Cationic lipid compound, preparation method therefor, composition comprising same, and use thereof

Abstract

A cationic lipid compound, a preparation method therefor, a composition comprising same, and a use thereof, relating to the technical field of biomedicine. The cationic lipid compound has relatively high transfection efficiency and relatively low cytotoxicity by introducing at least one protonatable polar headgroup containing a secondary or tertiary amine and at least two hydrophobic tails containing a specified number of carbons, and connecting the polar headgroup and the hydrophobic tails by means of a specific linker chain.

Description

Cationic lipid compound, preparation method thereof, composition containing same and application thereof Technical Field

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

[0002] Nucleic acid drugs hold great promise for treating major infectious diseases, tumors, and other diseases, but are hampered by the lack of effective delivery systems. Cationic lipid-based delivery systems have been widely used clinically. The head groups of cationic lipids generally contain amine groups (with the exception of one lipid that contains an amidine group), ranging from simple amino groups to quaternary ammonium salts substituted with methyl or hydroxyethyl groups. The polar head groups of cationic lipids facilitate the binding of lipids to nucleic acid molecules, and of lipid nanoparticle-nucleic acid complexes to cell membranes or other intracellular components.

[0003] In the related art, permanently charged cationic lipids include, for example, monovalent cationic lipids such as (2,3-dioleyloxypropyl)trimethylammonium chloride (DOTAP), dioleoylpropyltrimethylammonium chloride (DOTMA), dimethyl-2-hydroxyethyl-2,3-ditetradecyloxypropylammonium bromide (DMRIE) and 1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), and multivalent cationic lipids such as N-(2-sperminecarboxyl)-N',N'-dioctadecylglycinamide (DOGS) and dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate (DOSPA).

[0004] The drawbacks of these charged cationic lipids are their high toxicity and potential to induce immune responses. One solution is to focus on developing ionizable cationic lipids, incorporating protonable amine groups as head groups and utilizing unsaturated acyl chains to enhance transfection efficiency. The first-generation ionizable cationic lipid, 1,2-dihydroxy-3-dimethylaminopropane (DLin-DMA), with a pKa of 6.5, is positively charged at pH 4 and can effectively encapsulate negatively charged drugs. Following intravenous injection, its near-neutral pH at physiological pH 7.4 prevents nonspecific interactions with serum proteins and improves circulation time. It adsorbs endogenous apolipoprotein E (ApoE), triggering uptake by receptors on the surface of hepatocytes. It then enters hepatocytes via endocytosis, protonates in the acidic environment of endosomes, and interacts with negatively charged endogenous lipids, leading to endosomal membrane destabilization and escape into the cytoplasm. Based on DLin-DMA, modifications in length and unsaturation have yielded a variety of available cationic lipids. Among these, dioleomethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) is an effective lipid for siRNA delivery. Further optimization of the carrier can be achieved by incorporating endosomal escape enhancers (such as pH-sensitive polymers or peptides) or designing safer and more effective ionizable lipids. Lipid nanoparticle delivery systems based on ionizable cationic lipids 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 efficient nucleic acid delivery vectors.

[0005] Summary of the Invention

[0006] In view of this, the main purpose of the present invention is to provide a cationic lipid compound, a preparation method thereof, a composition comprising the same and its application, and to construct a lipid nanoparticle delivery system by using a cationic lipid compound with high transfection efficiency and low cytotoxicity, which is beneficial to improving the efficiency of nucleic acid delivery.

[0007] To achieve the above objectives, in one aspect of the present invention, a cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof is provided, wherein the cationic lipid compound has a structure shown in formula (IA):

[0008] Wherein, R1 and R2 are selected from C3~C 30 A straight or branched saturated or unsaturated hydrocarbon group; R3 is selected from H or C1~C 10 Straight or branched chain alkyl, or C2~C 10 A straight chain or branched alkenyl group; L is selected from C1 to C 10 Alkylene, or C2~C 10 Alkenylene; R4 and R5 are each independently selected from H, substituted or unsubstituted C1 to C 20 Alkyl, and R4 and R5 are selected from substituted or unsubstituted C1~C 20When the alkyl group is an alkyl group, R4 and R5 may form a ring; X1, X2, X3 and X4 are each independently selected from NH, S, O atoms; p is 1 or 2; wherein the substituted or unsubstituted C1 to C 20 When the alkyl group has a substituent, the substituent is selected from C1 to C 30 A combination of one or more of an alkyl group, a C3-C7 cycloalkyl group, a hydroxyl group, an amino group, an acyl group, an ether group, a carboxyl group, and a thiol group; wherein, when R4 and R5 can form a ring, R4, R5 and the nitrogen atom to which they are connected form a nitrogen heterocycle, or R4 or R5 forms a ring by itself to form a heterocyclic group connected to the nitrogen atom.

[0009] In another aspect of the present invention, there is provided a method for preparing the cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof as described above, comprising the following steps:

[0010] The compound represented by formula (1) is reacted with the compound represented by formula (2) to obtain the intermediate represented by formula (3);

[0011] The intermediate represented by formula (3) is reacted with the compound represented by formula (4) to obtain the intermediate represented by formula (5), wherein X1 is O, X2 is O, X3 is S, NH or O, or X1 is NH, X2 is O, and X3 is S or NH;

[0012] The intermediate represented by formula (5) is reacted with the compound represented by formula (7) to obtain the intermediate represented by formula (6);

[0013] The intermediate represented by formula (6) is reacted with the compound represented by formula (8) to obtain the intermediate represented by formula (9);

[0014] The intermediate represented by formula (9) is reacted with the compound represented by formula (10) to obtain the cationic lipid compound represented by formula (IA);

[0015] In another aspect of the present invention, there is provided a method for preparing the cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof as described above, comprising the following steps:

[0016] The compound represented by formula (1) is reacted with the compound represented by formula (2) to obtain the intermediate represented by formula (3);

[0017] The intermediate represented by formula (3) is reacted with the compound represented by formula (4') to obtain the intermediate represented by formula (5'), wherein X1 is O, X2 is NH or S, X3 is S or O, and R6 is a protecting group;

[0018] The intermediate represented by formula (5') is used as a reactant to react with the compound represented by formula (7) to obtain the intermediate represented by formula (6');

[0019] The intermediate represented by formula (6') is deprotected and then reacted with the compound represented by formula (8) to obtain the intermediate represented by formula (9);

[0020] The intermediate represented by formula (9) is reacted with the compound represented by formula (10) to obtain the cationic lipid compound represented by formula (IA);

[0021] In another aspect of the present invention, there is provided a method for preparing the cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof as described above, comprising the following steps:

[0022] The compound represented by formula (1) is reacted with the compound represented by formula (2) to obtain the intermediate represented by formula (3);

[0023] The compound represented by formula (4) is reacted with the compound represented by formula (7) to obtain the intermediate represented by formula (5"), wherein X1 is S, X2 is O, and X3 is S or O;

[0024] The intermediate represented by formula (5") is reacted with the intermediate represented by formula (3) to obtain the intermediate represented by formula (6);

[0025] The intermediate represented by formula (6) is reacted with the compound represented by formula (8) to obtain the intermediate represented by formula (9);

[0026] The intermediate represented by formula (9) is reacted with the compound represented by formula (10) to obtain the cationic lipid compound represented by formula (IA);

[0027] In yet another aspect of the present invention, a composition is provided, comprising the cationic lipid compound as described above or a pharmaceutically acceptable salt, solvate, nitrogen oxide, or stereoisomer thereof.

[0028] In another aspect of the present invention, there is provided a use of the cationic lipid compound as described above or its pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer or the composition as described above in the preparation of a delivery drug for nucleic acids, polypeptides or proteins.

[0029] The cationic lipid compound represented by the above formula (IA) provided by the present invention has significant structural differences compared to 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 therefrom can exhibit good nucleic acid delivery efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG1 is a particle size distribution diagram of lipid nanoparticles obtained by dynamic light scattering (DLS) in Example 60 of the present invention. DETAILED DESCRIPTION

[0031] To make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. However, the present invention is not limited to the following embodiments.

[0032] The terms used herein are only for describing specific embodiments and are not intended to limit the present invention. The terms "comprise", "include" and the like 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 meanings consistent with the context of this specification and should not be interpreted in an idealized or overly rigid manner.

[0033] It should be noted that, unless otherwise defined, technical or scientific terms used in this disclosure shall have the ordinary meanings understood by those skilled in the art to which this disclosure pertains. Where references to "first," "second," and the like are made throughout this disclosure, such references are intended solely to distinguish similar objects and are not to be construed as indicating or implying their relative importance, order of precedence, or implicitly specifying the quantity of the technical features being referred to. References to "first," "second," and the like are intended to be interchangeable where appropriate.

[0034] In the present invention, the term "pharmaceutically acceptable" refers to compounds, substances, compositions and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and are commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0035] 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 (e.g., ethanol or water). The term "hydrate" refers to the case where the solvent in the above-mentioned term "solvate" is water. Furthermore, the compound of formula (I) or a pharmaceutically acceptable salt thereof can be isolated as a solvate, and any solvate is included within the scope of protection of the present invention.

[0036] In the present invention, the term "pharmaceutically acceptable salt" refers to a relatively non-toxic organic or inorganic acid addition salt of a compound of formula (I), wherein inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, and the like, and organic acids include 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, dodecylsulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid, itaconic acid, naphthalene disulfonic acid, malic acid, adipic acid, alginic acid, maleic acid, D-gluconic acid, aspartic acid, and the like.

[0037] In the present invention, the term "stereoisomer" includes geometric isomers, diastereomers 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 chirally resolved by chromatographic resolution or chemical resolution.

[0038] In the present invention, the term "substituted or unsubstituted" group may be substituted with one substituent or multiple substituents. When there are multiple substituents, they may 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 the substituents is as shown above and will not be repeated one by one.

[0039] In the present invention, “each independently” means that when there are multiple subjects, they may be the same or different.

[0040] In the present invention, the expression of chemical elements, unless otherwise specified, generally includes the concept of isotopes with the same chemical properties. For example, the expression "hydrogen (H)" also includes isotopes with the same chemical properties. 1 H (hydrogen or H), 2 The concept of H (deuterium or D); carbon (C) includes 12 C. 13 C, etc., no further details.

[0041] In the present invention, C a ~C bThe expression "a" means that the number of carbon atoms in the group is a to b. Unless otherwise specified, the number of carbon atoms in the group generally does not include the number of carbon atoms in the substituent.

[0042] In the present invention, the term "alkyl" may include a branched or straight chain saturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. 20 Examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl.

[0043] In the present invention, the term "straight-chain or branched saturated or unsaturated hydrocarbon group" may include straight-chain or branched saturated aliphatic monovalent hydrocarbon groups or unsaturated aliphatic monovalent hydrocarbon groups having a specified number of carbon atoms. 30 Examples of the linear or branched saturated hydrocarbon group include propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, heptyl, octyl, isooctyl, nonyl, decyl, undecyl, and dodecyl. 30 The straight chain or branched unsaturated hydrocarbon group can be C3~C 30 One or more single bonds in a linear or branched saturated hydrocarbon group are replaced by double bonds, etc.

[0044] In the process of realizing the present invention, it was found that the cationic lipid compound of the present invention can be used to deliver nucleic acids, etc. Compared with some representative compounds in the related art, such as DLin-DMA, DLin-MC3-DMA, etc., it has a different structure and exhibits higher transfection efficiency and lower cytotoxicity. The lipid nanoparticle delivery system constructed therefrom is beneficial to improving the delivery efficiency of nucleic acids, etc.

[0045] According to an embodiment of one aspect of the present invention, a cationic lipid compound or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof is provided, wherein the cationic lipid compound has a structure represented by formula (IA):

[0046] Wherein, R1 and R2 are selected from C3~C 30 A straight or branched saturated or unsaturated hydrocarbon group, preferably C3 to C 30 A straight or branched saturated hydrocarbon group; R3 is selected from H or C1~C 10 Straight or branched chain alkyl, or C2~C 10 A straight-chain or branched alkenyl group, preferably H or a straight-chain alkyl group of C1 to C5; X1, X2, X3 and X4 are each independently selected from NH, S, O atoms; p is 1 or 2, wherein the substituted or unsubstituted C1 to C 20 When the alkyl group has a substituent, the substituent is selected from C1 to C30 A combination of one or more of an alkyl group, a C3-C7 cycloalkyl group, a hydroxyl group, an amino group, an acyl group, an ether group, a carboxyl group, and a thiol group; wherein when R4 and R5 can form a ring, R4, R5 and the nitrogen atom to which they are connected form a nitrogen heterocycle, or R4 or R5 form a ring by themselves to form a heterocyclic group connected to the nitrogen atom.

[0047] According to an embodiment of the present invention, by introducing at least one protonable polar head containing a secondary or tertiary amine and at least two hydrophobic tails containing a specified number of carbon atoms, the polar head and the hydrophobic tails are connected by a specific connecting chain, thereby achieving higher transfection efficiency and lower cytotoxicity.

[0048] According to an embodiment of the present invention, 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, and X4 can be O; or X1 can be O, X2 can be O, X3 can be O, and X4 can be O; or X1 can be O, X2 can be NH, X3 can be S, and X4 can be O; or X1 can be S, X2 can be NH, X3 can be S, and X4 can be O; or X1 can be N, X2 can be O, X3 can be NH, and X4 can be O; or X1 can be S, X2 can be O, X3 can be O, and X4 can be O, etc.; or X1 can be O, X2 can be O, X3 can be S, and X4 can be S; or X1 can be O, X2 can be O, X3 can be S, and X4 can be N. Preferably, X1 is O, X2 is O, X3 is O or S, and X4 is O.

[0049] According to an embodiment of the present invention, in the compound represented by formula (IA), when R4 and R5 can form a ring, R4, R5 and the nitrogen atom to which they are attached can form an nitrogen heterocyclic ring, or R4 or R5 can form a ring on their own to form a heterocyclic group connected to the nitrogen atom, and each is preferably an nitrogen heterocycloalkyl group. The ring formed can be, for example, a 5- to 8-membered ring, for example, a 5-membered ring, a 6-membered ring, a 7-membered ring, or an 8-membered ring.

[0050] According to an embodiment of the present invention, in the compound represented by formula (IA), R1 is selected from C3 to C 30 The linear or branched alkyl, alkene or alkyne group is preferably any of the following structures:

[0051] According to an embodiment of the present invention, in the compound represented by formula (IA), R2 is selected from C4 to C 30 The linear or branched alkyl, alkene or alkyne group is preferably any of the following structures:

[0052] According to an embodiment of the present invention, the aforementioned types of R1 and R2 can regulate lipid behavior, which is beneficial to improving transfection efficiency.

[0053] According to an embodiment of the present invention, in the compound represented by formula (IA), NR4R5 is selected from any of the following structures:

[0054] According to an embodiment of the present invention, the above-mentioned type of NR4R5 can improve transfection efficiency by regulating the lipid charge and affecting the binding ability with nucleic acids.

[0055] According to an embodiment of the present invention, in the compound represented by formula (IA), R3 may preferably be H, methyl, ethyl, vinyl or isopropyl, which simplifies the synthesis scheme without affecting the performance of the compound.

[0056] According to an embodiment of the present invention, the above-mentioned compound of the present invention is preferably selected from any of the following structures, but these compounds are only representative:

[0057] According to another embodiment of the present invention, there is also provided a method for preparing the cationic lipid compound or a pharmaceutically acceptable salt, solvate, nitrogen oxide or stereoisomer thereof as described above, comprising the following steps S101 to S105.

[0058] In step S101, the compound represented by formula (1) and the compound represented by formula (2) are reacted to obtain the intermediate represented by formula (3);

[0059] In step S102, the intermediate represented by formula (3) and the compound represented by formula (4) are reacted to obtain the intermediate represented by formula (5), wherein X1 is O, X2 is O, X3 is S, NH or O, or X1 is NH, X2 is O, and X3 is S or NH;

[0060] In step S103, the intermediate represented by formula (5) and the compound represented by formula (7) are reacted to obtain the intermediate represented by formula (6);

[0061] In step S104, the intermediate represented by formula (6) and the compound represented by formula (8) are reacted to obtain the intermediate represented by formula (9);

[0062] In step S105, the intermediate represented by formula (9) and the compound represented by formula (10) are reacted to obtain the cationic lipid compound represented by formula (IA);

[0063] According to the embodiments of the present invention, the preparation method has mild conditions and is simple to operate, and can effectively prepare the cationic lipid compound.

[0064] According to an embodiment of the present invention, step S101 may specifically include: dropwise adding the compound of formula (2) to a first organic solvent containing the compound of formula (1) and a first base, performing a reflux reaction, and after the reaction is completed, separating to obtain the intermediate of formula (3). 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.

[0065] According to an embodiment of the present invention, step S102 may specifically include: dropwise adding a compound of formula (4) to a second organic solvent containing the intermediate of formula (3) and a second base, reacting at room temperature, and separating to obtain the intermediate of formula (5) after the reaction is completed. 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, diisopropylethylamine, triethylamine, etc.

[0066] According to an embodiment of the present invention, in step S103, a compound represented by formula (7) is added dropwise to a third organic solvent containing the intermediate represented by formula (5) and a third base, and the mixture is reacted at room temperature. After the reaction is completed, the intermediate represented by formula (6) is separated. The third organic solvent may be, for example, dichloromethane, tetrahydrofuran, etc., and the third base may be, for example, an organic base or an inorganic base, specifically, triethylamine, diisopropylethylamine, etc.

[0067] In step S104, the compound represented by formula (8) is added dropwise to a fourth organic solvent containing the intermediate represented by formula (6), a condensation reagent, and a fourth base, and the mixture is reacted at room temperature. After the reaction is completed, the intermediate represented by formula (9) is separated. The condensation reagent may be, for example, N,N'-dicyclohexylcarbodiimide (DCC), N,N-carbonyldiimidazole (CDI), etc., the fourth organic solvent may be, for example, dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, tetrahydrofuran, etc., and the fourth base may be, for example, an organic base or an inorganic base, specifically, dimethylaminopyridine, diisopropylethylamine, triethylamine, etc.

[0068] In step S105, a compound represented by formula (10) is added to a fifth organic solvent containing the intermediate represented by formula (9) and a fifth base. After the reaction is completed, a cationic lipid compound represented by formula (IA) is separated. The fifth organic solvent may be, for example, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, etc., and the fifth base may be, for example, an organic base or an inorganic base, specifically, triethylamine, diisopropylethylamine, potassium carbonate, cesium carbonate, etc.

[0069] According to another embodiment of the present invention, there is also provided a method for preparing the cationic lipid compound or a pharmaceutically acceptable salt, solvate, nitrogen oxide or stereoisomer thereof as described above, comprising the following steps S201 to S205.

[0070] In step S201, the compound represented by formula (1) and the compound represented by formula (2) are reacted to obtain the intermediate represented by formula (3);

[0071] In step S202, the intermediate represented by formula (3) is reacted with the compound represented by formula (4') to obtain the intermediate represented by formula (5'), wherein X1 is O, X2 is NH or S, X3 is S or O, and R6 is a protecting group. For example, when X2 is NH, the protecting group may be a protecting group such as Boc, Bn or Cbz; when X2 is S, the protecting group may be a protecting group such as Trt;

[0072] In step S203, the intermediate represented by formula (5') and the compound represented by formula (7) are reacted to obtain the intermediate represented by formula (6');

[0073] In step S204, the intermediate represented by formula (6') is deprotected and then reacted with the compound represented by formula (8) to obtain the intermediate represented by formula (9);

[0074] In step S205, the intermediate represented by formula (9) and the compound represented by formula (10) are reacted to obtain the cationic lipid compound represented by formula (IA);

[0075] The reaction conditions of the above steps S201 to S205 are similar to those of steps S101 to S105. The main difference is that step S204 also includes a deprotection operation. The specific reaction conditions of the deprotection can be determined according to the selected protecting group. Since it is a well-known reaction, it will not be described here.

[0076] According to another embodiment of the present invention, there is also provided a method for preparing the cationic lipid compound or a pharmaceutically acceptable salt, solvate, nitrogen oxide or stereoisomer thereof as described above, comprising the following steps S301 to S305.

[0077] In step S301, the compound represented by formula (1) and the compound represented by formula (2) are reacted to obtain the intermediate represented by formula (3);

[0078] In step S302, the compound represented by formula (4) and the compound represented by formula (7) are reacted to obtain an intermediate represented by formula (5"), wherein X1 is S, X2 is O, and X3 is S or O;

[0079] In step S303, the intermediate represented by formula (5") and the intermediate represented by formula (3) are reacted to obtain the intermediate represented by formula (6);

[0080] In step S304, the intermediate represented by formula (6) and the compound represented by formula (8) are reacted to obtain the intermediate represented by formula (9);

[0081] In step S305, the intermediate represented by formula (9) and the compound represented by formula (10) are reacted to obtain the cationic lipid compound represented by formula (IA);

[0082] The reaction conditions of the above steps S301 to S305 are similar to those of steps S101 to S105. The difference lies mainly in the reaction order, so they are not described in detail.

[0083] According to another embodiment of the present invention, a composition is provided, comprising the cationic lipid compound as described above or a pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer thereof.

[0084] According to an embodiment of the present invention, the above-mentioned composition is a nanoparticle preparation to construct a lipid nanoparticle delivery system; the average particle size can be, for example, 10nm to 300nm, specifically, for example, 10nm, 20nm, 50nm, 100nm, 150nm, 200nm, 250nm, 300nm, etc.

[0085] According to an embodiment of the present invention, the above composition may further include a preventive agent or a therapeutic agent.

[0086] According to an embodiment of the present invention, the preventive or therapeutic agent may be at least one of a nucleic acid, a polypeptide, or a protein, and the mass ratio of the cationic lipid compound to the preventive or therapeutic agent is 2.5:1 to 50:1, for example, 2.5:1 to 15:1, 15:1 to 30:1, 30:1 to 40:1, or 40:1 to 50:1, and specifically, for example, 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, or 50:1. Nucleic acids include, but are not limited to, single-stranded DNA, double-stranded DNA, and RNA. The RNA includes, but is not limited to, small interfering RNA (siRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), messenger RNA (mRNA), small hairpin RNA, asymmetric interfering RNA, and mixtures thereof. The protein includes, but is not limited to, gentamicin, amikacin, insulin, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), interferon, hepatitis B surface antigen, or heparin.

[0087] According to an embodiment of the present invention, the composition may further include a neutral phospholipid, a steroidal lipid, and a PEGylated lipid; wherein the molar ratio of the cationic lipid compound to the neutral phospholipid, the steroidal lipid, and the PEGylated lipid is (45-55):(5-15):(35-45):(0.5-2.0). Alternatively, the molar ratio may be (48-52):(8-12):(36-40):(1.2-1.8), and specifically, for example, may be 50:10:38.5:1.5.

[0088] According to an embodiment of the present invention, phospholipids are suitable for improving the stability of lipid nanoparticles, regulating the fluidity of lipid nanoparticles, and may also affect target specificity. Neutral phospholipids may be, for example, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), diarachidoylphosphatidylcholine (DAPC), 1,2-behenoyl-phosphatidylcholine (DLPC), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), 1-stearoyl-2-myristoylphosphatidylcholine (SMPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoylphosphatidylcholine (POPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (D MPE), dilauroylphosphatidylethanolamine (DLPE), distearoylphosphatidyl sodium salt (DSPA), dipalmitoylphosphatidyl sodium salt (DPPA), dimyristoylphosphatidyl sodium salt (DMPA), dioleoylphosphatidyl sodium salt (DOPA), dilauroylphosphatidyl sodium salt (DLPA), distearoylphosphatidylglycerol sodium salt (DSPG), dipalmitoylphosphatidylglycerol sodium salt (DPPG), dimyristoylphosphatidylglycerol sodium salt (DMPG), dioleoylphosphatidylglycerol sodium salt (DOPG), dilauroylphosphatidylglycerol sodium salt (DLPG), distearoylphosphatidylserine sodium salt (DSPS), dipalmitoylphosphatidylserine sodium salt (DPPS), dimyristoylphosphatidylserine sodium salt (DMPS), dioleoylphosphatidylserine sodium salt (DOPS), dilauroylphosphatidylserine sodium salt (DLPS), etc.

[0089] According to an embodiment of the present invention, the steroidal lipid is selected from one or more of avenasterol, β-sitosterol, rapeseed sterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, melanosterol, epicholesterol, ergosterol, fuccasterol, hexahydroluminosterol, hydroxycholesterol, lanosterol, luminosterol, alginosterol, sitostanol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, and deoxycholic acid.

[0090] According to an embodiment of the present invention, the steroidal lipid is selected from cholesterol, which is a structural lipid. It regulates the fluidity of lipid nanoparticles by filling the gaps between phospholipids and enhances the stability of lipid nanoparticles.

[0091] According to embodiments of the present invention, PEGylated lipid (i.e. PEGylated lipid), can affect size, stability, distribution in vivo and transfection efficiency of lipid nanoparticle delivery system, in the preparation and storage process of lipid nanoparticle delivery system, PEGylated lipid can drive self-assembly and can prevent particle aggregation.PEGylated lipid such as can be distearoyl phosphatidylethanolamine polyethylene glycol (DSPE-PEG), dimyristoyl glycerol-rac-methoxy polyethylene glycol 2000 (DMG-PEG2000), methoxy polyethylene glycol ditetradecyl acetamide (ALC-0159), polyethylene glycol-diacylglycerol amide (PEG-DAG), polyethylene glycol-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), polyethylene glycol-disterol glycerol (PEG-DSG), polyethylene glycol-diacylglycerol amide (PEG-DAG) etc.

[0092] According to an embodiment of the present invention, the above-mentioned composition may further include pharmaceutically acceptable excipients or auxiliary ingredients, including but not limited to: binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, pigments, flavoring agents, etc. for oral preparations; preservatives, solubilizers, stabilizers, etc. for injectable preparations. The pharmaceutical preparation can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically).

[0093] According to an embodiment of the present invention, the above-mentioned composition can be formulated into several dosage forms according to the mode of administration, such as oral preparations (such as tablets, capsules, solutions or suspensions); injectable preparations (such as injectable solutions or suspensions, or injectable dry powders, which can be used immediately by adding injection water before injection), etc.

[0094] According to embodiments of the present invention, the compositions can be administered at a therapeutically effective dose, which can vary not only with the specific agent selected, but also with the route of administration, the nature of the disease being treated, and the age and condition of the patient. For example, a dose of about 0.001 mg / kg to about 10 mg / kg of nucleic acid can be administered to a subject, such as a mammal (e.g., a human).

[0095] According to another embodiment of the present invention, there is also provided the use of the cationic lipid compound as described above or its pharmaceutically acceptable salt or solvate or nitrogen oxide or stereoisomer or the composition as described above in the preparation of a delivery drug for nucleic acids, polypeptides or proteins.

[0096] According to embodiments of the present invention, due to the wide variety of nucleic acids, polypeptides, or proteins, the cationic lipid compounds of the present invention, or pharmaceutically acceptable salts or solvates or nitrogen oxides or stereoisomers thereof, or the above compositions can be used to treat or prevent a variety of diseases or conditions.

[0097] To make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific examples. The test materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. Where specific techniques or conditions are not specified in the examples, they are all conventional methods and can be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions.

[0098] Example 1: Synthesis of cationic lipid compound E10

[0099] Step 1, preparation of 2-bromopropionic acid 2-hexyl-1-decyl ester (1-3):

[0100] 41.2 mmol of 2-hexyl-1-decanol (1-1) was dissolved in 100 ml of dichloromethane, and then 61.8 mmol of triethylamine and 20.6 mmol of 4-dimethylaminopyridine were added. 61.8 mmol of 2-bromopropionyl bromide was added dropwise at 30-40°C. After the addition was complete, the reaction was stopped by stirring at reflux for 4 hours. After adding water, the liquid was extracted and separated. The organic phase was washed with saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate. The solvent was filtered, concentrated, and the residue (intermediate 1-3) was used directly in the next reaction without treatment.

[0101] Step 2: Preparation of 2-((2,3-dihydroxypropyl)thio)propionic acid-2-hexyl-1-decyl ester (1-5):

[0102] 26.5 mmol of 2-bromopropionic acid-2-hexyl-1-decyl ester (1-3) was dissolved in 100 ml of N,N-dimethylformamide, and 39.75 mmol of potassium carbonate was added. The mixture was stirred for 20-30 min, and 29.15 mmol of 1-thioglycerol was added dropwise below 30°C. After the addition was complete, the mixture was stirred at room temperature overnight. The reaction was stopped the next day. 300 ml of water was added and the mixture was extracted with ethyl acetate. The aqueous phase was back-extracted with ethyl acetate. The combined organic phases were washed with saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate. The solvent was filtered, concentrated, and the residue was separated and purified by column chromatography (dichloromethane:methanol = 95:5) to obtain a light yellow oily liquid (intermediate 1-5) in a yield of 56%.

[0103] Step 3, 3-((1-((2-hexyldecane)oxy)-1-oxoprop-2-yl)thio)-2-hydroxypropyl octanoate (1-6):

[0104] 24.7 mmol of 2-hexyl-1-decyl-2-((2,3-dihydroxypropyl)thio)propanoate (1-5) was dissolved in 100 ml of dichloromethane, and 37.05 mmol of triethylamine was added. The system was cooled to 0°C, and 27.2 mmol of octanoyl chloride (1-7) was added dropwise. After stirring for 3 hours after the addition, the reaction was stopped. Water was added and the liquid was extracted. The organic phase was washed with saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate. The solvent was filtered, concentrated, and the residue was separated by spin-drying. The residue was separated and purified by column chromatography (n-heptane:ethyl acetate = 90:10) to obtain a light yellow oily liquid (Intermediate 1-6) Intermediate #3 in a yield of 62%.

[0105] Step 4: Preparation of 2-((4-bromobutyryl)oxy)-3-((1-((2-hexyldecane)oxy)-1-oxoprop-2-yl)thio)octanoic acid propyl ester (1-9):

[0106] 18.8 mmol of 3-((1-((2-hexyldecane)oxy)-1-oxopropan-2-yl)thio)-2-hydroxypropyl octanoate (1-6) was dissolved in 100 ml of dichloromethane, followed by the addition of 28.2 mmol of N,N'-dicyclohexylcarbodiimide and 0.9 mmol of 4-dimethylaminopyridine. 22.6 mmol of 4-bromobutyric acid (1-8) was added dropwise below 35°C. After the addition was complete, the reaction was stopped by stirring for 4 hours. The system was filtered, the filtrate was concentrated, and the solvent was dried. The residue was separated and purified by column chromatography (n-heptane:ethyl acetate = 90:10) to obtain 8.3 g of a light yellow oily liquid (intermediate 1-9) in a yield of 65%.

[0107] Step 5, preparation of 3-((1-((2-hexyldecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)octanoic acid propyl ester (E10):

[0108] 7.35 mmol of 2-((4-bromobutyryl)oxy)-3-((1-((2-hexyldecane)oxy)-1-oxopropyl-2-yl)thio)octanoate (1-9) was dissolved in 25 ml of acetonitrile, and then 14.7 mmol of triethylamine and 12.5 mmol of 1-(3-(4-methylpiperazin-1-yl)propyl)amino)dec-2-ol (1-10) were added. The system was heated to 40° C. and stirred to react overnight. The reaction was stopped the next day, and the system was directly concentrated to dryness. The solvent was then separated and purified by column chromatography (DCM:MeOH=90:10) to obtain 2.1 g of a light yellow oily liquid (E10) in a yield of 40%.

[0109] The cationic lipid compound E10 was characterized and the results are as follows:

[0110] 1H NMR (400MHz, CDCl3) δ5.25-5.10(m,1H),4.38-4.27(m,1H),4.18-4.09(m,1H),4.03(d,J=5.6Hz,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=6.5Hz,12H).

[0111] [M+H] + :912.60,[1 / 2M+H] + :457.00.

[0112] Example 2: Synthesis of cationic lipid compound B2

[0113] The operation is similar to that of Example 1, except that the 2-hexyl-1-decanol (1-1) in step 1 is replaced with 9-heptadecanol (2-1), the octanoyl chloride (1-7) in step 3 is replaced with decanoyl chloride (2-7), and the 1-(3-(4-methylpiperazin-1-yl)propyl)amino)decan-2-ol (1-10) in step 5 is replaced with N-hydroxyethylpiperazine (2-10). Finally, 3-((1-(heptadecan-9-oxy)-1-oxopropyl-2-yl)thio)-2-((4-(4-(2-hydroxyethyl)piperazin-1-yl)butyryl)oxy)decanoic acid propyl ester (B2) is obtained.

[0114] The cationic lipid compound B2 was characterized and the results are as follows:

[0115] 1 H NMR (400MHz, CDCl3) δ5.26-5.11(m,1H),4.87(p,J=6.2Hz,1H),4.39-4.28(m,1 H),4.15(dd,J=11.8,5.6Hz,1H),3.62(t,J=5.3Hz,2H),3.47-3.38(m,1H),2.9 4-2.71(m,3H),2.70-2.42(m,10H),2.41-2.27(m,6H),1.86-1.76(m,2H),1.64 -1.47(m,6H),1.42(dd,J=9.8,7.2Hz,3H),1.25(s,36H),0.87(t,J=6.7Hz,9H).

[0116] [M+H] + :771.60.

[0117] Example 3: Synthesis of cationic lipid compound B3

[0118] The operation is similar to that of Example 1, except that the 2-hexyl-1-decanol (1-1) of step 1 is replaced with 9-heptadecanol (2-1), the octanoyl chloride (1-7) of step 3 is replaced with dodecanoyl chloride (3-7), and the 1-(3-(4-methylpiperazine-1-yl)propyl)amino)decane-2-ol (1-10) of step 5 is replaced with N-hydroxyethylpiperazine (2-10). Finally, 3-((1-(heptadecan-9-oxy)-1-oxopropyl-2-yl)thio)-2-((4-(4-(2-hydroxyethyl)piperazine-1-yl)butyryl)oxy)dodecanoic acid propyl ester (B3) is obtained.

[0119] The cationic lipid compound B3 was characterized and the results are as follows:

[0120] 1 H NMR (400MHz, CDCl3) δ5.26-5.10(m,1H),4.87(p,J=6.2Hz,1H),4.38-4.28(m,1 H),4.15(dd,J=11.9,5.6Hz,1H),3.61(t,J=5.3Hz,2H),3.47-3.38(m,1H),2.9 4-2.70(m,2H),2.70-2.42(m,10H),2.42-2.27(m,6H),1.86-1.75(m,2H),1.64 -1.47(m,6H),1.42(dd,J=9.8,7.2Hz,3H),1.25(s,40H),0.87(t,J=6.7Hz,9H).

[0121] [M+H] + :799.60.

[0122] Example 4: Synthesis of cationic lipid compound C1

[0123] The operation was similar to that of Example 1, except that 2-hexyl-1-decanol (1-1) in step 1 was replaced with 11-heneicosyl alcohol (4-1), and 1-(3-(4-methylpiperazin-1-yl)propyl)amino)decan-2-ol (1-10) in step 5 was replaced with N-hydroxyethylpiperazine (2-10). Finally, 3-((1-(heneicosane-11-oxy)-1-oxoprop-2-yl)thio)-2-((4-(4-(2-hydroxyethyl)piperazin-1-yl)butanoyl)oxy)octanoic acid propyl ester (C1) was obtained.

[0124] The cationic lipid compound C1 was characterized and the results are as follows:

[0125] 1 H NMR (400MHz, CDCl3) δ5.25-5.09(m,1H),4.87(p,J=6.2Hz,1H),4.39-4.26(m,1H),4.14(dd,J=11.6,5.3Hz,1H),3.60(t,J= 5.4Hz,2H),3.47-3.37(m,1H),2.95-2.70(m,2H),2.70-2.42(m,10H),2.39-2.26(m,6H),1.85-1 .75(m,2H),1.64-1.47(m,6H),1.41(dd,J=9.9,7.2Hz,3H),1.24(s,40H),0.86(t,J=6.8Hz,9H).

[0126] [M+H] + :799.60.

[0127] Example 5: Synthesis of cationic lipid compound C2

[0128] The operation was similar to that of Example 2, except that 9-heptadecanol (2-1) in step 1 was replaced with 11-heneicosyl (4-1). Finally, 3-((1-(heneicosane-11-oxy)-1-oxopropan-2-yl)thio)-2-((4-(4-(2-hydroxyethyl)piperazin-1-yl)butanoyl)oxy)decanoic acid propyl ester (C2) was obtained.

[0129] The cationic lipid compound C2 was characterized, and the results are as follows:

[0130] 1 H NMR (400MHz, CDCl3) δ5.25-5.09(m,1H),4.87(p,J=6.2Hz,1H),4.39-4.26(m,1 H),4.14(dd,J=11.6,5.3Hz,1H),3.60(t,J=5.4Hz,2H),3.47-3.37(m,1H),2.9 5-2.70(m,2H),2.70-2.42(m,10H),2.39-2.26(m,6H),1.85-1.75(m,2H),1.64 -1.47(m,6H),1.41(dd,J=9.9,7.2Hz,3H),1.24(s,44H),0.86(t,J=6.8Hz,9H).

[0131] [M+H] + :827.60.

[0132] Example 6: Synthesis of cationic lipid compound C3

[0133] The operation was similar to that of Example 4, except that octanoyl chloride (1-7) in step 3 was replaced with dodecanoyl chloride (3-7). Finally, 3-((1-(heneicosane-11-oxy)-1-oxoprop-2-yl)thio)-2-((4-(4-(2-hydroxyethyl)piperazin-1-yl)butanoyl)oxy)dodecanoic acid propyl ester (C3) was obtained.

[0134] The cationic lipid compound C3 was characterized, and the results are as follows:

[0135] 1 H NMR (400MHz, CDCl3) δ5.24-5.10(m,1H),4.87(p,J=6.2Hz,1H),4.39-4.27(m,1 H),4.14(dd,J=11.6,5.3Hz,1H),3.60(t,J=5.4Hz,2H),3.47-3.38(m,1H),2.9 4-2.70(m,2H),2.70-2.42(m,10H),2.39-2.26(m,6H),1.85-1.75(m,2H),1.64 -1.47(m,6H),1.41(dd,J=9.9,7.2Hz,3H),1.24(s,48H),0.86(t,J=6.8Hz,9H).

[0136] [M+H] + :855.60.

[0137] Example 7: Synthesis of cationic lipid compound D1

[0138] The operation was similar to that of Example 2, except that 9-heptadecanol (2-1) in step 1 was replaced with 13-pentacosanol (7-1), and decanoyl chloride (2-7) in step 3 was replaced with octanoyl chloride (1-7). Finally, 3-((1-(pentacosane-13-oxy)-1-oxoprop-2-yl)thio)-2-((4-(4-(2-hydroxyethyl)piperazin-1-yl)butanoyl)oxy)octanoic acid propyl ester (D1) was obtained.

[0139] The cationic lipid compound D1 was characterized, and the results are as follows:

[0140] 1H NMR (400MHz, CDCl3) δ5.25-5.10(m,1H),4.87(p,J=6.2Hz,1H),4.39-4.28(m,1 H),4.14(dd,J=11.6,5.3Hz,1H),3.60(t,J=5.4Hz,2H),3.48-3.37(m,1H),2.9 6-2.71(m,2H),2.70-2.43(m,10H),2.41-2.27(m,6H),1.86-1.76(m,2H),1.64 -1.48(m,6H),1.41(dd,J=9.9,7.2Hz,3H),1.24(s,48H),0.86(t,J=6.8Hz,9H).

[0141] [M+H] + :855.60.

[0142] Example 8: Synthesis of cationic lipid compound D2

[0143] The operation was similar to that of Example 2, except that 9-heptadecanol (2-1) in step 1 was replaced with 13-pentacosanol (7-1). Finally, 3-((1-(pentacosan-13-oxy)-1-oxopropan-2-yl)thio)-2-((4-(4-(2-hydroxyethyl)piperazin-1-yl)butanoyl)oxy)propyl decanoate (D2) was obtained.

[0144] The cationic lipid compound D2 was characterized and the results are as follows:

[0145] 1 H NMR (400MHz, CDCl3) δ5.25-5.10(m,1H),4.87(p,J=6.2Hz,1H),4.39-4.28(m,1 H),4.14(dd,J=11.6,5.3Hz,1H),3.60(t,J=5.4Hz,2H),3.47-3.38(m,1H),2.9 6-2.70(m,2H),2.70-2.42(m,10H),2.41-2.27(m,6H),1.86-1.76(m,2H),1.64 -1.48(m,6H),1.41(dd,J=9.9,7.2Hz,3H),1.24(s,52H),0.86(t,J=6.8Hz,9H).

[0146] [M+H] + :883.60.

[0147] Example 9: Synthesis of cationic lipid compound D3

[0148] The operation was similar to that of Example 2, except that 9-heptadecanol (2-1) in step 1 was replaced with 13-pentacosanol (7-1), and decanoyl chloride (2-7) in step 3 was replaced with dodecanoyl chloride (3-7). Finally, 3-((1-(pentacosan-13-oxy)-1-oxoprop-2-yl)thio)-2-((4-(4-(2-hydroxyethyl)piperazin-1-yl)butanoyl)oxy)dodecanoic acid propyl ester (D3) was obtained.

[0149] The cationic lipid compound D3 was characterized, and the results are as follows:

[0150] 1 H NMR (400MHz, CDCl3) δ5.25-5.11(m,1H),4.87(p,J=6.2Hz,1H),4.39-4.27(m,1 H),4.15(dd,J=11.6,5.3Hz,1H),3.60(t,J=5.4Hz,2H),3.47-3.38(m,1H),2.9 6-2.70(m,2H),2.69-2.42(m,10H),2.41-2.27(m,6H),1.86-1.75(m,2H),1.64 -1.48(m,6H),1.41(dd,J=9.9,7.2Hz,3H),1.24(s,56H),0.86(t,J=6.8Hz,9H).

[0151] [M+H] + :911.60.

[0152] Example 10: Synthesis of cationic lipid compound F1

[0153] The operation was similar to that of Example 7, except that 13-pentacosanol (7-1) in step 1 was replaced with 2-octyldodecanol (10-1). Finally, 3-((1-((2-octyldodecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-(4-(2-hydroxyethyl)piperazin-1-yl)butanoyl)oxy)octanoic acid propyl ester (F1) was obtained.

[0154] The cationic lipid compound F1 was characterized and the results are as follows:

[0155] 1H NMR (400MHz, CDCl3) δ5.25-5.10 (m, 1H), 4.33 (ddd, J = 15.3, 12.0, 3.6Hz, 1H), 4.1 4(dd,J=11.8,5.6Hz,1H),4.03(d,J=5.7Hz,2H),3.65(t,J=5.3Hz,2H),3.50-3.4 0(m,1H),2.95-2.72(m,2H),2.71-2.44(m,10H),2.45-2.26(m,7H),1.88-1.77(m ,2H),1.68-1.54(m,3H),1.46-1.39(m,3H),1.26(s,40H),0.87(t,J=6.7Hz,9H).

[0156] [M+H] + :785.40.

[0157] Example 11: Synthesis of cationic lipid compound G2

[0158] The operation was similar to that of Example 2, except that 9-heptadecanol (2-1) in step 1 was replaced with 2-decyltetradecyl-1-ol (11-1). Finally, 3-((1-((2-decyltetradecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-(4-(2-hydroxyethyl)piperazin-1-yl)butanoyl)oxy)octanoic acid propyl ester (G2) was obtained.

[0159] The cationic lipid compound G2 was characterized and the results are as follows:

[0160] 1 H NMR (400MHz, CDCl3) δ5.25-5.10 (m, 1H), 4.33 (ddd, J = 15.3, 12.0, 3.6Hz, 1H), 4.1 4(dd,J=11.8,5.6Hz,1H),4.03(d,J=5.7Hz,2H),3.61(t,J=5.3Hz,2H),3.50-3.4 0(m,1H),2.95-2.72(m,2H),2.71-2.44(m,10H),2.45-2.26(m,7H),1.86-1.76(m ,2H),1.66-1.54(m,3H),1.46-1.39(m,3H),1.26(s,52H),0.87(t,J=6.7Hz,9H).

[0161] [M+H] + :869.60.

[0162] Example 12: Synthesis of cationic lipid compound B4

[0163] The operation was similar to that of Example 2, except that decanoyl chloride (2-7) in step 3 was replaced with octanoyl chloride (1-7), and N-hydroxyethylpiperazine (2-10) in step 5 was replaced with N,N,N'-trimethylethylenediamine (12-10). 2-((4-((2-(dimethylamino)ethyl)(methyl)amino)butanoyl)oxy)-3-((1-(heptadecan-9-oxy)-1-oxopropyl-2-yl)thio)octanoate (B4) was finally obtained.

[0164] The cationic lipid compound B4 was characterized and the results are as follows:

[0165] 1 H NMR (400MHz, CDCl3) δ5.23-5.06(m,1H),4.92-4.82(m,1H),4.40-4.29(m,1 H),4.18-4.08(m,1H),3.55-3.31(m,4H),2.95-2.72(m,5H),2.58(s,4H),2 .51(td,J=7.6,2.6Hz,2H),2.41-2.26(m,6H),1.90-1.78(m,2H),1.64-1.4 8(m,6H),1.42(dd,J=9.4,7.2Hz,3H),1.25(s,32H),0.87(t,J=6.8Hz,9H).

[0166] [M+H] + :715.40.

[0167] Example 13: Synthesis of cationic lipid compound B5

[0168] The operation was similar to that of Example 2, except that N-hydroxyethylpiperazine (2-10) in step 5 was replaced with N,N,N'-trimethylethylenediamine (12-10). 2-((4-((2-(dimethylamino)ethyl)(methyl)amino)butanoyl)oxy)-3-((1-(heptadecan-9-yloxy)thio)propyl decanoate (B5) was finally obtained.

[0169] The cationic lipid compound B5 was characterized and the results are as follows:

[0170] 1H NMR (400MHz, CDCl3) δ5.23-5.06(m,1H),4.92-4.80(m,1H),4.40-4.26(m,1 H),4.16-4.09(m,1H),3.55-3.31(m,4H),2.95-2.70(m,5H),2.60(s,5H),2 .51(td,J=7.6,2.6Hz,2H),2.41-2.26(m,6H),1.88-1.78(m,2H),1.62-1.4 8(m,6H),1.41(dd,J=9.4,7.2Hz,3H),1.24(s,36H),0.86(t,J=6.8Hz,9H).

[0171] [M+H] + :743.60.

[0172] Example 14: Synthesis of cationic lipid compound B6

[0173] The operation was similar to that of Example 3, except that N-hydroxyethylpiperazine (2-10) in step 5 was replaced with N,N,N'-trimethylethylenediamine (12-10). 2-((4-((2-(dimethylamino)ethyl)(methyl)amino)butanoyl)oxy)-3-((1-(heptadecan-9-oxy)-1-oxoprop-2-yl)thio)dodecanoic acid propyl ester (B6) was finally obtained.

[0174] The cationic lipid compound B6 was characterized and the results are as follows:

[0175] 1 H NMR (400MHz, CDCl3) δ5.24-5.04(m,1H),4.90-4.80(m,1H),4.40-4.30(m,1 H),4.16-4.08(m,1H),3.55-3.36(m,4H),3.04-2.80(m,5H),2.60(s,4H),2 .56(td,J=7.6,2.6Hz,2H),2.42-2.26(m,6H),1.90-1.80(m,2H),1.63-1.4 8(m,6H),1.41(dd,J=9.4,7.2Hz,3H),1.24(s,40H),0.86(t,J=6.8Hz,9H).

[0176] [M+H] + :771.60.

[0177] Example 15: Synthesis of cationic lipid compound C4

[0178] The operation was similar to that of Example 5, except that decanoyl chloride (2-7) in step 3 was replaced with octanoyl chloride (1-7), and N-hydroxyethylpiperazine (2-10) in step 5 was replaced with N,N,N'-trimethylethylenediamine (12-10). Finally, propyl 2-((4-((2-(dimethylamino)ethyl)(methyl)amino)butanoyl)oxy)-3-((1-(heneicosane-11-oxy)-1-oxopropan-2-yl)thio)octanoate (C4) was obtained.

[0179] The cationic lipid compound C4 was characterized, and the results are as follows:

[0180] 1 H NMR (400MHz, CDCl3) δ5.25-5.07(m,1H),4.91-4.80(m,1H),4.72-4.26(m,3H),4.17-4.06(m,1H),3.47-3.38(m,2H),2.96- 2.82(m,5H),2.55(s,4H),2.49(td,J=7.6,2.6Hz,2H),2.40-2.26(m,6H),1.90-1.77(m,2H),1 .64-1.47(m,6H),1.41(dd,J=9.4,7.2Hz,3H),1.25(d,J=11.9Hz,40H),0.86(t,J=6.8Hz,9H).

[0181] [M+H] + :771.60.

[0182] Example 16: Synthesis of cationic lipid compound C5

[0183] The procedure was similar to that of Example 5, except that N-hydroxyethylpiperazine (2-10) in step 5 was replaced with N,N,N'-trimethylethylenediamine (12-10). Propyl 2-((4-((2-(dimethylamino)ethyl)(methyl)amino)butanoyl)oxy)-3-((1-(heneicosane-11-oxy)-1-oxopropan-2-yl)thio)decanoate (C5) was obtained.

[0184] The cationic lipid compound C5 was characterized, and the results are as follows:

[0185] 1H NMR (400MHz, CDCl3) δ5.25-5.07(m,1H),4.92-4.82(m,1H),4.40-4.29(m,1H),4.1 8-4.08(m,1H),3.47-3.38(m,2H),3.20-2.97(br,3H),2.95-2.72(m,5H),2.58(s, 4H),2.51(td,J=7.6,2.6Hz,2H),2.41-2.26(m,6H),1.90-1.77(m,2H),1.64-1.48 (m,6H),1.42(dd,J=9.4,7.2Hz,3H),1.26(d,J=9.2Hz,44H),0.87(t,J=6.8Hz,9H).

[0186] [M+H] + :799.60.

[0187] Example 17: Synthesis of cationic lipid compound C6

[0188] The procedure was similar to that of Example 6, except that N-hydroxyethylpiperazine (2-10) in step 5 was replaced with N,N,N'-trimethylethylenediamine (12-10). Propyl 2-((4-((2-(dimethylamino)ethyl)(methyl)amino)butanoyl)oxy)-3-((1-(heneicosane-11-oxy)-1-oxopropan-2-yl)thio)dodecanoate (C6) was obtained.

[0189] The cationic lipid compound C6 was characterized and the results are as follows:

[0190] 1 H NMR (400MHz, CDCl3) δ5.22-5.08(m,1H),4.91-4.82(m,1H),4.39-4.30(m,1H) ,4.27-4.07(m,4H),3.47-3.37(m,2H),3.08-2.83(m,5H),2.75(s,4H),2.57( td,J=7.4,3.6Hz,2H),2.42-2.26(m,6H),1.91-1.82(m,2H),1.63-1.48(m,6H) ),1.42(dd,J=9.4,7.2Hz,3H),1.24(d,J=8.9Hz,48H),0.86(t,J=6.8Hz,9H).

[0191] [M+H] + :827.60.

[0192] Example 18: Synthesis of cationic lipid compound D5

[0193] The operation was similar to that of Example 16, except that 11-heneicosol (4-1) in step 1 was replaced with 13-pentacosol (7-1). Propyl 2-((4-((2-(dimethylamino)ethyl)(methyl)amino)butanoyl)oxy)-3-((1-(pentacosan-13-oxy)-1-oxopropan-2-yl)thio)decanoate (D5) was finally obtained.

[0194] The cationic lipid compound D5 was characterized and the results are as follows:

[0195] 1 H NMR (400MHz, CDCl3) δ5.23-5.08(m,1H),4.91-4.81(m,1H),4.40-4.29(m,1H),4.1 7-4.09(m,1H),4.06-3.70(br,3H),3.47-3.36(m,2H),2.99-2.72(m,5H),2.68(s, 4H),2.55(td,J=7.6,2.6Hz,2H),2.41-2.26(m,6H),1.90-1.77(m,2H),1.64-1.48 (m,6H),1.42(dd,J=9.4,7.2Hz,3H),1.26(d,J=9.2Hz,52H),0.86(t,J=6.8Hz,9H).

[0196] [M+H] + :855.60.

[0197] Example 19: Synthesis of cationic lipid compound E5

[0198] The operation was similar to that of Example 16, except that 11-heneicosyl alcohol (4-1) in step 1 was replaced with 2-hexyl-1-decanol (1-1). Propyl 2-((4-((2-(dimethylamino)ethyl)(methyl)amino)butanoyl)oxy)-3-((1-((2-hexyldecyl)oxy)-1-oxopropan-2-yl)thio)decanoate (E5) was obtained.

[0199] The cationic lipid compound E5 was characterized and the results are as follows:

[0200] 1H NMR (400MHz, CDCl3) δ5.24-5.09(m,1H),4.38-4.27(m,1H),4.17-4.10(m,1H),4.01(d,J=5. 7Hz,2H),3.49-3.40(m,1H),3.25-3.05(br,1H),2.96-2.70(m,2H),2.55-2.37(m,6H),2.36 -2.23(m,12H),1.84-1.74(m,2H),1.68-1.54(m,3H),1.42(d,J=7.4Hz,3H),1.32-1.21(m,36H),0.86(t,J=6.6Hz,9H).

[0201] [M+H] + :729.40.

[0202] Example 20: Synthesis of cationic lipid compound F4

[0203] The operation was similar to that of Example 10, except that N-hydroxyethylpiperazine (2-10) in step 5 was replaced with N,N,N'-trimethylethylenediamine (12-10). 2-((4-((2-(dimethylamino)ethyl)(methyl)amino)butanoyl)oxy)-3-((1-((2-octyldodecyl)oxy)-1-oxoprop-2-yl)thio)propyl octanoate (F4) was finally obtained.

[0204] The cationic lipid compound F4 was characterized, and the results are as follows:

[0205] 1 H NMR (400MHz, CDCl3) δ5.20-5.06(m,1H),4.40-4.28(m,1H),4.16-4.08(m,2H),4.01(d,J=5.7Hz,2H),3.48-3.37(m,2H),3.05-2.76(m,5H),2 .75-2.50(m,6H),2.41-2.26(m,6H),1.91-1.73(m,2H),1.68-1.53(m, 3H), 1.43 (d, J = 7.4Hz, 3H), 1.32-1.17 (m, 40H), 0.86 (t, J = 6.7Hz, 9H).

[0206] [M+H] + :757.60.

[0207] Example 21: Synthesis of cationic lipid compound G4

[0208] The operation was similar to that of Example 12, except that 9-heptadecanol (2-1) in step 1 was replaced with 2-decyltetradecyl-1-ol (11-1). 2-((4-((2-(dimethylamino)ethyl)(methyl)amino)butanoyl)oxy)-3-((1-((2-decyltetradecyl)oxy)-1-oxopropyl-2-yl)thio)octanoate (G4) was finally obtained.

[0209] The cationic lipid compound G4 was characterized, and the results are as follows:

[0210] 1 H NMR (400MHz, CDCl3) δ5.20-4.96(m,1H),4.38-4.26(m,1H),4.16-4.06(m,2H),4.01(d,J=5.7Hz,2H) ,3.48-3.37(m,2H),2.93-2.72(m,5H),2.66-2.48(m,6H),2.40-2.26(m,6H),1.88-1.77(m,2H),1.67 -1.53(m,3H),1.41(t,J=7.7Hz,3H),1.34-1.16(m,48H),0.85(t,J=6.7Hz,9H).

[0211] [M+H]+:813.60.

[0212] Example 22: Synthesis of cationic lipid compound G5

[0213] The operation was similar to that of Example 13, except that 9-heptadecanol (2-1) in step 1 was replaced with 2-decyltetradecyl-1-ol (11-1). Finally, 2-((4-((2-(dimethylamino)ethyl)(methyl)amino)butanoyl)oxy)-3-((1-((2-decyltetradecyl)oxy)-1-oxopropan-2-yl)thio)propyl decanoate (G5) was obtained.

[0214] The cationic lipid compound G5 was characterized, and the results are as follows:

[0215] 1H NMR (400MHz, CDCl3) δ5.22-5.05(m,1H),4.75-4.55(br,1H),4.37-4.25(m,1H),4.16-4.08(m,1H),4.00(d,J=5 .7Hz,2H),3.47-3.38(m,2H),2.94-2.71(m,5H),2.58-2.46(m,6H),2.40-2.25(m,6H),1.87-1.76(m,2H),1.68 -1.53(m,3H),1.41(t,J=7.7Hz,3H),1.26-1.12(m,52H),0.86(t,J=6.8Hz,9H).

[0216] [M+H]+:841.60.

[0217] Example 23: Synthesis of cationic lipid compound G6

[0218] The operation was similar to that of Example 17, except that 11-heneicosyl alcohol (4-1) in step 1 was replaced with 2-decyltetradecyl-1-ol (11-1). 2-((4-((2-(dimethylamino)ethyl)(methyl)amino)butanoyl)oxy)-3-((1-((2-decyltetradecyl)oxy)-1-oxoprop-2-yl)thio)dodecanoic acid propyl ester (G6) was finally obtained.

[0219] The cationic lipid compound G6 was characterized, and the results are as follows:

[0220] 1 H NMR (400MHz, CDCl3) δ5.22-5.06(m,1H),4.36-4.19(m,2H),4.17-4.08(m,1H),4.01(d,J=5.7Hz,2H) ,3.48-3.37(m,2H),2.94-2.65(m,5H),2.52-2.42(m,6H),2.39-2.24(m,6H),1.87-1.76(m,2H),1.68 -1.53(m,3H),1.41(t,J=7.8Hz,3H),1.34-1.16(m,56H),0.86(t,J=6.8Hz,9H).

[0221] [M+H] + :869.60.

[0222] Example 24: Synthesis of cationic lipid compound A7

[0223] The operation was similar to that of Example 1, except that the 2-hexyl-1-decanol (1-1) in step 1 was replaced with 7-tridecanol (24-1), and the 1-(3-(4-methylpiperazin-1-yl)propyl)amino)decane-2-ol (1-10) in step 5 was replaced with 1-((2-hydroxyethyl)amino)decane-2-ol (24-10). 3-((1-(tridecane-7-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butyryl)oxy)octanoate (A7) was finally obtained.

[0224] Cationic lipid compound A7 was characterized, and the results are as follows:

[0225] 1 H NMR (400MHz, CDCl3) δ5.25-5.11(m,1H),4.88(p,J=6.1Hz,1H),4.46-4.32(m ,1H),4.18-4.09(m,1H),3.73-3.58(m,3H),3.43(p,J=7.8Hz,1H),2.94-2.52 (m,7H),2.51-2.35(m,4H),2.31(t,J=7.5Hz,2H),1.87-1.74(m,2H),1.66-1. 49(m,6H),1.43(dd,J=9.0,7.3Hz,4H),1.26(s,36H),0.87(t,J=6.7Hz,12H).

[0226] [M+H] + :774.40.

[0227] Example 25: Synthesis of cationic lipid compound A8

[0228] The operation was similar to that of Example 24, except that octanoyl chloride (1-7) in step 3 was replaced with decanoyl chloride (2-7). Finally, 3-((1-(tridecyl-7-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)decanoic acid propyl ester (A8) was obtained.

[0229] The cationic lipid compound A8 was characterized, and the results are as follows:

[0230] 1H NMR (400MHz, CDCl3) δ5.26-5.11(m,1H),4.88(p,J=6.1Hz,1H),4.46-4.29(m ,1H),4.18-4.09(m,1H),3.75-3.59(m,3H),3.43(p,J=7.8Hz,1H),2.94-2.52 (m,7H),2.51-2.35(m,4H),2.31(t,J=7.6Hz,2H),1.87-1.74(m,2H),1.66-1. 49(m,6H),1.43(dd,J=8.8,7.4Hz,4H),1.26(s,40H),0.87(t,J=6.7Hz,12H).

[0231] [M+H] + :802.40.

[0232] Example 26: Synthesis of cationic lipid compound A9

[0233] The operation was similar to that of Example 24, except that octanoyl chloride (1-7) in step 3 was replaced with dodecanoyl chloride (3-7). Finally, 3-((1-(tridecyl-7-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)dodecanoic acid propyl ester (A9) was obtained.

[0234] The cationic lipid compound A9 was characterized, and the results are as follows:

[0235] 1 H NMR (400MHz, CDCl3) δ5.26-5.10(m,1H),4.88(p,J=6.1Hz,1H),4.45-4.30(m,1H),4.19-4.09( m,1H),3.72-3.54(m,3H),3.43(p,J=7.8Hz,1H),2.94-2.53(m,7H),2.51-2.35(m,4H),2.31(t, J=7.5Hz,2H),1.87-1.74(m,2H),1.66-1.49(m,6H),1.43(dd,J=9.0,7.3Hz,4H),1.26(s,44H),0.87(t,J=6.7Hz,12H).

[0236] [M+H] + :830.60.

[0237] Example 27: Synthesis of cationic lipid compound B7

[0238] The operation was similar to that of Example 24, except that 7-tridecanol (24-1) in step 1 was replaced with 9-heptadecanol (2-1). Finally, 3-((1-(heptadecan-9-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)propyl octanoate (B7) was obtained.

[0239] The cationic lipid compound B7 was characterized and the results are as follows:

[0240] 1 H NMR (400MHz, CDCl3) δ5.25-5.11(m,1H),4.88(p,J=6.3Hz,1H),4.45-4.32(m ,1H),4.19-4.08(m,1H),3.72-3.53(m,3H),3.43(p,J=7.8Hz,1H),2.95-2.53 (m,7H),2.52-2.35(m,4H),2.31(t,J=7.5Hz,2H),1.87-1.76(m,2H),1.66-1. 49(m,6H),1.43(dd,J=9.0,7.3Hz,4H),1.26(s,44H),0.87(t,J=6.7Hz,12H).

[0241] [M+H] + :830.60.

[0242] Example 28: Synthesis of cationic lipid compound B8

[0243] The operation was similar to that in Example 2, except that the N-hydroxyethylpiperazine (2-10) in step 5 was replaced with 1-((2-hydroxyethyl)amino)decane-2-ol (24-10). 3-((1-(heptadecan-9-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butyryl)oxy)propyl decanoate (B8) was finally obtained.

[0244] The cationic lipid compound B8 was characterized and the results are as follows:

[0245] 1H NMR (400MHz, CDCl3) δ5.25-5.10(m,1H),4.88(p,J=6.1Hz,1H),4.46-4.29(m ,1H),4.18-4.09(m,1H),3.75-3.51(m,3H),3.43(p,J=7.8Hz,1H),2.94-2.56 (m,7H),2.55-2.36(m,4H),2.31(t,J=7.5Hz,2H),1.87-1.74(m,2H),1.65-1. 45(m,6H),1.43(dd,J=9.0,7.3Hz,4H),1.26(s,48H),0.87(t,J=6.7Hz,12H).

[0246] [M+H] + :858.60.

[0247] Example 29: Synthesis of cationic lipid compound B9

[0248] The operation was similar to that of Example 26, except that 7-tridecanol (24-1) in step 1 was replaced with 9-heptadecanol (2-1). Finally, 3-((1-(heptadecan-9-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)dodecanoic acid propyl ester (B9) was obtained.

[0249] The cationic lipid compound B9 was characterized and the results are as follows:

[0250] 1 H NMR (400MHz, CDCl3) δ5.25-5.10(m,1H),4.88(p,J=6.2Hz,1H),4.46-4.29(m ,1H),4.17-4.08(m,1H),3.72-3.56(m,3H),3.43(p,J=7.8Hz,1H),2.94-2.50 (m,7H),2.50-2.35(m,4H),2.31(t,J=7.5Hz,2H),1.87-1.76(m,2H),1.64-1. 48(m,6H),1.42(dd,J=9.2,7.3Hz,4H),1.26(s,52H),0.87(t,J=6.7Hz,12H).

[0251] [M+H] + :886.60.

[0252] Example 30: Synthesis of cationic lipid compound C7

[0253] The procedure was similar to that of Example 24, except that 7-tridecanol (24-1) in step 1 was replaced with 11-heneicostanol (4-1). Finally, 3-((1-(heneicosane-11-oxy)-1-oxoprop-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)octanoate (C7) was obtained.

[0254] The cationic lipid compound C7 was characterized and the results are as follows:

[0255] 1 H NMR (400MHz, CDCl3) δ5.26-5.10(m,1H),4.88(p,J=6.3Hz,1H),4.46-4.31(m ,1H),4.20-4.07(m,1H),3.74-3.58(m,3H),3.43(p,J=7.8Hz,1H),2.96-2.50 (m,7H),2.49-2.35(m,4H),2.31(t,J=7.5Hz,2H),1.89-1.75(m,2H),1.64-1. 48(m,6H),1.42(dd,J=9.2,7.3Hz,4H),1.25(s,52H),0.87(t,J=6.7Hz,12H).

[0256] [M+H] + :886.60.

[0257] Example 31: Synthesis of cationic lipid compound D8

[0258] The operation was similar to that of Example 25, except that 7-tridecanol (24-1) in step 1 was replaced with 13-pentacosanol (7-1). Finally, 3-((1-(pentacosane-13-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)propyl decanoate (D8) was obtained.

[0259] The cationic lipid compound D8 was characterized and the results are as follows:

[0260] 1H NMR (400MHz, CDCl3) δ5.25-5.10(m,1H),4.88(p,J=6.2Hz,1H),4.46-4.31(m ,1H),4.19-4.08(m,1H),3.72-3.56(m,3H),3.43(p,J=7.4Hz,1H),2.95-2.50 (m,7H),2.50-2.35(m,5H),2.31(t,J=7.5Hz,2H),1.86-1.75(m,2H),1.65-1. 49(m,6H),1.42(dd,J=9.2,7.3Hz,4H),1.25(s,64H),0.87(t,J=6.8Hz,12H).

[0261] [M+H] + :970.60.

[0262] Example 32: Synthesis of cationic lipid compound E7

[0263] The operation was similar to that of Example 24, except that 7-tridecanol (24-1) in step 1 was replaced with 2-hexyl-1-decanol (1-1). Finally, 3-((1-((2-hexyldecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)octanoic acid propyl ester (E7) was obtained.

[0264] The cationic lipid compound E7 was characterized and the results are as follows:

[0265] 1 H NMR (400MHz, CDCl3) δ5.24-5.10(m,1H),4.45-4.31(m,1H),4.18-4.09(m,1H ),4.03(d,J=5.7Hz,2H),3.72-3.57(m,3H),3.50-3.40(m,1H),2.96-2.51(m ,7H),2.50-2.36(m,5H),2.31(t,J=7.6Hz,2H),1.87-1.77(m,2H),1.69-1.5 5(m,3H),1.48-1.40(m,4H),1.27(d,J=7.7Hz,44H),0.87(t,J=6.5Hz,12H).

[0266] [M+H] + :816.60.

[0267] Example 33: Synthesis of cationic lipid compound F7

[0268] The operation was similar to that of Example 27, except that 9-heptadecanol (2-1) in step 1 was replaced with 2-octyldodecanol (10-1). Finally, 3-((1-((2-octyldodecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)octanoic acid propyl ester (F7) was obtained.

[0269] The cationic lipid compound F7 was characterized and the results are as follows:

[0270] 1 H NMR (400MHz, CDCl3) δ5.25-5.10(m,1H),4.44-4.31(m,1H),4.16-4.08(m, 1H),4.02(d,J=5.7Hz,2H),3.70-3.56(m,3H),3.50-3.41(m,1H),2.95-2. 50(m,7H),2.48-2.34(m,4H),2.31(t,J=7.6Hz,2H),1.84-1.74(m,2H),1. 68-1.55(m,3H),1.45-1.39(m,4H),1.26(s,52H),0.87(t,J=6.7Hz,12H).

[0271] [M+H] + :872.60.

[0272] Example 34: Synthesis of cationic lipid compound F8

[0273] The operation was similar to that of Example 28, except that 9-heptadecanol (2-1) in step 1 was replaced with 2-octyldodecanol (10-1). Finally, 3-((1-((2-octyldodecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)propyl decanoate (F8) was obtained.

[0274] The cationic lipid compound F8 was characterized and the results are as follows:

[0275] 1H NMR (400MHz, CDCl3) δ5.24-5.09(m,1H),4.45-4.31(m,1H),4.18-4.10(m, 1H), 4.03 (d, J = 5.7Hz, 2H), 3.72-3.56 (m, 3H), 3.50-3.40 (m, 1H), 2.95-2. 50(m,7H),2.49-2.35(m,5H),2.31(t,J=7.6Hz,2H),1.87-1.77(m,2H),1. 68-1.55(m,3H),1.48-1.40(m,4H),1.27(s,56H),0.87(t,J=6.5Hz,12H).

[0276] [M+H] + :900.60.

[0277] Example 35: Synthesis of cationic lipid compound F9

[0278] The operation was similar to that of Example 26, except that 7-tridecanol (24-1) in step 1 was replaced with 2-octyldodecanol (10-1). Finally, 3-((1-((2-octyldodecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)dodecanoic acid propyl ester (F9) was obtained.

[0279] The cationic lipid compound F9 was characterized and the results are as follows:

[0280] 1 H NMR (400MHz, CDCl3) δ5.23-5.09(m,1H),4.45-4.29(m,1H),4.19-4.10(m,1H),4.03(d,J=5.7H z,2H),3.71-3.56(m,3H),3.50-3.41(m,1H),2.95-2.50(m,7H),2.49-2.35(m,4H),2.30(t,J= 7.6Hz,2H),1.85-1.74(m,2H),1.68-1.54(m,3H),1.46-1.40(m,4H),1.25(s,60H),0.87(t,J=6.7Hz,12H).

[0281] [M+H] + :928.60.

[0282] Example 36: Synthesis of cationic lipid compound G7

[0283] The operation was similar to that of Example 24, except that 7-tridecanol (24-1) in step 1 was replaced with 2-decyltetradecyl-1-ol (11-1). Finally, 3-((1-((2-decyltetradecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)octanoic acid propyl ester (G7) was obtained.

[0284] The cationic lipid compound G7 was characterized and the results are as follows:

[0285] 1 H NMR (400MHz, CDCl3) δ5.25-5.08(m,1H),4.46-4.29(m,1H),4.20-4.10(m, 1H),4.03(d,J=5.7Hz,2H),3.75-3.56(m,3H),3.50-3.40(m,1H),2.95-2. 50(m,8H),2.49-2.35(m,4H),2.30(t,J=7.6Hz,2H),1.85-1.74(m,2H),1. 68-1.54(m,3H),1.46-1.40(m,4H),1.25(s,60H),0.87(t,J=6.7Hz,12H).

[0286] [M+H] + :928.60.

[0287] Example 37: Synthesis of cationic lipid compound G9

[0288] The operation was similar to that of Example 36, except that octanoyl chloride (1-7) in step 3 was replaced with dodecanoyl chloride (3-7). 3-((1-((2-decyltetradecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)dodecanoic acid propyl ester (G9) was finally obtained.

[0289] The cationic lipid compound G9 was characterized and the results are as follows:

[0290] 1H NMR (400MHz, CDCl3) δ5.24-5.10(m,1H),4.45-4.31(m,1H),4.16-4.09(m, 1H),4.03(d,J=5.7Hz,2H),3.72-3.58(m,3H),3.50-3.40(m,1H),2.95-2. 51(m,7H),2.50-2.35(m,5H),2.30(t,J=7.6Hz,2H),1.85-1.76(m,2H),1. 68-1.55(m,3H),1.46-1.40(m,4H),1.25(s,68H),0.87(t,J=6.7Hz,12H).

[0291] [M+H] + :984.60.

[0292] Example 38: Synthesis of cationic lipid compound H3

[0293] The procedure was similar to that of Example 28, except that 9-heptadecanol (2-1) in step 1 was replaced with n-octanol (38-1). Finally, 3-((1-(octyloxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)propyl decanoate (H3) was obtained.

[0294] The cationic lipid compound H3 was characterized and the results are as follows:

[0295] 1 H NMR (400MHz, CDCl3) δ5.25-5.11(m,1H),4.97-4.87(m,1H),4.46-4.31(m,1H ),4.14(dt,J=12.0,5.8Hz,1H),3.71-3.56(m,3H),3.46-3.37(m,1H),2.97-2 .51(m,7H),2.50-2.35(m,4H),2.31(t,J=7.5Hz,2H),1.87-1.77(m,2H),1.6 5-1.55(m,3H),1.53-1.39(m,6H),1.35-1.19(m,34H),0.87(t,J=6.7Hz,9H).

[0296] [M+H] + :732.40.

[0297] Example 39: Synthesis of cationic lipid compound H10

[0298] The procedure was similar to that of Example 24, except that 7-tridecanol (24-1) in step 1 was replaced with n-octanol (38-1). This yielded 3-((1-(octyloxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)propyl octanoate (H10).

[0299] The cationic lipid compound H10 was characterized and the results are as follows:

[0300] 1 H NMR (400MHz, CDCl3) δ5.24-5.10(m,1H),4.97-4.86(m,1H),4.44-4.32(m,1H ),4.13(dt,J=12.1,5.7Hz,1H),3.71-3.54(m,3H),3.46-3.35(m,1H),2.95-2 .50(m,7H),2.49-2.34(m,4H),2.31(t,J=7.5Hz,2H),1.84-1.74(m,2H),1.6 6-1.54(m,3H),1.53-1.37(m,6H),1.33-1.19(m,30H),0.87(t,J=6.5Hz,9H).

[0301] [M+H] + :704.40.

[0302] Example 40: Synthesis of cationic lipid compound H24

[0303] The procedure was similar to that of Example 24, except that 7-tridecanol (24-1) in step 1 was replaced with n-decanol (40-1). Finally, 3-((1-(decyloxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)octanoate (H24) was obtained.

[0304] The cationic lipid compound H24 was characterized and the results are as follows:

[0305] 1H NMR (400MHz, CDCl3) δ5.24-5.10(m,1H),4.44-4.29(m,1H),4.18-4.05(m,3H),3.72-3.56(m,3H),3.50-3.40(m,1H),2.96-2.51(m,7H),2. 50-2.35(m,4H),2.31(t,J=7.5Hz,2H),1.88-1.74(m,2H),1.69-1.55(m,3H),1.49-1.39(m,5H),1.35-1.11(m,34H),0.87(t,J=6.8Hz,9H).

[0306] [M+H] + :732.40.

[0307] Example 41: Synthesis of cationic lipid compound H18

[0308] The operation is similar to that of Example 38, except that n-octanol (38-1) is replaced by n-decanol (40-1) to finally obtain 3-((1-(decyloxy)-1-oxoprop-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)decanoic acid propyl ester (H18).

[0309] The cationic lipid compound H18 was characterized and the results are as follows:

[0310] 1 H NMR (400MHz, CDCl3) δ5.25-5.10(m,1H),4.45-4.29(m,1H),4.17-4.08(m,3H),3.72-3.57(m,3H),3.50-3.40(m,1H),2.96-2.52(m,7H),2. 50-2.35(m,4H),2.31(t,J=7.5Hz,2H),1.86-1.76(m,2H),1.68-1.55(m,3H),1.53-1.39(m,5H),1.35-1.19(m,38H),0.87(t,J=6.7Hz,9H).

[0311] [M+H] + :760.40.

[0312] Example 42: Synthesis of Cationic Lipid Compound A10

[0313] The operation was similar to that of Example 1, except that 2-hexyl-1-decanol (1-1) in step 1 was replaced with 7-tridecanol (24-1). Finally, 3-((1-(tridecyl-7-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)propyl octanoate (A10) was obtained.

[0314] The cationic lipid compound A10 was characterized, and the results are as follows:

[0315] 1 H NMR (400MHz, CDCl3) δ5.24-5.11(m,1H),4.88(p,J=6.2Hz,1H),4.39-4.28(m,1H),4.19-4.10(m,1H),3.57(s,1H),3.42(p,J=7.1Hz,1H),2.9 4-2.72(m,3H),2.69-2.25(m,21H),1.85-1.70(m,2H),1.67-1.49(m,8 H), 1.42 (dd, J = 10.3, 7.2Hz, 5H), 1.26 (s, 36H), 0.87 (t, J = 6.6Hz, 12H).

[0316] [M+H] + :870.60,[1 / 2M+H] + :436.00.

[0317] Example 43: Synthesis of Cationic Lipid Compound A11

[0318] The operation was similar to that of Example 42, except that octanoyl chloride (1-7) in step 3 was replaced with decanoyl chloride (2-7). Finally, 3-((1-(tridecyl-7-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)propyl decanoate (A11) was obtained.

[0319] The cationic lipid compound A11 was characterized, and the results are as follows:

[0320] 1H NMR (400MHz, CDCl3) δ5.26-5.11(m,1H),4.88(p,J=6.2Hz,1H),4.39-4.28(m,1H),4.19-4.10(m,1H),3.57(s,1H),3.42(p,J=7.1Hz,1H),2.9 4-2.69(m,3H),2.66-2.25(m,21H),1.85-1.69(m,2H),1.67-1.48(m,8 H), 1.42 (dd, J = 10.2, 7.3Hz, 5H), 1.26 (s, 40H), 0.87 (t, J = 6.6Hz, 12H).

[0321] [M+H] + :898.60,[1 / 2M+H] + :450.00.

[0322] Example 44: Synthesis of Cationic Lipid Compound A12

[0323] The operation was similar to that of Example 42, except that octanoyl chloride (1-7) in step 3 was replaced with dodecanoyl chloride (3-7). Finally, 3-((1-(tridecyl-7-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)dodecanoic acid propyl ester (A12) was obtained.

[0324] The cationic lipid compound A12 was characterized, and the results are as follows:

[0325] 1 H NMR (400MHz, CDCl3) δ5.25-5.10(m,1H),4.88(p,J=6.2Hz,1H),4.40-4.28(m,1H),4.18-4.07(m ,1H),3.60(s,1H),3.42(p,J=7.1Hz,1H),2.95-2.72(m,3H),2.71-2.25(m,21H),1.86-1.72(m, 2H),1.71-1.48(m,8H),1.42(dd,J=10.1,7.1Hz,5H),1.25(s,44H),0.87(t,J=6.6Hz,12H).

[0326] [M+H] + :926.60,[1 / 2M+H] + :464.00.

[0327] Example 45: Synthesis of cationic lipid compound B10

[0328] The operation was similar to that of Example 42, except that 7-tridecanol (24-1) in step 1 was replaced with 9-heptadecanol (2-1). Finally, 3-((1-(heptadecan-9-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)propyl octanoate (B10) was obtained.

[0329] The cationic lipid compound B10 was characterized and the results are as follows:

[0330] 1 H NMR (400MHz, CDCl3) δ5.25-5.10(m,1H),4.88(p,J=6.2Hz,1H),4.38-4.28(m,1H),4.17-4.10(m,1H),3.57(s,1H),3.42(p,J=7.1Hz,1H),2.9 4-2.71(m,3H),2.66-2.24(m,21H),1.85-1.70(m,2H),1.68-1.49(m,8 H), 1.42 (dd, J = 10.3, 7.3Hz, 5H), 1.25 (s, 44H), 0.87 (t, J = 6.6Hz, 12H).

[0331] [M+H] + :926.60,[1 / 2M+H] + :464.00.

[0332] Example 46: Synthesis of cationic lipid compound B11

[0333] The operation was similar to that of Example 43, except that 7-tridecanol (24-1) in step 1 was replaced with 9-heptadecanol (2-1). Finally, 3-((1-(heptadecan-9-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)propyl decanoate (B11) was obtained.

[0334] The cationic lipid compound B11 was characterized, and the results are as follows:

[0335] 1H NMR (400MHz, CDCl3) δ5.25-5.09(m,1H),4.88(p,J=6.2Hz,1H),4.38-4.28(m,1H),4.17-4.10(m,1H),3.57(s,1H),3.42(p,J=7.1Hz,1H),2.9 4-2.71(m,3H),2.65-2.24(m,21H),1.85-1.70(m,2H),1.68-1.49(m,8 H), 1.42 (dd, J = 10.3, 7.3Hz, 5H), 1.25 (s, 48H), 0.87 (t, J = 6.6Hz, 12H).

[0336] [M+H] + :954.60,[1 / 2M+H] + :478.00.

[0337] Example 47: Synthesis of Cationic Lipid Compound B12

[0338] The operation was similar to that of Example 46, except that decanoyl chloride (2-7) in step 3 was replaced with dodecanoyl chloride (3-7). 3-((1-(heptadecan-9-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)dodecanoic acid propyl ester (B12) was finally obtained.

[0339] The cationic lipid compound B12 was characterized and the results are as follows:

[0340] 1 H NMR (400MHz, CDCl3) δ5.26-5.10(m,1H),4.88(p,J=6.2Hz,1H),4.38-4.28(m,1H),4.17-4.10(m,1H),3.57(s,1H),3.42(p,J=7.1Hz,1H),2.9 4-2.70(m,3H),2.65-2.24(m,21H),1.85-1.70(m,2H),1.68-1.49(m,8 H), 1.42 (dd, J = 10.3, 7.3Hz, 5H), 1.26 (s, 52H), 0.87 (t, J = 6.6Hz, 12H).

[0341] [M+H] + :982.60,[1 / 2M+H] + :492.00.

[0342] Example 48: Synthesis of Cationic Lipid Compound C10

[0343] The operation was similar to that of Example 42, except that 7-tridecanol (24-1) in step 1 was replaced with 11-heneicosyl (4-1). Finally, 3-((1-(heneicosane-11-oxy)-1-oxopropan-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)octanoate (C10) was obtained.

[0344] The cationic lipid compound C10 was characterized, and the results are as follows:

[0345] 1 H NMR (400MHz, CDCl3) δ5.25-5.10 (m, 1H), 4.87 (dd, J = 12.5, 6.2Hz, 1H), 4.40-4.29 (m,1H),4.18-4.10(m,2H),3.56(s,1H),3.42(p,J=7.3Hz,1H),2.94-2.70(m,3H), 2.68-2.39(m,13H),2.38-2.22(m,7H),1.86-1.72(m,2H),1.70-1.58(m,4H),1.57 -1.49(m,4H),1.41(dd,J=10.6,7.2Hz,5H),1.25(s,52H),0.86(t,J=6.7Hz,12H).

[0346] [M+H] + :982.60,[1 / 2M+H] + :492.00.

[0347] Example 49 Synthesis of Cationic Lipid Compound C11

[0348] The operation was similar to that of Example 43, except that 7-tridecanol (24-1) in step 1 was replaced with 11-heneicosyl (4-1). 3-((1-(heneicosane-11-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)propyl decanoate (C11) was finally obtained.

[0349] The cationic lipid compound C11 was characterized, and the results are as follows:

[0350] 1H NMR (400MHz, CDCl3) δ5.26-5.10 (m, 1H), 4.87 (dd, J = 12.4, 6.2Hz, 1H), 4.41-4.28 (m,1H),4.23-4.05(m,2H),3.56(s,1H),3.42(p,J=7.1Hz,1H),2.94-2.70(m,3H), 2.65-2.34(m,13H),2.33-2.24(m,7H),1.84-1.68(m,2H),1.66-1.56(m,4H),1.56 -1.47(m,4H),1.41(dd,J=10.3,7.4Hz,5H),1.25(s,56H),0.86(t,J=6.6Hz,12H).

[0351] [M+H] + :1010.60,[1 / 2M+H] + :506.00.

[0352] Example 50: Synthesis of cationic lipid compound C12

[0353] The operation was similar to that of Example 44, except that 7-tridecanol (24-1) in step 1 was replaced with 11-heneicostanol (4-1). 3-((1-(heneicosane-11-oxy)-1-oxopropan-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)dodecanoic acid propyl ester (C12) was finally obtained.

[0354] The cationic lipid compound C12 was characterized, and the results are as follows:

[0355] 1 H NMR (400MHz, CDCl3) δ5.25-5.10 (m, 1H), 4.87 (dd, J = 12.4, 6.2Hz, 1H), 4.40-4.28 (m,1H),4.25-4.06(m,2H),3.60(s,1H),3.42(p,J=7.1Hz,1H),2.94-2.70(m,3H), 2.68-2.36(m,13H),2.33-2.24(m,7H),1.85-1.71(m,2H),1.69-1.58(m,4H),1.57 -1.49(m,4H),1.41(dd,J=10.6,7.2Hz,5H),1.25(s,60H),0.86(t,J=6.6Hz,12H).

[0356] [M+H] +:1038.60,[1 / 2M+H] + :520.20.

[0357] Example 51: Synthesis of cationic lipid compound D10

[0358] The same method as Example 42 was used, except that 7-tridecanol (24-1) in Example 1 was replaced with 13-pentacosanol (7-1). Finally, 3-((1-(pentacosan-13-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)propyl octanoate (D10) was obtained.

[0359] The cationic lipid compound D10 was characterized and the results are as follows:

[0360] 1 H NMR (400MHz, CDCl3) δ5.26-5.12(m,1H),4.86(dd,J=12.4,6.2Hz,1H),4.41-4.28(m,1H),4.23 -4.05(m,2H),3.56(s,1H),3.42(p,J=7.1Hz,1H),2.94-2.70(m,3H),2.65-2.34(m,13H),2.33 -2.24(m,7H),1.84-1.68(m,2H),1.66-1.56(m,4H),1.56-1.47(m,4H),1.41(dd,J=10.3,7.4Hz,5H),1.24(s,60H),0.86(t,J=6.6Hz,12H).

[0361] [M+H] + :1038.60,[1 / 2M+H] + :520.00.

[0362] Example 52: Synthesis of cationic lipid compound D12

[0363] The operation was similar to that of Example 47, except that 9-heptadecanol (2-1) in step 1 was replaced with 13-pentacosanol (7-1). Finally, 3-((1-(pentacosan-13-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)dodecanoic acid propyl ester (D12) was obtained.

[0364] The cationic lipid compound D12 was characterized, and the results are as follows:

[0365] 1 H NMR(400MHz, CDCl3) δ5.26-5.11(m,1H),4.86(dd,J=12.4,6.2Hz,1H),4.41-4.28 (m,1H),4.23-4.05(m,2H),3.56(s,1H),3.42(p,J=7.1Hz,1H),2.94-2.70(m,3H), 2.65-2.34(m,13H),2.33-2.24(m,7H),1.84-1.68(m,2H),1.67-1.56(m,4H),1.56 -1.47(m,4H),1.41(dd,J=10.3,7.4Hz,5H),1.25(s,68H),0.86(t,J=6.6Hz,12H).

[0366] [M+H] + :1094.60,[1 / 2M+H] + :548.20.

[0367] Example 53: Synthesis of cationic lipid compound E11

[0368] The operation was similar to that of Example 46, except that 9-heptadecanol (2-1) in step 1 was replaced with 2-hexyl-1-decanol (1-1). Finally, 3-((1-((2-hexyldecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)propyl decanoate (E11) was obtained.

[0369] The cationic lipid compound E11 was characterized, and the results are as follows:

[0370] 1 H NMR (400MHz, CDCl3) δ5.25-5.10(m,1H),4.38-4.27(m,1H),4.18-4.10(m,1H),4.03(d,J=5.6Hz,2H),3.57(s,1H),3.49-3.40(m,2H), 2.96-2.71(m,4H),2.70-2.22(m,20H),1.85-1.70(m,2H),1.68-1.53(m,5H),1.52-1.37(m,5H),1.26(s,48H),0.87(t,J=6.5Hz,12H).

[0371] [M+H] + :940.60,[1 / 2M+H] +:471.00.

[0372] Example 54: Synthesis of cationic lipid compound F10

[0373] The operation was similar to that of Example 42, except that 7-tridecanol (24-1) in step 1 was replaced with 2-octyldodecanol (10-1). Finally, 3-((1-((2-octyldodecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)propyl octanoate (F10) was obtained.

[0374] The cationic lipid compound F10 was characterized, and the results are as follows:

[0375] 1 H NMR (400MHz, CDCl3) δ5.25-5.10(m,1H),4.38-4.27(m,1H),4.18-4.09(m,1H),4.03(d,J=5.6Hz,2H),3.56(s,1H),3.49-3.39(m,2H), 2.95-2.70(m,3H),2.69-2.22(m,21H),1.85-1.70(m,2H),1.68-1.53(m,5H),1.52-1.36(m,5H),1.25(s,52H),0.87(t,J=6.5Hz,12H).

[0376] [M+H] + :968.60,[1 / 2M+H] + :485.00.

[0377] Example 55: Synthesis of cationic lipid compound F11

[0378] The operation was similar to that of Example 43, except that 7-tridecanol (24-1) in step 1 was replaced with 2-octyldodecanol (10-1). Finally, 3-((1-((2-octyldodecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)propyl decanoate (F11) was obtained.

[0379] The cationic lipid compound F11 was characterized, and the results are as follows:

[0380] 1H NMR (400MHz, CDCl3) δ5.23-5.08(m,1H),4.41-4.28(m,1H),4.17-4.09(m,1H),4.03(d,J=5.8Hz,2H),3.64(s,1H),3.50-3.39(m,2H), 2.95-2.80(m,3H),2.78-2.24(m,21H),1.88-1.76(m,2H),1.74-1.53(m,5H),1.50-1.34(m,5H),1.25(s,56H),0.87(t,J=6.5Hz,12H).

[0381] [M+H] + :996.60,[1 / 2M+H] + :499.00.

[0382] Example 56: Synthesis of cationic lipid compound F12

[0383] The operation was similar to that of Example 54, except that octanoyl chloride (1-7) in step 3 was replaced with dodecanoyl chloride (3-7). 3-((1-((2-octyldodecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)dodecanoic acid propyl ester (F12) was finally obtained.

[0384] The cationic lipid compound F12 was characterized, and the results are as follows:

[0385] 1 H NMR (400MHz, CDCl3) δ5.25-5.11(m,1H),4.38-4.27(m,1H),4.18-4.09(m,1H),4.03(d,J=5.6Hz,2H),3.56(s,1H),3.49-3.39(m,2H), 2.95-2.69(m,3H),2.69-2.22(m,21H),1.85-1.70(m,2H),1.68-1.53(m,5H),1.52-1.35(m,5H),1.26(s,60H),0.87(t,J=6.5Hz,12H).

[0386] [M+H] + :1024.60,[1 / 2M+H] + :513.00.

[0387] Example 57: Synthesis of cationic lipid compound G11

[0388] The operation was similar to that of Example 53, except that 2-hexyl-1-decanol (1-1) in step 1 was replaced with 2-decyltetradecyl-1-ol (11-1). Finally, 3-((1-((2-decyltetradecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)propyl decanoate (G11) was obtained.

[0389] The cationic lipid compound G11 was characterized, and the results are as follows:

[0390] 1 H NMR (400MHz, CDCl3) δ5.24-5.08(m,1H),4.38-4.28(m,1H),4.17-4.09(m,1H),4.02(d,J=5.6Hz,2H),3.59(s,1H),3.50-3.41(m,2H), 2.95-2.70(m,3H),2.70-2.25(m,21H),1.87-1.71(m,2H),1.70-1.55(m,5H),1.48-1.38(m,5H),1.25(s,64H),0.87(t,J=6.6Hz,12H).

[0391] [M+H] + :1052.80,[1 / 2M+H] + :527.00.

[0392] Example 58: Synthesis of cationic lipid compound G12

[0393] The operation was similar to that of Example 47, except that 9-heptadecanol (2-1) in step 1 was replaced with 2-decyltetradecyl-1-ol (11-1). Finally, 3-((1-((2-decyltetradecyl)oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(3-(4-methylpiperazin-1-yl)propyl)amino)butanoyl)oxy)dodecanoic acid propyl ester (G12) was obtained.

[0394] The cationic lipid compound G12 was characterized, and the results are as follows:

[0395] 1H NMR (400MHz, CDCl3) δ5.26-5.10(m,1H),4.37-4.28(m,1H),4.20-4.05(m,1H),4.03(d,J=5.6 Hz,2H),3.56(s,1H),3.50-3.40(m,2H),2.95-2.70(m,3H),2.70-2.22(m,21H),1.84-1.70(m, 2H),1.68-1.54(m,5H),1.50-1.38(m,5H),1.26(s,68H),0.87(t,J=6.5Hz,12H).

[0396] [M+H] + :1080.60,[1 / 2M+H] + :541.00.

[0397] Example 59: Synthesis of cationic lipid compound P12

[0398] The operation was similar to that of Example 25, except that 7-tridecanol (24-1) in step 1 was replaced with 2-octen-1-ol (59-1). Finally, 3-((1-(2-octen-1-oxy)-1-oxopropyl-2-yl)thio)-2-((4-((2-hydroxydecyl)(2-hydroxyethyl)amino)butanoyl)oxy)propyl decanoate (P12) was obtained.

[0399] The cationic lipid compound P12 was characterized, and the results are as follows:

[0400] 1 H NMR (400MHz, CDCl3) δ5.85-5.75(m,1H),5.61-5.51(m,1H),5.25-5.10(m,1H),4.57(d,J=6.5Hz,2H), 4.37(ddd,J=25.6,13.5,3.5Hz,1H),4.13(dt,J=10.3,5.1Hz,1H),3.75-3.60(m,3H),3.51-3.41(m,1H ),2.95-2.68(m,4H),2.67-2.45(m,4H),2.40(dd,J=12.3,6.7Hz,2H),2.32(t,J=7.5Hz,2H),2.04(q, J=6.8Hz,2H),1.85(dd,J=14.1,7.0Hz,2H),1.65-1.55(m,3H),1.50-1.20(m,36H),0.91-0.85(m,9H).

[0401] [M+H] +:730.40.

[0402] Example 60: Construction of lipid nanoparticle (LNP) delivery system

[0403] The cationic lipid compound E10, phospholipid DSPC, cholesterol, and DMG-PEG2000 prepared in Example 1 were dissolved in ethanol at a molar ratio of 50 / 10 / 38.5 / 1.5, respectively. Firefly luciferase mRNA (Fluc mRNA) was diluted in citrate buffer (10mM, pH=4). Finally, the mass ratio of cationic lipid to mRNA was set to 10 / 1, and the calculated amount of ethanol phase and aqueous phase solution were quickly mixed at a volume ratio of 1 / 3, and the LNP was prepared by standing for 20 minutes. Finally, 1×PBS was added to the required sample volume to obtain the LNP delivery system. Dynamic light scattering was used to detect the particle size distribution of the LNP delivery system. As shown in Figure 1, five sets of parallel experiments were used, and the measured average particle size was 93.91±0.83nm.

[0404] Examples 61-118 and Comparative Example 1: Construction of Lipid Nanoparticle (LNP) Delivery System

[0405] The LNP delivery systems of Examples 61 to 118 and Comparative Example 1 were obtained in the same manner as in Example 60, except that the cationic lipid compound E10 in Example 60 was replaced by the corresponding compounds in Table 1.

[0406] The LNP delivery system constructed by the above process was tested as follows:

[0407] 1. Determination of the biological toxicity of the LNP delivery system:

[0408] Human ovarian cancer cells IGROV1 were cultured in 1640 medium containing 10% fetal bovine serum, seeded in 96-well plates at a density of 10,000 cells / well, and incubated in a CO2 cell culture incubator for 24 hours. Then, prepared LNPs loaded with Fluc mRNA were added to the cells, with 25 ng of mRNA per well, and incubated for a further 24 hours. Cell viability was determined using an alamarBlue detection kit. After adding the alamarBlue detection reagent, the cells were incubated for another 2 hours and detected using a multifunctional microplate reader with the excitation wavelength set to 530 nm and the emission wavelength set to 590 nm. Cell viability was calculated using cells without LNPs as a reference. When the cell viability was greater than 80%, the LNP delivery system was evaluated as non-cytotoxic and marked as ○. Otherwise, the LNP delivery system was evaluated as cytotoxic and marked as ×.

[0409] 2. Determination of in vitro mRNA delivery efficiency of the LNP delivery system:

[0410] Human ovarian cancer cells IGROV1 were cultured in 1640 medium containing 10% fetal bovine serum, seeded in 96-well plates at a density of 10,000 cells / well, and incubated in a CO2 cell culture incubator for 24 hours. Then, the prepared LNPs loaded with Fluc mRNA were added to the cells, with 25 ng of mRNA per well, and incubation continued. After 24 hours, the cells were analyzed using a multifunctional microplate reader and Bio-Lumi TM The luciferase reporter gene detection kit was used to detect the expression efficiency of Fluc mRNA. 4 ~5*10 4 C; 5*10 4 ~4*10 5 B; greater than 4.0*10 5 It is A.

[0411] Table 1

[0412] From the analysis of the results in the above table, it can be seen that, on the one hand, the cationic lipid compounds of Examples 1 to 59 of the present invention are used to construct LNP delivery systems. Compared with the existing DLin-MC3-DMA cationic lipid, the survival rates of human ovarian cancer cells IGROV1 detected are all greater than 80%, showing lower cytotoxicity.

[0413] On the other hand, the cationic lipid compounds of Examples 1 to 59 of the present invention were used to construct LNP delivery systems, which achieved delivery effects similar to or even better than those of the DLin-MC3-DMA cationic lipids. In particular, it was found that the cationic lipid compounds of the present invention, when using specific amine groups as polar heads, generally exhibited better delivery effects, especially A10, B11, C11, D10, F10, and G12. It is speculated that the specific amine groups of the present invention, when bound to the hydrophobic tail through the connecting structure provided by the present invention, are more conducive to regulating the interaction between the delivered molecules and endogenous cellular compounds, thereby improving the delivery effect.

[0414] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cationic lipid compound or a pharmaceutically acceptable salt thereof, characterized in that: The cationic lipid compound has a structure shown in formula (IA): Wherein, R1 is selected from C3~C 30 straight-chain alkyl groups; R2 is selected from C4~C 30 a straight-chain or branched alkyl group, or R3 is selected from H, methyl or ethyl; L is selected from C1~C 10 alkylene; NR4R5 is selected from any of the following structures: X1 is an O atom, X2 is an O atom, X3 is a S atom, and X4 is an O atom; p is 1.

2. The cationic lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that R1 is selected from any of the following structures:

3. The cationic lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R2 is selected from any of the following structures:

4. The cationic lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein The cationic lipid compound is selected from any one of the following structures:

5. A method for preparing the cationic lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that: The following steps are involved: The compound represented by formula (1) is reacted with the compound represented by formula (2) to obtain the intermediate represented by formula (3); The intermediate represented by formula (3) is reacted with the compound represented by formula (4) to obtain the intermediate represented by formula (5); The intermediate represented by formula (5) is reacted with the compound represented by formula (7) to obtain the intermediate represented by formula (6); The intermediate represented by formula (6) is reacted with the compound represented by formula (8) to obtain the intermediate represented by formula (9); The intermediate represented by formula (9) is reacted with the compound represented by formula (10) to obtain the cationic lipid compound represented by formula (IA); 6. A composition, characterized in that The composition comprises the cationic lipid compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

7. The composition according to claim 13, characterized in that The composition also includes a prophylactic or therapeutic agent, which is a nucleic acid.

8. The composition according to claim 7, characterized in that The mass ratio of the cationic lipid compound to the preventive agent or therapeutic agent in the composition is 2.5:1 to 50:

1.

9. The composition according to claim 16, characterized in that: The nucleic acid is selected from one or more of single-stranded DNA, double-stranded DNA, small interfering RNA, microRNA, Dicer-substrate RNA, messenger RNA, small hairpin RNA, and asymmetric interfering RNA.

10. The composition according to claim 6 or 7, characterized in that The composition also includes neutral phospholipids, steroidal lipids, and pegylated lipids.

11. The composition according to claim 10, characterized in that: The neutral phospholipid is selected from diarachidylphosphatidylcholine, 1,2-behenoyl-phosphatidylcholine, 1-stearoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, 1-stearoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, distearoylphosphatidylethanol Amine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, distearoylphosphatidic acid sodium salt, dipalmitoylphosphatidic acid sodium salt, dimyristoylphosphatidic acid sodium salt, dioleoylphosphatidic acid sodium salt, dilauroylphosphatidic acid sodium salt, distearoylphosphatidylglycerol sodium salt, dipalmitoylphosphatidylglycerol sodium salt, dimyristoylphosphatidylglycerol sodium salt, dioleoylphosphatidylglycerol sodium salt, dilauroylphosphatidylglycerol One or more of sodium oil, sodium distearoylphosphatidylserine, sodium dipalmitoylphosphatidylserine, sodium dimyristoylphosphatidylserine, sodium dioleoylphosphatidylserine, and sodium dilauroylphosphatidylserine; The steroidal lipid is selected from one or more of avenasterol, β-sitosterol, rapeseed sterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, melanosterol, epicholesterol, ergosterol, fuccasterol, hexahydroluminosterol, hydroxycholesterol, lanosterol, luminosterol, alginosterol, sitostanol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, and deoxycholic acid; The PEGylated lipid is selected from one or more of distearoylphosphatidylethanolamine polyethylene glycol, dimyristoylglycerol-rac-methoxy polyethylene glycol 2000, methoxy polyethylene glycol ditetradecyl acetamide, polyethylene glycol-diacylglyceramide, polyethylene glycol-dipalmitoylphosphatidylethanolamine, polyethylene glycol-disterol glycerol, and polyethylene glycol-diacylglyceramide.

12. The composition according to claim 10, characterized in that The molar ratio of the cationic lipid compound to the neutral phospholipid, the steroid lipid and the PEGylated lipid is (45-55):(5-15):(35-45):(0.5-2.0).

13. Use of the cationic lipid compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, or the composition according to any one of claims 6 to 12, in the preparation of a nucleic acid delivery drug.

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