, the substituent is selected from one or more combinations of hydroxyl group, mercapto group, C1-C 10 One or more combinations of alkyl groups; X1' and X2' are each independently selected from -O-, -S- or -NH-.

[0034] According to an embodiment of the present invention, the cationic lipid compound of the present invention has the structure shown in formula (III-A):

[0035]

[0036] Among them, X4’ is selected from -O-, -S- or -NH-; R4 and R7 are selected from H or C1-C 14 alkyl, and when both R4 and R7 are selected from C1-C 14 alkyl, R4 and R7 can form a ring. When R4 and R7 form a ring, for example, they can form a C3-C8 cycloalkyl; R5 and R6 are each independently selected from H, substituted or unsubstituted C1-C 20 alkyl, and when both R5 and R6 are selected from substituted or unsubstituted C1-C 20 alkyl, R5 and R6 can form a ring; among them, when the substituted or unsubstituted C1-C 20 alkyl has a substituent, the substituent is selected from C1-C 30 alkyl, C3-C7 cycloalkyl, hydroxyl, amino, acyl, ether, carboxyl, sulfhydryl, or a combination of one or more of them, and m is an integer from 0 to 9.

[0037] According to an embodiment of the present invention, the cationic lipid compound of the present invention has the structure shown in formula (II-B):

[0038]

[0039] Among them, R2 and R3 are selected from C3-C 30 linear or branched saturated or unsaturated hydrocarbon groups; L1 is selected from substituted or unsubstituted C1-C 20 saturated or unsaturated alkylene groups, q methylene groups in L1 are replaced by -O-, -C(O)- or -C(O)O-, and when the substituted or unsubstituted C1-C 20 saturated or unsaturated alkylene group has a substituent, the substituent is selected from hydroxyl, mercapto, C1-C 10 alkyl, or a combination of one or more of them; X1’ and X2’ are each independently selected from -O-, -S- or -NH-.

[0040] According to an embodiment of the present invention, the cationic lipid compound of the present invention has the structure shown in formula (III-B):

[0041]

[0042] Among them, X4’ is selected from -O-, -S- or -NH-; R4 and R7 are each independently selected from H or C1-C 14 alkyl, and when both R4 and R7 are selected from C1-C 14 alkyl, R4 and R7 can form a ring. When R4 and R7 form a ring, for example, they can form a C3-C8 cycloalkyl; R5 and R6 are each independently selected from H, substituted or unsubstituted C1-C 20alkyl, and when both R5 and R6 are selected from substituted or unsubstituted C1-C 20 alkyl, R5 and R6 can form a ring; wherein, when the substituted or unsubstituted C1-C 20 alkyl has a substituent, the substituent is selected from C1-C 30 alkyl, C3-C7 cycloalkyl, hydroxyl, amino, acyl, ether, carboxyl, sulfhydryl, or a combination of one or more thereof, and m is an integer from 0 to 9.

[0043] According to an embodiment of the present invention, in the compounds represented by the above formulas (III-A) and (III-B), when R5 and R6 can form a ring, it may be that R5, R6 and the connected N atom form a nitrogen heterocycle, or it may be that R5 or R6 forms a ring by itself to form a heterocyclic group connected to the N atom, and each is preferably azacycloalkyl. The formed ring can be, for example, a 5- to 8-membered ring, such as a 5-membered ring, 6-membered ring, 7-membered ring or 8-membered ring.

[0044] According to an embodiment of the present invention, in the compounds represented by formula (I), formula (II-A) or (III-A), R1 can further be selected from C3-C 15 linear or branched alkyl, alkenyl or alkynyl, and can further be selected from C3-C12 linear or branched alkyl, C6-C12 linear alkenyl, C6-C12 linear alkynyl, etc., and is preferably any of the following structures:

[0045]

[0046] According to an embodiment of the present invention, in the compounds represented by formula (I), formula (II-B) or formula (III-B), R2 is selected from C3-C 15 linear or branched alkyl, alkenyl or alkynyl, and can further be selected from C3-C12 linear or branched alkyl, C6-C12 linear alkenyl, C6-C12 linear alkynyl, etc., and is preferably any of the following structures:

[0047]

[0048] According to an embodiment of the present invention, in the compounds represented by formula (I), formula (II-A), formula (III-A), formula (II-B) and formula (III-B), R3 is selected from C4-C 25 linear or branched alkyl, alkenyl or alkynyl, and can further be C 10 -C 25 branched alkyl, C4-C 18 linear alkyl, C 10 - 20 branched alkenyl, C6-C 15 linear alkenyl, C 10 -C 25Branched alkenyl group, C6-C 12 Linear alkynyl group, etc., preferably any of the following structures:

[0049]

[0050] According to an embodiment of the present invention, R1, R2, and R3 of the above types can regulate lipid behavior, which is beneficial to improving transfection efficiency.

[0051] According to an embodiment of the present invention, in the compounds represented by formula (III-A) or formula (III-B), NR5R6 is selected from any of the following structures:

[0052]

[0053] According to an embodiment of the present invention, NR5R6 of the above types can affect the binding ability with nucleic acids by regulating the lipid charge situation, thereby improving transfection efficiency.

[0054] According to an embodiment of the present invention, in the compounds represented by formula (III-A) and formula (III-B), the number of NH, O, and S in X1’, X2’, X3, and X4’ can be one or more. For example, X1’ can be O, X2’ can be O, X3 can be S, X4’ can be O; or X1’ can be O, X2’ can be O, X3 can be O, X4’ can be O; or X1’ can be O, X2’ can be NH, X3 can be S, X4’ can be O; or X1’ can be S, X2’ can be NH, X3 can be S, X4’ can be O; or X1’ can be N, X2’ can be O, X3 can be NH, X4’ can be O; or X1’ can be S, X2’ can be O, X3 can be O, etc., X4’ can be O; or X1’ can be O, X2’ can be O, X3 can be S, X4’ can be S. Preferably, X1’ is O, X2’ is O, X3 is O or S, X4’

[0055] is O.

[0056] According to an embodiment of the present invention, in the compounds represented by formula (II-A), formula (III-A), formula (II-B), and formula (III-B), L1 or L2 is selected from any of the following structures:

[0057]

[0058] Among them, in the groups represented by V17 and V18, the serial number 1 represents the connection site with the N atom, and the serial number 2 represents the connection site with the carbonyl group.

[0059] According to an embodiment of the present invention, in L1 or L2, the introduction of the substituent hydroxyl group will enhance the hydrogen bonding between the lipid and the nucleic acid molecule, which is beneficial to protecting the nucleic acid from enzymatic degradation and improving the delivery efficiency. The replacement of the methylene group by an ester group helps to enhance the degradability of the lipid itself, thus facilitating the release of the nucleic acid molecule in cells and reducing the toxicity in vivo and in vitro.

[0060] According to an embodiment of the present invention, in the compound represented by formula (I), formula (II-A) or formula (II-B), L3 is selected from any one of the following structures:

[0061]

[0062] According to an embodiment of the present invention, in L3, the selection of the above groups, especially the extension or cyclization of the carbon chain, is beneficial to enhancing the hydrophobicity of the lipid molecule, thereby improving the stability of the entire nanoparticle.

[0063] According to an embodiment of the present invention, the above compounds of the present invention are preferably selected from any one of the structures shown in Table 1 below, but these compounds are only representative:

[0064] Table 1

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] It should be noted that in the above compound R3, W7 / W8 represents the simultaneous connection of the groups represented by W7 and W8, and the specific structural formula is as follows:

[0089] Moreover, the subunits in T43 to T51 indicate that in the compounds of general formula (III-A) or (IIII-B), R4 and R7 are connected to form a ring. Exemplarily, taking T43 as an example, its structural formula is:

[0090]

[0091] According to an embodiment of another aspect of the present invention, there is also provided a method for preparing the above-mentioned cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof. Taking the preparation of the compound shown in formula (III-A) as an example, the preparation method includes the following steps S101 to step S104.

[0092] In step S101, the compound shown in formula (1) and the compound shown in formula (2) are reacted to obtain the intermediate shown in formula (3);

[0093]

[0094] In step S102, the intermediate shown in formula (3) and the compound shown in formula (4) are reacted to obtain the intermediate shown in formula (5), where X1 is O, X2 is O, X3 is S, NH or O, or X1 is NH, X2 is O, X3 is S or NH;

[0095]

[0096] In step S103, the intermediate shown in formula (5) and the compound shown in formula (7) are reacted to generate the intermediate shown in formula (6);

[0097]

[0098] In step S104, the intermediate shown in formula (6) is used as a reactant to generate the cationic lipid compound shown in formula (III-A).

[0099]

[0100] According to an embodiment of the present invention, the conditions of the above preparation method are mild and the operation is simple, and the cationic lipid compound can be prepared more effectively.

[0101] According to an embodiment of the present invention, in step S101, it may specifically include: dropping the compound shown in formula (2) into the first organic solvent containing the compound shown in formula (1) and the first base, and performing a reflux reaction. After the reaction is completed, the intermediate shown in formula (3) is separated. Among them, the first organic solvent may be, for example, dichloromethane (DCM), tetrahydrofuran (THF), etc.; the first base may be an organic base or an inorganic base, specifically, for example, a mixed base of triethylamine and 4-dimethylaminopyridine, diisopropylethylamine, pyridine, etc.

[0102] According to an embodiment of the present invention, in step S102, it may specifically include: dropping the compound shown in formula (4) into the second organic solvent containing the intermediate shown in formula (3) and the second base, and reacting at room temperature. After the reaction is completed, the intermediate shown in formula (5) is separated. Among them, the second organic solvent may be, for example, N,N-dimethylformamide (DMF), acetone, acetonitrile, N,N-dimethylacetamide, etc., and the second base may be an organic base or an inorganic base, specifically, for example, potassium carbonate, sodium carbonate, diisopropylethylamine, triethylamine, etc.

[0103] According to an embodiment of the present invention, in step S103, the compound shown in formula (7) is dropped into the third organic solvent containing the intermediate shown in formula (5) and the third base, and the reaction is carried out at room temperature. After the reaction is completed, the intermediate shown in formula (6) is separated. Among them, the third organic solvent may be, for example, dichloromethane, tetrahydrofuran, etc., and the third base may be an organic base or an inorganic base, specifically, for example, triethylamine, diisopropylethylamine, etc.

[0104] In step S104, different synthesis strategies can be selected according to different L2 to generate the cationic lipid compound shown in formula (III-A).

[0105] For example, when L2 is an unsubstituted C1-C 20 saturated alkylene group, step S104 includes sub-steps S1041 to sub-steps S1042.

[0106] In sub-step S1041, the compound shown in formula (8) is added dropwise to a fourth organic solvent containing the intermediate shown in formula (6), a condensing agent, and a fourth base, and the reaction is carried out at room temperature. After the reaction is completed, the intermediate shown in formula (9) is separated. Among them, the condensing agent can be, for example, N,N'-dicyclohexylcarbodiimide (DCC), etc., the fourth organic solvent can be, for example, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, etc., and the fourth base can be an organic base or an inorganic base. Specifically, it can be, for example, dimethylaminopyridine, diisopropylethylamine, triethylamine, etc.

[0107]

[0108] In sub-step S1042, the compound shown in formula (10) is added to a fifth organic solvent containing the intermediate shown in formula (9) and a fifth base, and after the reaction is completed, the cationic lipid compound shown in formula (III-A) is separated. Among them, the fifth organic solvent can be, for example, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, etc., and the fifth base can be an organic base or an inorganic base. Specifically, it can be, for example, triethylamine, diisopropylethylamine, potassium carbonate, cesium carbonate, etc.

[0109]

[0110] It can be understood that a reaction similar to the above can be adopted to obtain the cationic lipid compound shown in formula (III-B), which will not be elaborated here.

[0111] Again, when L2 is the case, step S104 may include sub-step S1041' to sub-step S1042'.

[0112] In sub-step S1041', the operation is similar to that of sub-step S1041, and the main difference is that the compound shown in formula (8) is replaced by the compound shown in formula (8').

[0113]

[0114] In sub-step S1042', the operation is similar to that of sub-step S1042, and the difference is that the intermediate shown in formula (9) is replaced by the intermediate shown in formula (9'), and the cationic lipid compound shown in formula (III-A-1) is generated.

[0115]

[0116] Again, when L2 is the case, step S104 may include sub-step S1041" to sub-step S1042".

[0117] In sub-step S1041”, the operation is similar to that of sub-step S1041, and the main difference is that the compound shown in formula (8) is replaced by the compound shown in formula (8”).

[0118]

[0119] In sub-step S1042”, a Michael addition reaction is carried out with the intermediate shown in formula (9”) and the compound shown in formula (10) to obtain the cationic lipid compound shown in formula (III-A-2).

[0120]

[0121] According to an embodiment of still another aspect of the present invention, a composition is provided, which comprises the above-mentioned cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof.

[0122] According to an embodiment of the present invention, the above composition is a nanoparticle preparation for constructing a lipid nanoparticle delivery system; the average particle size can be, for example, 10 nm to 300 nm, specifically, for example, 10 nm, 20 nm, 50 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, etc.

[0123] According to an embodiment of the present invention, the above composition may further comprise a prophylactic agent or a therapeutic agent.

[0124] According to an embodiment of the present invention, the prophylactic or therapeutic agent may be at least one of nucleic acid, polypeptide or protein. Further, the therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response. For example, the therapeutic or prophylactic agent may be ribonucleic acid or deoxyribonucleic acid. The mass ratio of the cationic lipid compound to the prophylactic or therapeutic agent is 2.5:1 to 50:1. For example, it may be 2.5:1 to 15:1, 15:1 to 30:1, 30:1 to 40:1, 40:1 to 50:1. Specifically, for example, it may be 2.5:1, 5:1, 6:1, 8:1, 10:1, 12:1, 14:1, 15:1, 16:1, 18:1, 20:1, 22:1, 24:1, 25:1, 26:1, 28:1, 30:1, 32:1, 34:1, 35:1, 36:1, 38:1, 40:1, 42:1, 44:1, 45:1, 46:1, 48:1, 50:1, etc. Among them, the nucleic acid includes but is not limited to single-stranded DNA, double-stranded DNA and RNA. Among them, RNA includes but is not limited to small interfering RNA (siRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), messenger RNA (mRNA), small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA) and mixtures thereof. Proteins include but are not limited to gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), interferon, hepatitis B surface antigen or heparin.

[0125] According to an embodiment of the present invention, the above composition may further include neutral phospholipids, sterol lipids and polyethylene glycolated lipids; wherein, the molar ratio of the cationic lipid compound to the neutral phospholipids, sterol lipids and polyethylene glycolated lipids is (45-55):(5-15):(35-45):(0.5-2.0). It may be (48-52):(8-12):(36-40):(1.2-1.8). Specifically, for example, it may be 50:10:38.5:1.5, etc.

[0126] According to an embodiment of the present invention, neutral phospholipids are suitable for improving the stability of lipid nanoparticles, regulating the fluidity of lipid nanoparticles, and may also affect the target specificity. Phospholipids can be, for example, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dipalmitoyl phosphatidylcholine (DAPC), 1,2-dilauroyl phosphatidylcholine (DLPC), 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC), 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), dioleoyl phosphatidylcholine (DOPC), 1-stearoyl-2-myristoyl phosphatidylcholine (SMPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE), distearoyl phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), dilauroyl phosphatidylethanolamine (DLPE), sodium distearoyl phosphatide (DSPA), sodium dipalmitoyl phosphatide (DPPA), sodium dimyristoyl phosphatide (DMPA), sodium dioleoyl phosphatide (DOPA), sodium dilauroyl phosphatide (DLPA), sodium distearoyl phosphatidylglycerol (DSPG), sodium dipalmitoyl phosphatidylglycerol (DPPG), sodium dimyristoyl phosphatidylglycerol (DMPG), sodium dioleoyl phosphatidylglycerol (DOPG), sodium dilauroyl phosphatidylglycerol (DLPG), sodium distearoyl phosphatidylserine (DSPS), sodium dipalmitoyl phosphatidylserine (DPPS), sodium dimyristoyl phosphatidylserine (DMPS), sodium dioleoyl phosphatidylserine (DOPS), sodium dilauroyl phosphatidylserine (DLPS), etc.

[0127] According to an embodiment of the present invention, the sterol lipids are selected from one or more of avenasterol, β-sitosterol, campesterol, ergocalciferol, stigmasterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, desmosterol, dihydroergocalciferol, marasmanol, epicholesterol, ergosterol, fucosterol, hexahydro-lumisterol, hydroxycholesterol, lanosterol, lumisterol, saringosterol, sitostanol, stigmasterol, stigmasterol, cholanic acid, glycochenodeoxycholic acid, taurocholic acid, deoxycholic acid.

[0128] According to an embodiment of the present invention, the sterol lipid is selected from cholesterol, which belongs to the structural lipid and regulates the fluidity of lipid nanoparticles and enhances the stability of lipid nanoparticles by filling the gaps between phospholipids.

[0129] According to an embodiment of the present invention, polyethylene glycolylated lipids (i.e., PEGylated lipids) can affect the size, stability, in vivo distribution, and transfection efficiency of lipid nanoparticle delivery systems. During the preparation and storage of lipid nanoparticle delivery systems, PEGylated lipids can both drive self-assembly and prevent particle aggregation. PEGylated lipids can be, for example, distearoyl phosphatidylethanolamine polyethylene glycol (DSPE-PEG), dimyristoyl glycerol-rac-methoxypolyethylene glycol 2000 (DMG-PEG2000), methoxypolyethylene glycol ditetradecylacetamide (ALC-0159), polyethylene glycol-diacylglycerol amide (PEG-DAG), polyethylene glycol-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), polyethylene glycol-distearylglycerol (PEG-DSG), polyethylene glycol-diacylglycerol amide (PEG-DAG), etc.

[0130] According to an embodiment of the present invention, the above composition may further include pharmaceutically acceptable excipients or auxiliary components, including but not limited to: binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, colorless agents, flavoring agents, etc. for oral preparations; preservatives, solubilizing agents, stabilizers, etc. for injectable preparations. The pharmaceutical preparation can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically).

[0131] According to an embodiment of the present invention, the above composition can be formulated into several dosage forms according to the administration route, such as oral preparations (e.g., tablets, capsules, solutions, or suspensions); injectable preparations (e.g., injectable solutions or suspensions, or injectable dry powders that can be immediately used after adding water for injection before injection), etc.

[0132] According to an embodiment of the present invention, the above composition can be administered at a therapeutically effective dose, which can vary not only with the specific reagent selected but also with the administration route, the nature of the disease being treated, and the age and condition of the patient. For example, a dose of nucleic acid from about 0.001 mg / Kg to about 10 mg / Kg can be administered to a subject, which can be a mammal (such as a human), etc.

[0133] According to an embodiment of another aspect of the present invention, there is also provided the use of the above-mentioned cationic lipid compound or its pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer, or the above-mentioned composition in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptides, or protein drugs.

[0134] According to an embodiment of the present invention, due to the wide variety of nucleic acids, polypeptides, or proteins, the above-mentioned cationic lipid compound or its pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer, or the above-mentioned composition can be used to treat or prevent a variety of diseases or disorders.

[0135] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the following provides a further detailed description of the present invention in conjunction with specific embodiments. The test materials, reagents, etc. used in the following embodiments can be obtained from commercial sources without special instructions. For those not specifying specific technologies or conditions in the embodiments, they are all conventional methods and can be carried out according to the technologies or conditions described in the literature in this field or according to the product instructions.

[0136] Example 1: Synthesis of cationic lipid compound E10

[0137]

[0138] Step 1: Preparation of 2-bromo-2-hexyldecyl propionate (1-3):

[0139] Dissolve 41.2 mmol of 2-hexyldecanol (1-1) in 100 ml of dichloromethane, then add 61.8 mmol of triethylamine and 20.6 mmol of 4-dimethylaminopyridine. Dropwise add 61.8 mmol of 2-bromopropionyl bromide at 30 - 40 °C. After the addition is complete, reflux and stir for 4 h to stop the reaction. Add water and then perform extraction and liquid separation. Wash the organic phase with saturated sodium chloride aqueous solution and then dry it with anhydrous sodium sulfate. Filter, concentrate, and rotary evaporate the solvent. The residue (intermediate 1-3) can be directly used in the next reaction without treatment.

[0140] Step 2: Preparation of 2-((2,3-dihydroxypropyl)thio)propionic acid 2-hexyldecyl ester (1-5):

[0141] Dissolve 26.5 mmol of 2-bromo-2-hexyldecyl propionate (1-3) in 100 ml of N,N-dimethylformamide, then add 39.75 mmol of potassium carbonate and stir for 20 - 30 min. Dropwise add 29.15 mmol of 1-thioglycerol (1-4) below 30 °C. After the addition is complete, stir at room temperature overnight. Stop the reaction the next day. Add 300 ml of water and then extract with ethyl acetate. Back-extract the aqueous phase with ethyl acetate. Combine the organic phases, wash with saturated sodium chloride aqueous solution, and then dry with anhydrous sodium sulfate. Filter, concentrate, and rotary evaporate the solvent. Purify the residue by column chromatography (dichloromethane:methanol = 95:5) to obtain a light yellow oily liquid (intermediate 1-5) with a yield of 56%.

[0142] Step 3: 3-((1-((2-hexyldecyloxy)-1-oxopropan-2-yl)thio)-2-hydroxypropyl octanoate (1-6):

[0143] Dissolve 24.7 mmol of 2-((2,3-dihydroxypropyl)thio)propanoic acid 2-hexyldecyl ester (1-5) in 100 ml of dichloromethane, add 37.05 mmol of triethylamine, cool the system to 0 °C, and dropwise add 27.2 mmol of octanoyl chloride (1-7). After the addition is complete, stir for 3 h and then stop the reaction. Add water and perform extraction and liquid separation. Wash the organic phase with saturated sodium chloride aqueous solution and then dry it over anhydrous sodium sulfate. Filter, concentrate, and rotary evaporate the solvent. Purify the residue by column chromatography (n-heptane:ethyl acetate = 90:10) to obtain a light yellow oily liquid (intermediate 1-6) intermediate #3 with a yield of 62%.

[0144] Step 4, Preparation of 2-((4-bromobutyryl)oxy)-3-((1-((2-hexyldecyloxy)-1-oxopropan-2-yl)thio)octanoic acid propyl ester (1-9):

[0145] Dissolve 18.8 mmol of 3-((1-((2-hexyldecyloxy)-1-oxopropan-2-yl)thio)-2-hydroxypropyl octanoate (1-6) in 100 ml of dichloromethane, add 28.2 mmol of N,N'-dicyclohexylcarbodiimide and 0.9 mmol of 4-dimethylaminopyridine, and dropwise add 22.6 mmol of 4-bromobutyric acid (1-8) below 35 °C. After the addition is complete, stir for 4 h and then stop the reaction. Filter the system, concentrate the filtrate, and rotary evaporate the solvent. Purify the residue by column chromatography (n-heptane:ethyl acetate = 90:10) to obtain 8.3 g of a light yellow oily liquid (intermediate 1-9) with a yield of 65%.

[0146] Step 5, Preparation of 3-((1-((2-hexyldecyloxy)-1-oxopropan-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butyryl)oxy)octanoic acid propyl ester (E10):

[0147] Dissolve 7.35 mmol of 2-((4-bromobutyryl)oxy)-3-((1-((2-hexyldecyloxy)-1-oxopropan-2-yl)thio)octanoic acid propyl ester (1-9) in 25 ml of acetonitrile, add 14.7 mmol of triethylamine and 12.5 mmol of 1-(3-(4-methylpiperazin-1-yl)propyl)amino)dec-2-ol (1-10), heat the system to 40 °C, stir and react overnight, stop the reaction the next day, directly concentrate the system and rotary evaporate the solvent. Purify the residue by column chromatography (DCM:MeOH = 90:10) to obtain 2.1 g of a light yellow oily liquid (E10) with a yield of 40%.

[0148] Characterize the cationic lipid compound E10, and the results are as follows:

[0149] 11H NMR (400 MHz, CDCl3) δ 5.25–5.10 (m, 1H), 4.38–4.27 (m, 1H), 4.18–4.09 (m, 1H), 4.03 (d, J = 5.6 Hz, 2H), 3.56 (s, 1H), 3.49–3.39 (m, 2H), 2.96–2.71 (m, 4H), 2.69–2.22 (m, 20H), 1.85–1.70 (m, 2H), 1.68–1.53 (m, 5H), 1.52–1.36 (m, 5H), 1.25 (s, 44H), 0.87 (t, J = 6.5 Hz, 12H).

[0150] [M+H] + : 912.60, [1 / 2M+H] + : 457.00。

[0151] Examples 2 to 59: Synthesis of cationic lipid compounds

[0152] The operation was similar to that of Example 1, except that according to each example shown in Table 3, Compound 1-1, Compound 1-7 and Compound 1-10 were replaced with the raw materials shown in Table 2, and the structures and characterization results of the finally obtained cationic lipid compounds are shown in Table 3.

[0153] Table 2

[0154]

[0155]

[0156] Table 3

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] Note: The compounds represented by the header marks 1, 7, and 10 respectively correspond to the compounds represented by the general formulas 1, 7, and 10 in the preparation method of the cationic lipid compound.

[0167] Example 72: Synthesis of the cationic lipid compound S124

[0168]

[0169] Step 1: Dissolve 10 g of 9-heptadecanol (2-1) in 100 ml of dichloromethane, then add 1.5 molar equivalents of triethylamine and 0.5 molar equivalent of 4-dimethylaminopyridine. Dropwise add 1.5 molar equivalents of 2-bromopropionyl bromide at 30 - 40 °C. After the addition is complete, reflux and stir for 4 h to stop the reaction. Add water and then perform extraction and liquid separation. Wash the organic phase with saturated sodium chloride aqueous solution and then dry it with anhydrous sodium sulfate. Filter, concentrate, and rotary evaporate the solvent. The residue can be directly used in the next step without treatment.

[0170] Step 2: Dissolve 10 g of intermediate (2-3) in 100 ml of N,N-dimethylformamide, then add 1.5 molar equivalents of potassium carbonate and stir for 20 - 30 min. Dropwise add 1.1 molar equivalents of 1-thioglycerol (1-4) below 30 °C. After the addition is complete, stir overnight at room temperature. Stop the reaction the next day. Add 300 ml of water and then extract with ethyl acetate. Back-extract the aqueous phase with ethyl acetate. Combine the organic phases, wash with saturated sodium chloride aqueous solution, and then dry with anhydrous sodium sulfate. Filter, concentrate, and rotary evaporate the solvent. Purify the residue by column chromatography (dichloromethane:methanol = 95:5) to obtain a light yellow oily liquid intermediate (2-5) with a yield of 53%.

[0171] Step 3: Dissolve 10 g of intermediate (2-5) in 100 ml of dichloromethane, then add 1.5 molar equivalents of triethylamine. Cool the system to 0 °C and dropwise add 1.1 molar equivalents of octanoyl chloride (1-7). After the addition is complete, stir for 3 h and then stop the reaction. Add water and then perform extraction and liquid separation. Wash the organic phase with saturated sodium chloride aqueous solution and then dry it with anhydrous sodium sulfate. Filter, concentrate, and rotary evaporate the solvent. Purify the residue by column chromatography (n-heptane:ethyl acetate = 90:10) to obtain a light yellow oily liquid intermediate (4-6) with a yield of 66%.

[0172] Step 4: Dissolve 10 g of intermediate (4-6) in 100 ml of dichloromethane, then add 2.0 molar equivalents of N,N'-dicyclohexylcarbodiimide and 0.05 molar equivalent of 4-dimethylaminopyridine. Dropwise add 1.5 molar equivalents of ethylene oxide pentanoic acid (4-8’) below 35 °C. After the addition is complete, stir for 4 h and then stop the reaction. Filter the system. Concentrate the filtrate and rotary evaporate the solvent. Purify the residue by column chromatography (n-heptane:ethyl acetate = 90:10) to obtain a light yellow oily liquid intermediate (4-9’) with a yield of 60%.

[0173] Step 5: Dissolve 5 g of intermediate (4-9’) in 25 ml of acetonitrile, then add 1.1 equivalents of N-hydroxyethylpiperazine (2-10). Heat the system to 60 °C and stir the reaction overnight. Stop the reaction the next day. Directly concentrate the system to dry the solvent by rotary evaporation. Purify the residue by column chromatography (DCM:MeOH = 90:10) to obtain the final product (S124) as a light yellow oily liquid with a yield of 57%.

[0174] Characterize the cationic lipid compound S124, and the results are as follows:

[0175] 1 . 1H NMR (400 MHz, CDCl3) δ 5.27–5.11 (m, 1H), 4.92–4.83 (m, 1H), 4.38–4.28 (m, 1H), 4.19–4.12 (m, 1H), 3.71–3.59 (m, 3H), 3.47–3.39 (m, 1H), 2.94–2.22 (m, 20H), 1.69–1.48 (m, 8H), 1.45–1.38 (m, 5H), 1.34–1.18 (m, 34H), 0.87 (t, J = 6.6 Hz, 9H).

[0176] [M+H] + : 801.4

[0177] Examples 73-126: Synthesis of cationic lipid compounds

[0178] Similar to the operation of Example 72, except that according to the raw materials shown in Table 2 for each example shown in Table 4, replace compound 2-1, compound 1-7 and compound 2-10. The structures and characterization results of the finally obtained cationic lipid compounds are shown in Table 4.

[0179] Table 4

[0180]

[0181]

[0182]

[0183]

[0184] Example 127: Synthesis of cationic lipid compound S111

[0185]

[0186] Step 1: Dissolve 10 g of 11 - hentriacontanol (4 - 1) in 100 ml of dichloromethane, then add 1.5 molar equivalents of triethylamine and 0.5 molar equivalent of 4 - dimethylaminopyridine. Dropwise add 1.5 molar equivalents of 2 - bromopropionyl bromide at 30 - 40 °C. After the addition is complete, reflux and stir for 4 h to stop the reaction. Add water and then extract and separate the layers. Wash the organic phase with saturated sodium chloride aqueous solution and then dry it over anhydrous sodium sulfate. Filter, concentrate, and rotary - evaporate the solvent. The residue containing intermediate (4 - 3) can be directly used for the next reaction without further treatment.

[0187] Step 2: Dissolve 10 g of intermediate (4 - 3) in 100 ml of N,N - dimethylformamide, then add 1.5 molar equivalents of potassium carbonate, and stir for 20 - 30 min. Dropwise add 1.1 molar equivalents of 1 - thioglycerol (1 - 4) below 30 °C. After the addition is complete, stir overnight at room temperature. Stop the reaction the next day. Add 300 ml of water and then extract with ethyl acetate. Back - extract the aqueous phase with ethyl acetate. Combine the organic phases, wash with saturated sodium chloride aqueous solution, and then dry over anhydrous sodium sulfate. Filter, concentrate, and rotary - evaporate the solvent. Purify the residue by column chromatography (dichloromethane:methanol = 95:5) to obtain the intermediate (4 - 5) as a light - yellow oily liquid with a yield of 50%.

[0188] Step 3: Dissolve 10 g of intermediate (4 - 5) in 100 ml of dichloromethane, then add 1.5 molar equivalents of triethylamine. Cool the system to 0 °C, and dropwise add 1.1 molar equivalents of octanoyl chloride (1 - 7). After the addition is complete, stir for 3 h and then stop the reaction. Add water and then extract and separate the layers. Wash the organic phase with saturated sodium chloride aqueous solution and then dry over anhydrous sodium sulfate. Filter, concentrate, and rotary - evaporate the solvent. Purify the residue by column chromatography (n - heptane:ethyl acetate = 90:10) to obtain the intermediate (4 - 6) as a light - yellow oily liquid with a yield of 63%.

[0189] Step 4: Dissolve 10 g of intermediate (4 - 6) in 100 ml of dichloromethane, then add 2.0 molar equivalents of N,N'-dicyclohexylcarbodiimide and 0.05 molar equivalent of 4 - dimethylaminopyridine. Dropwise add 1.5 molar equivalents of β - (acryloyloxy)propionic acid (4 - 8”) below 35 °C. After the addition is complete, stir for 4 h and then stop the reaction. Filter the system, concentrate the filtrate, and rotary - evaporate the solvent. Purify the residue by column chromatography (n - heptane:ethyl acetate = 90:10) to obtain the intermediate (4 - 9”) as a light - yellow oily liquid with a yield of 42%.

[0190] Step 5: Dissolve 5 g of intermediate (4 - 9”) in 25 ml of acetonitrile, then add 1.1 equivalents of N - hydroxyethylpiperazine (2 - 10). Heat the system to 60 °C and stir the reaction overnight. Stop the reaction the next day. Directly concentrate the system and rotary - evaporate the solvent. Purify the residue by column chromatography (DCM:MeOH = 90:10) to obtain the final product (S111) as a light - yellow oily liquid with a yield of 55%.

[0191] The cationic lipid compound S111 was characterized, and the results are as follows:

[0192] 1 H NMR (400 MHz, CDCl3) δ 5.29–5.14 (m, 1H), 4.92–4.84 (m, 1H), 4.41–4.30 (m, 3H), 4.19–4.12 (m, 1H), 3.64–3.57 (m, 2H), 3.46–3.38 (m, 1H), 2.96–2.37 (m, 18H), 2.31 (q, J = 7.5 Hz, 2H), 1.65–1.48 (m, 6H), 1.46–1.38 (m, 3H), 1.35–1.18 (m, 40H), 0.87 (t, J = 6.7 Hz, 9H).

[0193] [M + H] + : 857.6.

[0194] Examples 128 - 179: Synthesis of Cationic Lipid Compounds

[0195] The operation was similar to that of Example 127, except that according to the raw materials shown in Table 2 for each example shown in Table 5, Compound 2 - 1, Compound 1 - 7, and Compound 2 - 10 were replaced. The structures and characterization results of the finally obtained cationic lipid compounds are shown in Table 5.

[0196] Table 5

[0197]

[0198]

[0199]

[0200]

[0201]

[0202] Example 180: Construction of Lipid Nanoparticle (LNP) Delivery System

[0203] Dissolve the cationic lipid compound E10, phospholipid DSPC, cholesterol, and DMG-PEG2000 prepared in Example 1 in ethanol at a molar ratio of 50 / 10 / 38.5 / 1.5. Dilute firefly luciferase mRNA (Fluc mRNA) in citrate buffer (10 mM, pH = 4). Finally, set the mass ratio of cationic lipid to mRNA to 10 / 1, and quickly mix the calculated amount of ethanol phase and aqueous solution at a volume ratio of 1 / 3, and let it stand for 20 min to prepare LNP. Finally, add 1×PBS to the required sample volume to obtain the LNP delivery system. The particle size distribution of the LNP delivery system was detected by dynamic light scattering method, as Figure 1 shown. Five groups of parallel experiments were carried out, and the measured average particle size was 93.91 ± 0.83 nm.

[0204] Examples 181 to 346 and Comparative Example 1: Construction of lipid nanoparticle (LNP) delivery system

[0205] Except that the cationic lipid compound E10 in Example 180 was replaced with the corresponding compounds in Table 6 respectively, the LNP delivery systems of Examples 181 to 346 and Comparative Example 1 were obtained in the same manner as in Example 180.

[0206] The following determinations were carried out on the LNP delivery system constructed by the above process:

[0207] 1. Determine the biotoxicity of the LNP delivery system:

[0208] Human ovarian cancer cells IGROV1 were cultured in 1640 medium containing 10% fetal bovine serum, seeded in a 96-well plate at a density of 10000 cells / well, and incubated in a CO2 cell incubator for 24 h. Then, the prepared LNP encapsulating Fluc mRNA was added to the cells, 25 ng of mRNA per well, and incubation was continued for 24 h. The alamarBlue detection kit was used to measure the cell viability. After adding the alamarBlue detection reagent, incubation was continued for 2 h, and detection was carried out using a multifunctional microplate reader. The excitation wavelength was set at 530 nm, and the emission wavelength was set at 590 nm. The cells without adding LNP were used as a reference to calculate the cell viability. Among them, when the cell viability was greater than 80%, the LNP delivery system was evaluated as having no cytotoxicity and marked as ○, otherwise, the LNP delivery system was evaluated as having cytotoxicity and marked as ×.

[0209] 2. Determine the in vitro mRNA delivery efficiency of the LNP delivery system:

[0210] The human ovarian cancer cell line IGROV1 was cultured in RPMI-1640 medium containing 10% fetal bovine serum, seeded in a 96-well plate at a density of 10,000 cells / well, and incubated in a CO2 cell incubator for 24 h. Then, the prepared LNP encapsulating Fluc mRNA was added to the cells at 25 ng of mRNA per well, and incubation was continued. After 24 h, a multifunctional microplate reader and a Bio-Lumi TM luciferase reporter gene detection kit were used to detect the expression efficiency of Fluc mRNA. Among them, a fluorescence detection result of 10 4 ~5×10 4 was rated as C; 5×10 4 ~4×10 5 was rated as B; greater than 4.0×10 5 was rated as A.

[0211] Table 6

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] Analysis of the results in the above table shows that, on the one hand, when the LNP delivery system was constructed using the cationic lipid compounds of Examples 1 to 179 of the present invention, compared with the existing DLin-MC3-DMA cationic lipid, the survival rate of human ovarian cancer cell line IGROV1 detected was greater than 80%, showing low cytotoxicity.

[0218] On the other hand, when the LNP delivery system was constructed using the cationic lipid compounds of Examples 1 to 179 of the present invention, a delivery effect similar to or even better than that of the DLin-MC3-DMA cationic lipid could be achieved. In particular, it was found that the cationic lipid compounds with specific amino groups as polar heads in the present invention generally showed better delivery effects, especially A10, B11, C11, D10, F10, G12, S199, S213. It is speculated that when the specific amino groups of the present invention are combined with the hydrophobic tail through the linking structure provided by the present invention, it is more conducive to regulating the interaction forces between the delivery molecule and the intracellular endogenous compounds, thereby improving the delivery effect.

[0219] The specific embodiments described above further elaborate on the objective, technical solution, and beneficial effects of the present disclosure. It should be understood that the above are only specific embodiments of the present disclosure and are not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included within the protection scope of the present disclosure.

Claims

1. A cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof, characterized in that, The cationic lipid compound has the structure shown in formula (I): Among them, one or more of R1, R2, and R3 are each independently selected from secondary amines or tertiary amines, and the rest are each independently selected from C3-C 30 linear or branched saturated or unsaturated hydrocarbon groups, and not all of R1, R2, and R3 are secondary amines or tertiary amines; L1 and L2 are each independently selected from a single bond, or a substituted or unsubstituted C1-C 20 saturated or unsaturated alkylene group, and q methylene groups of L1 and / or L2 are each independently replaced by -O-, -C(O)- or -C(O)O-, and when the substituted or unsubstituted C1-C 20 saturated or unsaturated alkylene group has a substituent, the substituent is selected from one or more combinations of hydroxy, mercapto, C1-C 20 alkyl groups; L3 is selected from substituted or unsubstituted C1-C 10 alkylene, and when the substituted or unsubstituted C1-C 10 alkylene has a substituent, the substituent is selected from C1-C 20 alkyl, a combination of one or more of C3-C8 cycloalkyl; X1, X2 and X4 are each independently selected from -C(O)-, -C(O)-NH-, -C(O)-S-, -C(O)-O-, -O-, -S- or -NH-, and X3 is selected from -NH-, -S- or -O-; p is 1 or 2; q is an integer from 0 to 10.

2. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to claim 1, wherein, The cationic lipid compound has the structure shown in formula (II-A): Among them, R1 and R3 are selected from C3-C 30 a linear or branched saturated or unsaturated hydrocarbon group; L2 is selected from substituted or unsubstituted C1-C 20 saturated or unsaturated alkylene, q methylene groups in L2 are replaced by -O-, -C(O)- or -C(O)O-, and when there are substituents on the substituted or unsubstituted C1-C 20 saturated or unsaturated alkylene, the substituents are selected from one or more combinations of hydroxy, mercapto, C1-C 10 alkyl; X1 ’ and X2 ’ are each independently selected from -O-, -S- or -NH-.

3. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to claim 2, wherein The cationic lipid compound has the structure shown in formula (III-A): Among them, X4 ’ is selected from -O-, -S- or -NH-; R4 and R7 are each independently selected from H, C1-C 14 alkyl groups, and when both R4 and R7 are selected from C1-C 14 alkyl groups, R4 and R7 may form a ring; R5 and R6 are each independently selected from H, substituted or unsubstituted C1-C 20 alkyl, and when both R5 and R6 are selected from substituted or unsubstituted C1-C 20 alkyl, R5 and R6 may form a ring; Among them, the substituted or unsubstituted C1-C 20 When the alkyl group has a substituent, the substituent is selected from one or more combinations of C1-C 30 alkyl group, C3-C7 cycloalkyl group, hydroxyl group, amino group, acyl group, ether group, carboxyl group, mercapto group; Wherein, when R5 and R6 can form a ring, R5, R6 and the N atom to which they are connected form a nitrogen heterocycle, or R5 or R6 forms a ring by itself to form a heterocyclic group connected to the N atom-containing group; m is an integer from 0 to 9.

4. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to claim 1, characterized in that, The cationic lipid compound has the structure shown in formula (II-B): Among them, R2 and R3 are selected from C3-C 30 a linear or branched saturated or unsaturated hydrocarbon group; L1 is selected from substituted or unsubstituted C1-C 20 saturated or unsaturated alkylene, q methylenes in L1 are replaced by -O-, -C(O)- or -C(O)O-, and when there are substituents on the substituted or unsubstituted C1-C 20 saturated or unsaturated alkylene, the substituents are selected from one or more combinations of hydroxyl, mercapto, C1-C 10 alkyl; X1' and X2' are each independently selected from -O-, -S- or -NH-.

5. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to claim 4, wherein, The cationic lipid compound has the structure shown in formula (III-B): Wherein, X4' is selected from -O-, -S- or -NH-; R4 and R7 are each independently selected from H, C1-C 14 alkyl groups, and when both R4 and R7 are selected from C1-C 14 alkyl groups, R4 and R7 may form a ring; R5 and R6 are each independently selected from H, substituted or unsubstituted C1-C 20 alkyl, and when both R5 and R6 are selected from substituted or unsubstituted C1-C 20 alkyl, R5 and R6 may form a ring; Among them, the substituted or unsubstituted C1-C 20 When the alkyl group has a substituent, the substituent is selected from one or more combinations of C1-C 30 alkyl group, C3-C7 cycloalkyl group, hydroxyl group, amino group, acyl group, ether group, carboxyl group, mercapto group; Wherein, when R5 and R6 can form a ring, R5, R6 and the N atom to which they are connected form a nitrogen heterocycle, or R5 or R6 forms a ring by itself to form a heterocyclic group connected to the N atom-containing group; m is an integer from 0 to 9.

6. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to any one of claims 1 to 3, characterized in that, R1 is selected from C3-C 30 linear or branched alkyl, alkenyl or alkynyl groups.

7. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to claim 6, wherein, R1 is selected from any of the following structures:

8. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to claim 1, 4 or 5, characterized in that, R2 is selected from C3-C 30 linear or branched alkyl, alkenyl or alkynyl groups.

9. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to claim 8, characterized in that, R2 is selected from any of the following structures:

10. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to any one of claims 1 to 5, characterized in that, R3 is selected from C4-C 25 linear or branched alkyl, alkenyl or alkynyl groups.

11. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to claim 10, characterized in that, R3 is selected from any of the following structures:

12. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to claim 3 or 5, characterized in that, NR5R6 is selected from any one of the following structures:

13. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to claim 3 or 5, characterized in that, X1 ’ X1 is an O atom, X2’ is an O atom, X3 is an O or S atom, and X4’ is an O atom.

14. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to any one of claims 1 to 5, characterized in that, L1 or L2 is selected from any of the following structures:

15. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to claim 1, 2 or 4, characterized in that, L3 is selected from any of the following structures; 16. The cationic lipid compound or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof according to claim 3 or 5, characterized in that, The cationic lipid compound is selected from any of the following structures shown:

17. A composition, characterized in that, The composition comprises the cationic lipid compound according to any one of claims 1 to 16 or a pharmaceutically acceptable salt or solvate or N-oxide or stereoisomer thereof.

18. The composition according to claim 17, wherein The composition further comprises a prophylactic or therapeutic agent; The mass ratio of the cationic lipid compound to the prophylactic or therapeutic agent in the composition is 2.5:1 to 50:1; The prophylactic or therapeutic agent is one or more of nucleic acid, polypeptide or protein; The therapeutic or prophylactic agent is a vaccine or compound capable of eliciting an immune response.

19. The composition according to any one of claims 17 or 18, characterized in that, The composition further comprises neutral phospholipid, sterol lipid and polyethylene glycolated lipid; The molar ratio of the cationic lipid compound to neutral phospholipid, sterol lipid and polyethylene glycolated lipid is (45 - 55):(5 - 15):(35 - 45):(0.5 - 2.0).

20. Use of a cationic lipid compound according to any one of claims 1 to 16 or a pharmaceutically acceptable salt or solvate or stereoisomer thereof or a composition according to any one of claims 17 to 19 in the preparation of nucleic acid drugs, gene vaccines, small molecule drugs, polypeptide or protein drugs.

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