Ionizable lipid as well as preparation method, application, drug carrier and drug composition thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to ionizable lipid and a preparation method and application thereof, a drug carrier and a drug composition. According to the ionizable lipid provided by the invention, hydroxyl is introduced to the head, meanwhile, the overall structure is similar to a cone, the head is small, the tail is large, and through structural improvement cooperation, lipid nanoparticles prepared from the ionizable lipid have higher in-vivo mRNA transfection efficiency, and an unexpected technical effect is achieved. The ionizable lipid provided by the invention has a nitrogen branching structure, and the amino group at the branching center is easy to protonate under the physiological pH condition to form part of positive charges, so that the ionizable lipid can be combined with nucleic acid with negative charges, the loading rate of nucleic acid drugs is improved, and the ionizable lipid is suitable for preparing drug carriers or pharmaceutical compositions.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a class of ionizable lipids and a preparation method, application, drug carrier and drug composition thereof. Background Art

[0002] In recent years, with the development of molecular biology technology, the innovative research and development of mRNA drugs has made unprecedented progress, especially in the fields of vaccines and protein replacement therapies. The core advantage of mRNA drugs is that they can effectively shorten the time for drug development and production, and can provide personalized treatment for a variety of diseases. Compared with traditional vaccines or drugs, mRNA drugs do not need to directly introduce antigens or drug molecules, but achieve therapeutic effects by providing genetic information encoding specific proteins and stimulating the body's immune response. This method shows great potential in responding to emerging infectious diseases, cancers, and genetic diseases.

[0003] However, the effectiveness and safety of mRNA drugs largely depend on the design of the delivery system. The mRNA molecule itself is very unstable in the body and is easily degraded by enzymes, so an effective delivery system is needed to protect the mRNA and deliver it safely to the target cells. The ideal delivery system should not only ensure the stable presence of mRNA in the body, but also improve its uptake and intracellular protein expression capabilities for specific cell populations, ensuring that the mRNA can effectively enter the target cells and be translated into protein.

[0004] Among the many delivery systems, lipid nanoparticles have become the first choice for mRNA drug delivery due to their multiple advantages. Lipid nanoparticles are microvesicles formed by encapsulating drugs in lipid bilayers. They can effectively encapsulate mRNA and protect it from degradation. At the same time, through the fusion of cell membranes, they can increase the cellular uptake rate of mRNA.

[0005] The researchers have synthesized some lipid nanoparticles with good biocompatibility, which can gradually release mRNA in the body and reduce side effects on the body. However, their in vivo mRNA transfection efficiency needs to be further improved to meet the higher requirements of mRNA drugs. Summary of the invention

[0006] The purpose of the present invention is to provide a class of ionizable lipids and a preparation method, application, drug carrier and pharmaceutical composition thereof. The lipid nanoparticles prepared from the ionizable lipids provided by the present invention have higher in vivo mRNA transfection efficiency.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides an ionizable lipid, including a compound of formula A, a stereoisomer of the compound of formula A, a tautomer of the compound of formula A, a solvate of the compound of formula A, an N-oxide of the compound of formula A, a pharmaceutically acceptable salt of the compound of formula A, a deuterated product of the compound of formula A, a prodrug of the compound of formula A, a compound of formula B, a stereoisomer of the compound of formula B, a tautomer of the compound of formula B, a solvate of the compound of formula B, an N-oxide of the compound of formula B, a pharmaceutically acceptable salt of the compound of formula B, a deuterated product of the compound of formula B, a prodrug of the compound of formula B, a compound of formula C, a stereoisomer of the compound of formula C, a tautomer of the compound of formula C, a solvate of the compound of formula C, an N-oxide of the compound of formula C, a pharmaceutically acceptable salt of the compound of formula C, a deuterated product of the compound of formula C, or a prodrug of the compound of formula C;

[0009]

[0010] In formula A, G is HO(CH2)2NCH3CH2CH(OH)CH2-; G1 is an alkylene group or R1-G3-O-CO-G1- is a methyl group as a whole; G2 is an alkylene group; G3 is an alkylene group; R is -H, a straight-chain alkyl group, an acyl group or an ester group; R1 is a branched alkyl group;

[0011] In formula B, G4 is an alkylene group or an ether group; G5 is an alkylene group; G6 is an alkylene group; R2 is a branched alkyl group;

[0012] In formula C, R5 is hydroxyl, R6 is -O-, G7 is C4 alkylene, G8 is C5 alkylene, R3 is alkyl or alcoholic hydroxyl, and R4 is a branched alkyl; or, R5 is H, R6 is -CH2-, G7 is C5 alkylene, G8 is C5 alkylene, R3 is alcoholic hydroxyl, and R4 is a branched alkyl.

[0013] Preferably, the ionizable lipid is any one of the compounds of formula Li-01 to formula Li-47.

[0014] The present invention also provides a method for preparing the ionizable lipid described in the above scheme, comprising the following steps:

[0015] Compound D and compound E are mixed and subjected to deoxygenation reaction to obtain compound F; the structure of compound D is shown in formula D; the structure of compound E is shown in formula E; the structure of compound F is shown in formula F;

[0016]

[0017] The compound F and the compound containing an R group are subjected to a substitution reaction to obtain a compound of formula A; the structure of the compound containing an R group is shown in formula G or formula H;

[0018]

[0019] Compound J and compound K are mixed to perform N-substitution reaction to obtain a compound of formula B; the structure of compound J is shown in formula J; the structure of compound K is shown in formula K;

[0020]

[0021] Compound L and compound M are mixed to perform N-substitution reaction to obtain a compound of formula C; the structure of compound L is shown in formula L; the structure of compound M is shown in formula M;

[0022]

[0023] Or, compound N and compound P are mixed to perform N substitution reaction to obtain a compound of formula C; the structure of compound N is shown in formula N; the structure of compound P is shown in formula P;

[0024]

[0025] In Formula D, Formula E, Formula F, Formula G, Formula H, Formula J, Formula K, Formula L, Formula M, Formula N or Formula P, G, G1, G2, G3, G4, G5, G6, G7, G8, R, R1, R2, R3, R4, R5 and R6 have the same meanings as in Formula A, Formula B or Formula C.

[0026] The present invention also provides the use of the ionizable lipid described in the above scheme or the ionizable lipid obtained by the preparation method described in the above scheme in the preparation of a drug carrier or a drug composition.

[0027] The present invention also provides a drug carrier, comprising an ionizable lipid; the ionizable lipid is the ionizable lipid described in the above scheme or the ionizable lipid obtained by the preparation method described in the above scheme.

[0028] Preferably, the drug carrier is in the form of a preparation including lipid nanoparticles, polymer nanoparticles, lactic acid-glycolic acid copolymer nanoparticles, core-shell nanoparticles, liposomes or micelles.

[0029] The present invention also provides a method for preparing the drug carrier described in the above scheme, comprising the following steps:

[0030] The ionizable lipid and the alcohol solvent are mixed to obtain the drug carrier in the alcohol solvent.

[0031] The present invention also provides a pharmaceutical composition, comprising a drug carrier and a carrier carried by the drug carrier; the drug carrier is the drug carrier described in the above scheme or the drug carrier obtained by the preparation method described in the above scheme; the carrier comprises one or more of a drug reagent and a drug adjuvant.

[0032] The present invention also provides a method for preparing the pharmaceutical composition of the above scheme, comprising the following steps:

[0033] The drug carrier and the alcohol solvent are mixed to obtain an alcohol phase, the carrier and the buffer are mixed to obtain an aqueous phase, and the alcohol phase and the aqueous phase are mixed to perform a replacement reaction under dialysis conditions to obtain a drug composition.

[0034] The present invention also provides the use of the ionizable lipid, drug carrier, drug composition described in the above scheme or the ionizable lipid, drug carrier or drug composition obtained by the preparation method described in the above scheme in the preparation of targeted drugs.

[0035] The present invention provides an ionizable lipid. The ionizable lipid provided by the present invention introduces a hydroxyl group at the head, and the overall structure is similar to a cone, with a small head and a large tail. Through the coordination of structural improvement, the lipid nanoparticles prepared by the ionizable lipid of the present invention have a higher in vivo mRNA transfection efficiency, and an unexpected technical effect is achieved. The lipid nanoparticles prepared by the ionizable lipid of the present invention have a stable nanostructure, can be stored at low temperature for a long time, prolong the shelf life of the drug, and reduce the transportation conditions of the drug.

[0036] The present invention also provides a method for preparing the ionizable lipids described in the above scheme. The preparation method provided by the present invention has a simple and easy synthesis route, and the raw materials are cheap and readily available, which is conducive to industrial production.

[0037] The present invention also provides the use of the ionizable lipid described in the above scheme or the ionizable lipid obtained by the preparation method described in the above scheme in the preparation of a drug carrier or a pharmaceutical composition. The ionizable lipid provided by the present invention has a nitrogen branched structure, wherein the amino group at the branching center is easily protonated under physiological pH conditions to form a partial positive charge, thereby being able to bind to a negatively charged nucleic acid. The ionizable lipid provided by the present invention has an average loading rate of more than 97% for nucleic acid drugs after being prepared into lipid nanoparticles, and is suitable for use in the preparation of a drug carrier or a pharmaceutical composition.

[0038] The present invention also provides a drug carrier. The drug carrier provided by the present invention has high in vivo mRNA transfection efficiency and is suitable for the preparation of drugs.

[0039] The present invention also provides a method for preparing the drug carrier described in the above scheme. The preparation method provided by the present invention has simple steps and is easy to operate.

[0040] The present invention also provides a pharmaceutical composition. The pharmaceutical composition provided by the present invention has a positive charge on its surface, is adsorbed to the negatively charged cell surface through electrostatic interaction, enters the cell by endocytosis, and forms endosomes. The pharmaceutical composition provided by the present invention has high serum stability, strong gene complexing ability, excellent biocompatibility and significant gene transfection effect, and can effectively improve the gene therapy effect of the drug, especially in the gene therapy of nucleic acid drugs.

[0041] The present invention also provides a method for preparing the pharmaceutical composition of the above scheme. The preparation method provided by the present invention has low cost, high efficiency and is suitable for industrial production.

[0042] The present invention also provides the use of the ionizable lipid, drug carrier, pharmaceutical composition described in the above scheme or the ionizable lipid, drug carrier or pharmaceutical composition obtained by the preparation method described in the above scheme in the preparation of targeted drugs. The ionizable lipid, drug carrier or pharmaceutical composition provided by the present invention can effectively improve the therapeutic effect of the drug and is used to target at least one of the heart, liver, spleen, lungs and kidneys, especially in the gene therapy of nucleic acid drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 H of the compound of formula Li-01 1 -NMR;

[0045] Figure 2 is a high-resolution mass spectrum (ESI) of the compound of formula Li-01;

[0046] Figure 3 H of the compound of formula Li-29 1 -NMR;

[0047] Figure 4 is a high resolution mass spectrum (ESI) of the compound of formula Li-29;

[0048] Figure 5 H of the compound of formula Li-42 1 -NMR;

[0049] Figure 6 is a high resolution mass spectrum (ESI) of the compound of formula Li-42;

[0050] Figure 7 H of the compound of formula Li-46 1 -NMR;

[0051] Figure 8 is a high resolution mass spectrum (ESI) of the compound of formula Li-46;

[0052] Fig. 9 H of the compound of formula Li-47 1 -NMR;

[0053] Fig.10 is a high resolution mass spectrum (ESI) of the compound of formula Li-47;

[0054] Fig.11 is the particle size of the lipid nanoparticles synthesized in Example 8;

[0055] Fig.12 is the potential of the lipid nanoparticles synthesized in Example 8;

[0056] Fig.13 The bioluminescence results of mice in vivo and various organs in vitro of the LNP preparation of Fluc-mRNA prepared by ionizable lipids in Example 4;

[0057] Fig.14 The bioluminescence results of mice in vivo and various organs in vitro of the LNP preparation of Fluc-mRNA prepared by ionizable lipids in Example 1;

[0058] Fig.15 The bioluminescence results of mice in vivo and various organs in vitro of the LNP preparation of Fluc-mRNA prepared by ionizable lipids in Example 5;

[0059] Fig.16 The bioluminescence results of mice in vivo and various organs in vitro of the LNP preparation of Fluc-mRNA prepared by ionizable lipids in Example 6;

[0060] Fig.17 The bioluminescence results of mice in vivo and various organs of the LNP preparation of Fluc-mRNA prepared with ionizable lipids in Example 7. DETAILED DESCRIPTION

[0061] The present invention provides an ionizable lipid, including a compound of formula A, a stereoisomer of the compound of formula A, a tautomer of the compound of formula A, a solvate of the compound of formula A, an N-oxide of the compound of formula A, a pharmaceutically acceptable salt of the compound of formula A, a deuterated product of the compound of formula A, a prodrug of the compound of formula A, a compound of formula B, a stereoisomer of the compound of formula B, a tautomer of the compound of formula B, a solvate of the compound of formula B, an N-oxide of the compound of formula B, a pharmaceutically acceptable salt of the compound of formula B, a deuterated product of the compound of formula B, a prodrug of the compound of formula B, a compound of formula C, a stereoisomer of the compound of formula C, a tautomer of the compound of formula C, a solvate of the compound of formula C, an N-oxide of the compound of formula C, a pharmaceutically acceptable salt of the compound of formula C, a deuterated product of the compound of formula C, or a prodrug of the compound of formula C;

[0062]

[0063] In formula A, G is HO(CH2)2NCH3CH2CH(OH)CH2-; G1 is an alkylene group or R1-G3-O-CO-G1- is a methyl group as a whole; G2 is an alkylene group; G3 is an alkylene group; R is -H, a straight-chain alkyl group, an acyl group or an ester group; R1 is a branched alkyl group;

[0064] In formula B, G4 is an alkylene group or an ether group; G5 is an alkylene group; G6 is an alkylene group; R2 is a branched alkyl group;

[0065] In formula C, R5 is hydroxyl, R6 is -O-, G7 is C4 alkylene, G8 is C5 alkylene, R3 is alkyl or alcoholic hydroxyl, and R4 is a branched alkyl; or, R5 is H, R6 is -CH2-, G7 is C5 alkylene, G8 is C5 alkylene, R3 is alcoholic hydroxyl, and R4 is a branched alkyl.

[0066] In the present invention, G1 in formula A, the alkylene group is preferably a C8 alkylene group; the C8 alkylene group preferably includes -(CH2)8-, -CH2CH(CH3)(CH2)5-, -(CH2)2CH(CH3)(CH2)4-, -(CH2)3CH(CH3)(CH2)3-, -CH2C(CH3)2(CH2)4- or -(CH2)2C(CH3)2(CH2)3-.

[0067] In the present invention, G2 in formula A, the alkylene group is preferably a C8 alkylene group; the C8 alkylene group preferably includes -(CH2)8-, -CH2CH(CH3)(CH2)5-, -(CH2)2CH(CH3)(CH2)4-, -(CH2)3CH(CH3)(CH2)3-, -CH2C(CH3)2(CH2)4- or -(CH2)2C(CH3)2(CH2)3-.

[0068] In the present invention, in formula A, G3, the alkylene group is preferably a C1-C2 alkylene group; the C1-C2 alkylene group preferably includes -CH2- or -CH2CH2-.

[0069] In the present invention, in Formula A, the linear alkyl group R is preferably a C1-C10 linear alkyl group; the C1-C10 linear alkyl group preferably includes methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl or nonyl.

[0070] In the present invention, in Formula A, R, the acyl group is preferably an alkyl acyl group; the alkyl acyl group is preferably a C5-C10 alkyl acyl group; the C5-C10 alkyl acyl group is preferably -CO(CH2)6CH3.

[0071] In the present invention, in Formula A, R, the ester group is preferably a C10-C20 ester group; the C10-C20 ester group is preferably CH3(CH2)8COO(CH2)5-.

[0072] In the present invention, in formula A, R1, the branched alkyl is preferably a C5-C20 branched alkyl; the C5-C20 branched alkyl preferably includes

[0073] In the present invention, G4 in formula B, the alkylene group is preferably a C4~C5 alkylene group; the C4~C5 alkylene group preferably includes -(CH2)4-, -CH2CH(CH3)CH2-, -(CH2)5-, -CH2CH2CH(CH3)CH2-, -CH(CH3)CH(CH3)CH2-, or -CH2CH(CH3)2CH2-.

[0074] In the present invention, in G4 in Formula B, the ether group is preferably a C2-C10 ether group; the C2-C10 ether group preferably includes -CH2O(CH2)2- or -(CH2)2O(CH2)2O(CH2)2-.

[0075] In the present invention, G5 in formula B, the alkylene group is preferably a C8 alkylene group; the C8 alkylene group preferably includes -(CH2)8-, -CH2CH(CH3)(CH2)5-, -(CH2)2CH(CH3)(CH2)4-, -(CH2)3CH(CH3)(CH2)3-, -CH2C(CH3)2(CH2)4- or -(CH2)2C(CH3)2(CH2)3-.

[0076] In the present invention, in formula B, G6, the alkylene group is preferably a C1-C2 alkylene group; the C1-C2 alkylene group preferably includes -CH2- or -CH2CH2-.

[0077] In the present invention, in Formula B, R2, the branched alkyl is preferably a C10-C20 branched alkyl; the C10-C20 branched alkyl preferably includes the following structural groups:

[0078]

[0079] In the present invention, when R5 is hydroxyl and R6 is -O-, in G7 of Formula C, the C4 alkylene group preferably includes -(CH2)4- or -CH2CH(CH3)CH2-.

[0080] In the present invention, when R5 is hydroxyl and R6 is -O-, G8 in formula C, the C5 alkylene group preferably includes -(CH2)2CH(CH3)CH2-, -CH(CH3)CH(CH3)CH2-, -CH2CH(CH3)2CH2- or -(CH2)5-.

[0081] In the present invention, when R5 is a hydroxyl group and R6 is -O-, in Formula C, R3, the alkyl group is preferably a C1-C5 alkyl group; the C1-C5 alkyl group preferably includes CH3, CH3CH2 or (CH3)2CH-; the alcoholic hydroxyl group is preferably a C1-C5 alcoholic hydroxyl group; the C1-C5 alcoholic hydroxyl group is preferably HO(CH2)2.

[0082] In the present invention, when R5 is a hydroxyl group and R6 is -O-, R4 in Formula C, the branched alkyl group is preferably a C10-C20 branched alkyl group; the C10-C20 branched alkyl group preferably includes the following structural groups:

[0083]

[0084] In the present invention, when R5 is H and R6 is -CH2-, G7 in formula C, the C5 alkylene group preferably includes -(CH2)2CH(CH3)CH2-, -CH(CH3)CH(CH3)CH2-, -CH2CH(CH3)2CH2- or -(CH2)5-.

[0085] In the present invention, when R5 is H and R6 is -CH2-, G8 in formula C, the C5 alkylene group preferably includes -(CH2)2CH(CH3)CH2-, -CH(CH3)CH(CH3)CH2-, -CH2CH(CH3)2CH2- or -(CH2)5-.

[0086] In the present invention, when R5 is H and R6 is -CH2-, in Formula C, the alcoholic hydroxyl group of R3 is preferably a C1-C3 alcoholic hydroxyl group; the C1-C3 alcoholic hydroxyl group is preferably HO(CH2)2.

[0087] In the present invention, when R5 is H and R6 is -CH2-, R4 in Formula C, the branched alkyl is preferably a C10-C20 branched alkyl; the C10-C20 branched alkyl preferably includes the following structural groups:

[0088]

[0089] In the present invention, the ionizable lipid may be any one of the compounds of formula Li-01 to formula Li-47:

[0090]

[0091]

[0092]

[0093]

[0094] The present invention also provides a method for preparing the ionizable lipid described in the above scheme, comprising the following steps:

[0095] Compound D and compound E are mixed and subjected to deoxygenation reaction to obtain compound F; the structure of compound D is shown in formula D; the structure of compound E is shown in formula E; the structure of compound F is shown in formula F;

[0096]

[0097] The compound F and the compound containing an R group are subjected to a substitution reaction to obtain a compound of formula A; the structure of the compound containing an R group is shown in formula G or formula H;

[0098]

[0099] Compound J and compound K are mixed to perform N-substitution reaction to obtain a compound of formula B; the structure of compound J is shown in formula J; the structure of compound K is shown in formula K;

[0100]

[0101] Compound L and compound M are mixed to perform N-substitution reaction to obtain a compound of formula C; the structure of compound L is shown in formula L; the structure of compound M is shown in formula M;

[0102]

[0103] Or, compound N and compound P are mixed to perform N substitution reaction to obtain a compound of formula C; the structure of compound N is shown in formula N; the structure of compound P is shown in formula P;

[0104]

[0105] In Formula D, Formula E, Formula F, Formula G, Formula H, Formula J, Formula K, Formula L, Formula M, Formula N or Formula P, G, G1, G2, G3, G4, G5, G6, G7, G8, R, R1, R2, R3, R4, R5 and R6 have the same meanings as in Formula A, Formula B or Formula C.

[0106] In the present invention, compound D and compound E are mixed for deoxygenation reaction to obtain compound F. In the present invention, the molar ratio of compound D to compound E is preferably 0.8-1:1-2, specifically 0.8:1.2, 0.8:1.5, 0.8:1.7, 0.8:2, 0.9:1.2, 0.9:1.5, 0.9:1.7, 0.9:2, 1:1.2, 1:1.7 or 1:2.

[0107] In the present invention, the temperature of the deoxygenation reaction is preferably 25-40°C, specifically 25°C, 28°C, 30°C, 33°C, 35°C or 40°C, and the insulation reaction time is preferably 12-48h, specifically 12h, 24h, 36h or 48h; the deoxygenation reaction is preferably carried out under stirring conditions.

[0108] After obtaining compound F, the present invention performs a substitution reaction between the compound F and the compound containing an R group to obtain a compound of formula A. In the present invention, the molar ratio of the compound F to the compound containing an R group is preferably 0.13-1:0.46-3.5, specifically 0.13:0.46, 0.13:1, 0.13:2, 0.13:3, 0.13:3.5, 0.4:0.46, 0.4:1, 0.4:2, 0.4:3, 0.4:3.5, 0.7:0.46, 0.7:1, 0.7:2, 0.7:3, 0.7:3.5, 1:0.46, 1:1, 1:2, 1:3 or 1:3.5.

[0109] In the present invention, the temperature of the substitution reaction is preferably 25-40°C, specifically 25°C, 28°C, 30°C, 33°C, 35°C or 40°C, and the insulation reaction time is preferably 12-48h, specifically 12h, 24h, 36h or 48h; the substitution reaction is preferably carried out under stirring conditions.

[0110] In the present invention, compound J and compound K are mixed to carry out an N-substitution reaction (referred to as the first N-substitution reaction) to obtain a compound of formula B. In the present invention, the molar ratio of compound J to compound K is preferably 0.5-1:0.75-1.5, specifically 0.5:0.75, 0.5:1, 0.5:1.25, 0.5:1.5, 0.75:0.75, 0.75:1, 0.75:1.25, 0.75:1.5, 1:0.75, 1:1, 1:1.25 or 1:1.5.

[0111] In the present invention, the temperature of the first N-substitution reaction is preferably 25-40°C, specifically 25°C, 28°C, 30°C, 33°C, 35°C or 40°C, and the insulation reaction time is preferably 24-48h, specifically 24h, 30h, 36h, 40h, 45h or 48h; the first N-substitution reaction is preferably carried out under stirring conditions.

[0112] In the present invention, compound L and compound M are mixed to carry out an N-substitution reaction (referred to as the second N-substitution reaction) to obtain a compound of formula C. In the present invention, the molar ratio of compound L to compound M is preferably 0.8 to 1:1.2 to 1.5, specifically 0.8:1.2, 0.8:1.3, 0.8:1.4, 0.8:1.5, 0.9:1.2, 0.9:1.3, 0.9:1.4, 0.9:1.5, 1:1.2, 1:1.3, 1:1.4 or 1:1.5.

[0113] In the present invention, the preparation method of the compound L is preferably: mixing compound L-1 and compound L-2 to perform a ring-opening reaction to obtain the compound L; the structure of the compound L-1 is shown in formula L-1; the structure of the compound L-2 is shown in formula L-2;

[0114]

[0115] In Formula L-1 and Formula L-2, G7 and R3 have the same meanings as in Formula A, Formula B or Formula C.

[0116] In the present invention, the molar ratio of compound L-1 to compound L-2 is preferably 0.8-1.5:1.2-2.3, specifically 0.8:1.2, 0.8:1.55, 0.8:1.95, 0.8:2.3, 1:1.2, 1:1.55, 1:1.95, 1:2.3, 1.3:1.2, 1.3:1.55, 1.3:1.95, 1.3:2.3, 1.5:1.2, 1.5:1.55, 1.5:1.95 or 1.5:2.3.

[0117] In the present invention, the preparation method of the compound L-1 is preferably: Compound L-11 and compound L-12 are mixed and subjected to esterification reaction to obtain the compound L-1; the structure of the compound L-11 is shown in formula L-11; the structure of the compound L-12 is shown in formula L-12;

[0118]

[0119] In formula L-11, G7 has the same meaning as in formula L-1.

[0120] In the present invention, the molar ratio of compound L-11 to compound L-12 is preferably 2-10:2.2-11, specifically 2:2.2, 2:3, 2:7, 2:11, 2.4:2.6, 4:2.2, 4:3, 4:7, 4:11, 7:2.2, 7:3, 7:7, 7:11, 10:2.2, 10:3, 10:7 or 10:11.

[0121] In the present invention, the preparation method of the compound L-11 is preferably: Compound L-111 and compound L-112 are mixed to carry out a substitution reaction (referred to as the second substitution reaction) to obtain the compound L-11; the structure of the compound L-111 is shown in formula L-111; the structure of the compound L-112 is shown in formula L-112;

[0122]

[0123] In formula L-111, G7 has the same meaning as in formula L-11.

[0124] In the present invention, the molar ratio of compound L-111 to compound L-112 is preferably 5-20:5-20, specifically 5:5, 5:10, 5:15, 5:20, 10:5, 10:15, 15:5, 15:10, 15:20, 20:5, 20:15 or 20:20.

[0125] In the present invention, the temperature of the second substitution reaction is preferably 25-45°C, specifically 25°C, 30°C, 35°C, 40°C or 45°C, and the insulation reaction time is preferably 12-24h, specifically 12h, 15h, 18h, 21h or 24h; the second substitution reaction is preferably carried out under stirring conditions.

[0126] In the present invention, the temperature of the esterification reaction is preferably 0-40°C, specifically 0°C, 5°C, 10°C, 20°C, 25°C, 30°C or 40°C, and the insulation reaction time is preferably 12-48h, specifically 12h, 18h, 24h, 30h, 36h, 42h or 48h; the esterification reaction is preferably carried out under stirring conditions.

[0127] In the present invention, the temperature of the ring-opening reaction is preferably 25-45°C, specifically 25°C, 30°C, 35°C, 40°C or 45°C, and the insulation reaction time is preferably 12-48h, specifically 12h, 24h, 36h or 48h; the ring-opening reaction is preferably carried out under stirring conditions.

[0128] In the present invention, the temperature of the second N-substitution reaction is preferably 0-40°C, specifically 0°C, 5°C, 10°C, 20°C, 25°C, 30°C or 40°C, and the insulation reaction time is preferably 24-48h, specifically 24h, 30h, 36h, 42h or 48h; the second N-substitution reaction is preferably carried out under stirring.

[0129] The present invention mixes compound N and compound P to carry out N substitution reaction (referred to as the third N substitution reaction) to obtain a compound of formula C. In the present invention, the molar ratio of compound N to compound P is preferably 0.45-5:0.9-10, specifically 0.45:0.9, 0.45:1.5, 0.45:4, 0.45:7, 0.45:10, 0.47:0.94, 0.47:1.5, 0.47:4, 0.47:7, 0.47:10, 1:0.9, 1:1.5, 1:4, 1:7, 1:10, 3:0.9, 3:1.5, 3:4, 3:7, 3:10, 5:0.9, 5:1.5, 5:4, 5:7 or 5:10.

[0130] In the present invention, the preparation method of the compound N is preferably: mixing compound N-1 and compound N-2 to perform an N-substitution reaction (referred to as the fourth N-substitution reaction) to obtain the compound N; the structure of the compound N-1 is shown in formula N-1; the structure of the compound N-2 is shown in formula N-2;

[0131]

[0132] In formula N-2, G8 and R4 have the same meanings as in formula N.

[0133] In the present invention, the molar ratio of compound N-1 to compound N-2 is preferably 1-5:1.5-7.5, specifically 1:1.5, 1:3.5, 1:5.5, 1:7.5, 1.5:2.3, 1.5:3.5, 1.5:5.5, 1.5:7.5, 3:1.5, 3:3.5, 3:5.5, 3:7.5, 5:1.5, 5:3.5, 5:5.5 or 5:7.5.

[0134] In the present invention, the temperature of the fourth N-substitution reaction is preferably 25-70°C, specifically 25°C, 35°C, 45°C, 55°C, 65°C or 70°C, and the insulation reaction time is preferably 12-48h, specifically 12h, 18h, 24h, 30h, 36h or 48h; the fourth N-substitution reaction is preferably carried out under stirring.

[0135] In the present invention, the preparation method of the compound P is preferably: mixing compound P-1 and compound P-2 to carry out a substitution reaction (referred to as the third substitution reaction) to obtain the compound P; the structure of the compound P-1 is shown in formula P-1; the structure of the compound P-2 is shown in formula P-2;

[0136]

[0137] In formula P-2, G7 has the same meaning as in formula P.

[0138] In the present invention, the molar ratio of compound P-1 to compound P-2 is preferably 5 to 20:5.5 to 22, specifically 5:5.5, 5:8.5, 5:10, 5:13, 5:15, 5:17, 5:20, 5:22, 10:5.5, 10:8.5, 10:10, 10:13, 10:15, 10:17, 10:20, 10:22, 15:5.5, 15:8.5, 15:10, 15:13, 15:15, 15:17, 15:20, 15:22, 20:5.5, 20:8.5, 20:10, 20:13, 20:15, 20:17, 20:20 or 20:22.

[0139] In the present invention, the temperature of the third substitution reaction is preferably 25-40°C, specifically 25°C, 30°C, 35°C or 40°C, and the insulation reaction time is preferably 6-18h, specifically 6h, 9h, 12h, 15h or 18h; the third substitution reaction is preferably carried out under stirring conditions.

[0140] In the present invention, the temperature of the third N-substitution reaction is preferably 25-70°C, specifically 25°C, 35°C, 45°C, 55°C, 65°C or 70°C, and the insulation reaction time is preferably 12-48h, specifically 12h, 18h, 24h, 30h, 36h, 45h or 48h; the third N-substitution reaction is preferably carried out under stirring.

[0141] The present invention also provides the use of the ionizable lipid described in the above scheme or the ionizable lipid obtained by the preparation method described in the above scheme in the preparation of a drug carrier or a drug composition.

[0142] The present invention also provides a drug carrier, comprising an ionizable lipid; the ionizable lipid is the ionizable lipid described in the above scheme or the ionizable lipid obtained by the preparation method described in the above scheme.

[0143] In the present invention, the drug carrier preferably also includes a neutral lipid; the neutral lipid preferably includes one or more of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE) and dieucoylphosphatidylcholine (DEPC); the molar ratio of the ionizable lipid to the neutral lipid is preferably not more than 50:5, specifically 50:5, 50:8, 50:10, 50:15, 50:20, 50:25, 50:30, 50:35, 50:40, 50:45 or 50:50.

[0144] In the present invention, the drug carrier preferably also includes a steroid; the steroid preferably includes one or more of cholesterol, ergosterol and vitamin D; the molar ratio of the ionizable lipid to the steroid is preferably not more than 50:10, specifically 50:10, 50:15, 50:18, 50:20, 50:25, 50:30, 50:35, 50:40, 50:45, 50:50, 50:55 or 50:60.

[0145] In the present invention, the drug carrier preferably also includes polyethylene glycol-lipid (PEG-lipid); the PEG-lipid preferably includes one or more of dimethyl glutarate-polyethylene glycol and 1,2-dimyristyl glycerol-polyethylene glycol; the molar ratio of the ionizable lipid to the PEG-lipid is preferably not more than 50:0.01, and specifically can be 50:0.01, 50:0.05, 50:0.08, 50:0.1, 50:0.3, 50:0.5, 50:0.8, 50:1, 50:3, 50:5, 50:8 or 50:10.

[0146] In a specific embodiment of the present invention, the dimethyl glutarate-polyethylene glycol is specifically DMG-PEG2k purchased from Shanghai Avituo Pharmaceutical Technology Co., Ltd.

[0147] In the present invention, the drug carrier preferably has a formulation form including lipid nanoparticles, polymer nanoparticles, lactic acid-glycolic acid copolymer nanoparticles (PLGA nanoparticles), core-shell nanoparticles, liposomes or micelles, and more preferably lipid nanoparticles.

[0148] The present invention also provides a method for preparing the drug carrier described in the above scheme, comprising the following steps:

[0149] The ionizable lipid and the alcohol solvent are mixed to obtain the drug carrier in the alcohol solvent.

[0150] The present invention mixes an ionizable lipid and an alcohol solvent to obtain an ionizable lipid solution. In the present invention, the alcohol solvent is preferably anhydrous ethanol; the mass concentration of the ionizable lipid in the ionizable lipid solution is preferably 1 to 10 mg / mL, specifically 1 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 7 mg / mL, 8 mg / mL or 10 mg / mL.

[0151] In the present invention, when the drug carrier includes a neutral lipid, the method for preparing the drug carrier is preferably: mixing an ionizable lipid, a neutral lipid and an alcohol solvent to obtain the drug carrier in the alcohol solvent.

[0152] In the present invention, when the drug carrier includes a steroid, the method for preparing the drug carrier is preferably: mixing an ionizable lipid, a neutral lipid, a steroid and an alcohol solvent to obtain the drug carrier in the alcohol solvent.

[0153] In the present invention, when the drug carrier includes PEG-lipid, the preparation method of the drug carrier is preferably: mixing ionizable lipids, neutral lipids, steroids, PEG-lipids and alcohol solvents to obtain the drug carrier in the alcohol solvent.

[0154] The present invention also provides a pharmaceutical composition, comprising a drug carrier and a carrier carried by the drug carrier; the drug carrier is the drug carrier described in the above scheme or the drug carrier obtained by the preparation method described in the above scheme; the carrier comprises one or more of a drug reagent and a drug adjuvant.

[0155] In the present invention, the pharmaceutical agent is preferably a therapeutic or preventive agent; the therapeutic or preventive agent preferably includes one or more of small nucleic acids and messenger nucleic acids; the pharmaceutical adjuvant preferably includes one or more of macrolide immunosuppressants and glucocorticoids; the macrolide immunosuppressant is preferably rapamycin; and the glucocorticoid is preferably dexamethasone.

[0156] In the present invention, the mass ratio of the drug carrier to the carrier is preferably 5 to 50:1, specifically 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1 or 50:1.

[0157] In the present invention, the preparation form of the pharmaceutical composition is preferably a nanoparticle preparation, and the average particle size of the nanoparticle preparation is preferably 10 to 150 nm, specifically 10 nm, 30 nm, 50 nm, 70 nm, 90 nm, 110 nm, 130 nm or 150 nm; the polydispersity index of the nanoparticle preparation is preferably ≤0.2.

[0158] The present invention also provides a method for preparing the pharmaceutical composition of the above scheme, comprising the following steps:

[0159] The drug carrier and the alcohol solvent are mixed to obtain an alcohol phase, the carrier and the buffer are mixed to obtain an aqueous phase, and the alcohol phase and the aqueous phase are mixed to perform a replacement reaction under dialysis conditions to obtain a drug composition.

[0160] In the present invention, the alcohol solvent is preferably anhydrous ethanol; the mass concentration of the drug carrier in the alcohol phase is preferably 1-10 mg / mL, specifically 1 mg / mL, 4 mg / mL, 5 mg / mL, 7 mg / mL, 8 mg / mL or 10 mg / mL.

[0161] In the present invention, the buffer is preferably a citrate buffer; the concentration of the citrate buffer is preferably 25-75 mmol / L, specifically 25 mmol / L, 35 mmol / L, 50 mmol / L, 60 mmol / L or 75 mmol / L, and the pH value is preferably 3-5, specifically 4.

[0162] In the present invention, the mass concentration of the carrier in the aqueous phase is preferably 0.5-1.5 mg / mL, specifically 1 mg / mL.

[0163] In the present invention, the volume ratio of the alcohol phase to the water phase is preferably 1:2.5-3.5, specifically 1:3.

[0164] In the present invention, the replacement reaction time is preferably 12 hours. In the present invention, the alcohol in the product is replaced by a phosphate buffer treated with diethyl pyrocarbonate (DEPC) through dialysis.

[0165] The present invention also provides the use of the ionizable lipid, drug carrier, drug composition described in the above scheme or the ionizable lipid, drug carrier or drug composition obtained by the preparation method described in the above scheme in the preparation of targeted drugs.

[0166] In the present invention, the targeted drug is preferably used to target at least one of the heart, liver, spleen, lung and kidney, and more preferably used to target the liver.

[0167] In order to further illustrate the present invention, the scheme of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0168] Example 1 Synthesis of Ionizable Lipid - Formula Li-01 Compound

[0169]

[0170] (1) Synthesis of Compound I

[0171]

[0172] The raw material 1 (40 mmol, 1 eq) was dissolved in 30 mL of dichloromethane, triethylamine (44 mmol, 1.1 eq) was added and stirred for 5 min, and a DCM solution of the raw material 2 (44 mmol, 1 eq) was added dropwise at room temperature and reacted for 5 h; after the reaction, water was added to the mixture and extracted with DCM; the organic phases were combined, dried over anhydrous sodium sulfate, and the organic phases were spin-dried and passed through a column to obtain the raw material 3 (8.1 g, yield 96%); the raw material 3 (10 mmol, 1 eq) and 2-methylaminoethanol (10 mmol, 1 eq) were dissolved in 3 0mL acetonitrile, potassium carbonate (30mmol, 3eq) and potassium iodide (2mmol, 0.2eq) were added at room temperature; after the reaction was completed, the mixture was filtered, the filtrate was dried by rotary evaporation, and the raw material 4 (2.2g, yield 87%) was obtained; the raw material 4 (4mmol, 1eq) was dissolved in 4mol / LHCl / 1,4-Dioxane (6mL) and stirred at room temperature for 10h, and monitored by TLC; after the reaction was completed, the solvent was dried by rotary evaporation to obtain an oily substance, which was dissolved in methanol, and triethylamine was added dropwise to adjust the pH value to 7 to obtain compound I with a yield of 70%.

[0173] (2) Synthesis of Compound II

[0174]

[0175] The raw material 5 (15mmol, 1eq) was dissolved in dichloromethane at room temperature, and ethanol (18mmol, 1.2eq), N,N'-dicyclohexylcarboximide DCC (21mmol, 1.4eq), and 4-dimethylaminopyridine DMAP (6mmol, 0.4eq) were added in sequence. The mixture was reacted at room temperature for 4h. The mixture was filtered, the filtrate was washed with water and extracted with dichloromethane, the organic layers were combined, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure using a rotary evaporator, and the raw material 6 was obtained by column separation and purification; diethyl 1,3-acetonedicarboxylate (10mmol, 1eq) was dissolved in 5mL of anhydrous ethanol, sodium ethoxide (10mmol, 1eq) was added, the temperature was raised to 95°C and stirred for 30min to obtain a mixture; raw material 6 (10mmol, 1eq) was added dropwise to the above mixture to react for 2h; sodium ethoxide (10mmol, 1eq) of ethanol solution; raw material 6 (10mmol, 1eq) was added dropwise to the reaction solution and refluxed for 18h. The obtained raw material 7 was directly used in the experiment without purification; 10mL of ethanol and 10mL of purified water were added, and NaOH (90mmol, 9eq) was added and heated to reflux for 6h; 50mL of water was added, EA was extracted, and the aqueous phase was collected; the pH value of the aqueous phase was adjusted to 1-2 at 0°C with concentrated hydrochloric acid (solid precipitated) to obtain crude product 8; the crude product 8 (5mmol, 1eq) was dissolved in DCM and placed at 0°C; raw material 9 (11mmol, 2.2eq), DMAP (2mmol, 0.4eq) and EDCI (15mmol, 3eq) were added in sequence at room temperature; the mixture was returned to room temperature and stirred for 48h, monitored by TLC, and after the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure, and column separation and purification were performed to obtain compound II with a yield of 50%.

[0176] (3) Synthesis of Compound III

[0177] Compound I (1.2 mmol, 1.5 eq) and compound II (0.8 mmol, 1 eq) were dissolved in 5 mL of DCE, and NaH(BOAc)3 (1.2 mmol, 1.5 eq) and acetic acid (4 mmol, 5 eq) were added at room temperature. The reaction was stirred for 48 h to obtain compound III.

[0178] (4) Synthesis of Li-01 compound

[0179] Compound III (0.13 mmol, 1 eq) was dissolved in 1 mL of DCE, and nonanal (0.46 mmol, 3.5 eq) and NaH(BOAc)3 (0.46 mmol, 3.5 eq) were added at room temperature, and the reaction was stirred for 48 h. After the reaction, the mixture was extracted with ethyl acetate, and the organic layers were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure using a rotary evaporator, and the mixture was purified by column separation (silica gel column, eluent: DCM:MeOH=10:1) to obtain a compound of formula Li-01 as a yellow oily liquid with a yield of 30%.

[0180] By H 1 -NMR and HRMS were used to analyze the product structure, and the results are shown in 1-2; 1 HNMR (400MHz, CDCl3) δ (ppm): 4.89-4.83 (m, 2H), 3.77-3.74 (m, 3H), 3.65 (t, J = 5.4Hz, 2H), 2.70-2.67 (m, 2H), 2.52-2.38 (m, 9H),2.29-2.25(m,5H),1.62-1.59(m,4H),1.52-1.47(m,8H),1.28-1.25(m,70H),0.88-0.85(m,16H); HRMS(ESI)calcd.for C 60 H 121 N2O6[M+H + ]:965.9219,found:965.9220.

[0181] Example 2 Synthesis of Ionizable Lipids - Formula Li-13 Compounds

[0182]

[0183] (1) Synthesis of Compound I-1

[0184] Compound I (5 mmol, 1 eq) was dissolved in DCM (20 mL), cooled to 0°C, 2,6-lutidine (20 mmol, 4 eq) was added, tert-butyldimethylsilyl trifluoromethanesulfonate (15 mmol, 3 eq) was added dropwise and stirred for 18 h. The reaction was monitored by TLC, water was added to quench the reaction, and DCM was used for extraction. The organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure using a rotary evaporator, and the mixture was purified by column separation to obtain compound I-1.

[0185] (2) Synthesis of Compound III-1

[0186] Compound I-1 (2 mmol, 1 eq) and compound II (2.4 mmol, 1.2 eq) were dissolved in DCE, and NaH(BOAc)3 (3 mmol, 1.5 eq) and acetic acid (10 eq, 5 eq) were added at room temperature. The reaction was stirred for 48 h, and the reaction was monitored by TLC. The solvent was removed under reduced pressure using a rotary evaporator, and the mixture was purified by column separation to obtain compound III-1.

[0187] (3) Synthesis of Compound IV

[0188] Compound III-1 (1 mmol, 1 eq) was dissolved in DCM, triethylamine (1.5 mmol, 1.5 eq) was added at 0°C and stirred for 10 min, then hexanoyl chloride (1.2 mmol, 1.2 eq) was added dropwise. The reaction was returned to room temperature and stirred for 5 h, and monitored by TLC. After the reaction was completed, the solvent was removed under reduced pressure using a rotary evaporator, and the mixture was separated and purified by column to obtain compound IV.

[0189] (4) Synthesis of Li-13 compound

[0190] Compound IV (0.5 mmol, 1 eq) was dissolved in DMF, TBAF (0.75 mmol, 1.5 eq) was slowly added dropwise, stirred at room temperature for 2 h, and the reaction was monitored by TLC; after the reaction, it was diluted with 20 mL of Et2O / EtOAc (volume ratio 1:1) solution, the organic layer was separated, and washed with water (3×50 mL); the organic layers were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure using a rotary evaporator, and separated and purified on a silica gel column to obtain a compound of formula Li-13.

[0191] Example 3 Synthesis of Ionizable Lipids - Formula Li-26 Compound

[0192]

[0193] (1) Synthesis of Compound II-1

[0194]

[0195] Diethyl 1,3-acetone dicarboxylate (5 mmol, 1 eq) and raw material 6 (5 mmol, 1 eq) were dissolved in 40 mL of anhydrous ethanol, potassium carbonate (5.5 mmol, 1.1 eq) and potassium iodide (5.5 mmol, 1.1 eq) were added at room temperature, and the reaction temperature was raised to 80°C for 18 h. The obtained raw material 10 was directly used in the experiment without purification; the raw material 10 (5 mmol, 1 eq) was dissolved in ethanol aqueous solution (volume ratio, EtOH:H2O=1:2), NaOH (45 mmol, 9 eq) was added at room temperature and heated to reflux 6h, the mixture was extracted with ethyl acetate, and the pH of the aqueous phase was adjusted to 1-2 with concentrated hydrochloric acid at 0°C (solid precipitated) to obtain a crude product 11; the crude product 11 (3mmol, 1eq) was dissolved in DCM, placed at 0°C, and raw material 9 (4.5mmol, 1.5eq), DMAP (1.2mmol, 0.4eq) and EDCI (9mmol, 3eq) were added in sequence at room temperature, and the mixture was returned to room temperature and stirred for 48h, monitored by TLC, and after the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure, and the compound II-1 was obtained by column separation and purification in a yield of 50%.

[0196] (2) Synthesis of Li-26 compound

[0197] Compound I and compound II-1 were dissolved in DCE, NaH(BOAc)3 (1.5eq) and acetic acid (5eq) were added at room temperature, and the reaction was stirred for 48h and monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure, and column separation and purification were performed to obtain compound Li-26.

[0198] By H 1 -NMR analysis of the product structure, 1 HNMR (400MHz, CDCl3) δ (ppm): 4.89-4.83 (m, 2H), 3.77-3.74 (m, 3H), 3.65 (t, J = 5.4Hz, 2H), 2.70-2.67 (m, 2H), 2.5 2-2.38(m,9H),2.29-2.25(m,5H),1.62-1.59(m,4H),1.52-1.47(m,8H),1.28-1.25(m,70H),0.88-0.85(m,16H).

[0199] Example 4 Synthesis of Ionizable Lipids - Formula Li-29 Compounds

[0200]

[0201] (1) Synthesis of Compound V

[0202]

[0203] The raw material 12 (11 mmol, 1.1 eq) was dissolved in 50 mL of anhydrous ethanol, and the raw material 13 (10 mmol, 1 eq) and EEDQ (12 mmol, 1.2 eq) were added in turn at room temperature, and the reaction temperature was raised to 60°C. After the reaction was completed, the filtrate was dried by rotary evaporation and passed through a column to obtain the raw material 14; the raw material 14 (8.8 mmol, 1 eq) was dissolved in dichloromethane, and 4 mol / L HCl / 1,4-Dioxane was added dropwise. The reaction was continued for 12 h and monitored by TLC. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure to obtain compound V, which was directly used for the next step without purification.

[0204] (2) Synthesis of Compound VI

[0205]

[0206] Under nitrogen protection, raw material 15 (10 mmol, 1 eq) was dissolved in tetrahydrofuran and cooled to 0°C; NaH (11 mmol, 1.1 eq) and LDA (11 mmol, 1.1 eq) were then added, and the mixture was reacted at room temperature for 30 min before raw material 16 (20 mmol, 2 eq) was added, and the temperature was raised to 45°C, and the reaction was monitored by TLC; saturated ammonium chloride was quenched, 1 mol / L HCl was added to adjust the pH value, EA was extracted, the organic phase was collected, dried over anhydrous sodium sulfate, and filtered to obtain raw material 17; under nitrogen protection, Raw material 17 (9.6 mmol, 1 eq) was dissolved in tetrahydrofuran, cooled to 0°C, LAH (24 mmol, 2.5 eq) was slowly added, stirred for 30 min, then returned to room temperature and stirred, and the reaction was monitored by TLC to obtain raw material 18; raw material 18 (7.5 mmol, 1.5 eq) and raw material 19 (5 mmol, 1 eq) were dissolved in DCM, EDCI (7.5 mmol, 1.5 eq) and DMAP (1 mmol, 0.2 eq) were added in sequence, stirred for 24 h, the solvent was spin-dried, and passed through a column to obtain compound VI.

[0207] (3) Synthesis of Li-29 compound

[0208] Compound V (1 mmol, 1 eq) was dissolved in methanol, and triethylamine was added dropwise to adjust the pH value to 7; after the solvent was spin-dried, an acetonitrile solution of compound VI (1.5 mmol, 1.5 eq) was added at room temperature, followed by potassium carbonate (3 mmol, 3 eq) and potassium iodide (0.2 mmol, 0.2 eq) were added in sequence to react; after the reaction was completed, ethyl acetate was used for extraction, the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure using a rotary evaporator, and purified by column separation to obtain a compound of formula Li-29 as a yellow oily liquid.

[0209] By H 1-NMR and HRMS were used to analyze the product structure. Figures 3-4 As shown; 1 HNMR (400MHz, CDCl3) δ (ppm): 4.89-4.83 (m, 2H), 3.77-3.74 (m, 3H), 3.65 (t, J = 5.4Hz, 2H), 2.70-2.67 (m, 2H), 2.56 (br s,3H),2.52-2.38(m,9H),2.29-2.25(m,5H),1.62-1.59(m,4H),1.52-1. 47(m,8H),1.28-1.25(m,70H),0.88-0.85(m,16H); HRMS(ESI)m / z:[M+H] + calcd.forC 57 H 113 N2O8953.8491, found 953.8488.

[0210] Example 5 Synthesis of Ionizable Lipid - Formula Li-42 Compound

[0211]

[0212] (1) Synthesis of Compound VII

[0213] The raw material 25 (20 mmol, 1 eq) was dissolved in chloroform, sodium hydroxide (20 mmol, 1 eq) was added at room temperature and stirred, the reaction temperature was raised to 45°C, and the raw material 26 (20 mmol, 1 eq) was added dropwise, and the reaction was stirred for 24 h; after the reaction, the mixture was washed with water, extracted with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure using a rotary evaporator, and the raw material 27 was obtained by column separation and purification; the raw material 27 (2.4 mmol, 1 eq) was dissolved in DCM, DMAP (3.6 mmol, 1.5 eq) was added ) was added in batches of p-nitrophenyl chloroformate (2.9 mmol, 1.2 eq) and stirred at room temperature for 3 h; raw material 28 (β-citronellol, 2.6 mmol, 1.1 eq) was added and stirred at room temperature for 12 h, then DCM was added to dilute, washed with brine, the organic phase was collected, dried over anhydrous sodium sulfate, concentrated and passed through a column to obtain raw material 29; raw material 29 (1.3 mmol, 1 eq) and raw material 30 (ethylamine, 1.95 mmol, 1.5 eq) were dissolved in anhydrous ethanol and stirred at room temperature for 18 h. The solvent was spin-dried and separated by column chromatography to obtain compound VII.

[0214] (2) Synthesis of Li-42 compound

[0215] Compound VI (1.2 mmol, 1.5 eq) and compound VII (0.8 mmol, 1 eq) were dissolved in acetonitrile, potassium carbonate (2.4 mmol, 3 eq) and iodomethane (0.32 mmol, 0.4 eq) were added at room temperature, the reaction temperature was raised to 70°C, and the reaction was continued for 24 hours; the solvent was removed under reduced pressure using a rotary evaporator, and the mixture was separated and purified by column to obtain a compound of formula Li-42.

[0216] By H 1 -NMR and HRMS were used to analyze the product structure. Figures 5-6 As shown; 1 HNMR(400MHz, CDCl3)δ(ppm):5.06-5.02(m,1H),4.86-4.79(m,1H),4.14-4.06(m,4H),3.77-3.75(m,1H),3.70(br s,1H),3.43(t,J=6.6Hz,2H),3.37(d,J=4.8Hz,2H),2.63(dd,J=13.2,7.2Hz, 1H),2.55-2.47(m,2H),2.45-2.40(m,2H),2.24(t,J=7.4Hz,2H),2.00-1.87( m,1H),1.68-1.44(m,13H),1.42-1.37(m,9H),1.33-1.26(m,23H),0.99(t,J= 7.2Hz,3H),0.87(d,J=6.6Hz,3H),0.83(t,J=6.8Hz,6H); HRMS(ESI)m / z:[M+H] + calcd.for C 40 H 78 NO7684.5773,found:696.5775.

[0217] Example 6 Synthesis of Ionizable Lipid - Formula Li-46 Compound

[0218]

[0219] (1) Synthesis of raw material 32

[0220] The raw material 31 (5 mmol, 1 eq) was dissolved in 20 mL of DCM, and DMAP (7.5 mmol, 1.5 eq) was added. p-Nitrophenyl chloroformate (6 mmol, 1.2 eq) was added in batches, and the mixture was stirred at room temperature for 3 h. The raw material 28 (β-citronellol, 5.5 mmol, 1.1 eq) was added, and the mixture was stirred at room temperature for 12 h. DCM was added to dilute the mixture, and the mixture was washed with brine. The organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and passed through a column to obtain the raw material 32.

[0221] (2) Synthesis of Compound VIII

[0222] The raw material 33 (2.3 mmol, 1.5 eq) and compound VI (1.5 mmol, 1 eq) were dissolved in acetonitrile, potassium carbonate (3 mmol, 3 eq) and iodomethane (0.4 mmol, 0.4 eq) were added at room temperature, the reaction temperature was raised to 70°C, the reaction was carried out for 24 hours, the solvent was removed under reduced pressure using a rotary evaporator, and the mixture was separated and purified by a column to obtain compound VIII.

[0223] (3) Synthesis of Li-46 compound

[0224] Compound VIII (0.47 mmol, 1 eq) and raw material 32 (0.94 mmol, 2 eq) were dissolved in acetonitrile, potassium carbonate (1.41 mmol, 3 eq) and iodomethane (0.19 mmol, 0.4 eq) were added at room temperature, the reaction temperature was raised to 70°C, the reaction was continued for 24 hours, the solvent was removed under reduced pressure using a rotary evaporator, and the mixture was separated and purified by a column to obtain a compound of formula Li-46.

[0225] By H 1 -NMR and HRMS were used to analyze the product structure. Figures 7-8 As shown; 1 H NMR (400MHz, CDCl3) δ (ppm): 5.06-5.04 (m, 1H), 4.17-4.09 (m, 4H), 3.94 (d, J = 5.8Hz, 2H), 3.72 (t, J = 5.0Hz, 2H), 2.84 (t, J = 5.0Hz, 2H), 2.7 5-2.71(m,4H),2.30(t,J=7.4Hz,2H),2.02-1.91(m,2H),1.71-1.54(m,17H),1.48-1.18(m,36H),0.90-0.84(m,9H); HRMS(ESI)m / z:[M+H] + calcd.for C 42 H 82 NO6696.6137, found 696.6130.

[0226] Example 7 Synthesis of Ionizable Lipid - Formula Li-47 Compound

[0227] The preparation method of this embodiment is the same as that of embodiment 6, except that: compound VI-1 and raw material 33 are subjected to substitution reaction to obtain intermediate VIII-1, and the intermediate VIII-1 is reacted with raw material 32 to obtain compound of formula Li-47;

[0228]

[0229] By H 1 -NMR and HRMS were used to analyze the product structure. Figures 9-10 As shown; 1 HNMR (400MHz, CDCl3) δ (ppm): 5.06-5.02 (m, 1H), 4.85-4.79 (m, 1H), 4.14-4.07 (m, 4H), 3.56 (d, J = 5.0Hz, 2H), 3.35 (br s,1H),2.64(t,J=5.2Hz,2H),2.54-2.51(m,3H),2.31-2.29(m,2H),1.96-1.91(m,2H),1.70-1 .52(m,12H),1.47-1.42(m,10H),1.33-1.11(m,22H),0.88-0.82(m,9H); HRMS(ESI)m / z:[M+H] + calcd.for C 37 H 32 NO6626.5360, found 626.5365.

[0230] Example 8 Synthesis of lipid nanoparticles

[0231] The ionizable lipids synthesized in Examples 1 and 4 to 7 of the present invention were dissolved in anhydrous ethanol with DSPC, cholesterol, and DMG-PEG2k (Shanghai Avituo Pharmaceutical Technology Co., Ltd.) at a specific molar ratio (as shown in Table 1) as the ethanol phase; mRNA was diluted in a citrate buffer solution with a pH value of 4 and a concentration of 50 mmol / L as the aqueous phase; the ethanol phase and the aqueous phase were mixed at a volume ratio of 1:3 using a quantitative mixing device; the resulting product was then placed in a dialysis bag for overnight dialyzation so that the ethanol in the product was fully replaced by a DEPC-treated PBS solution, and stored in a refrigerator at 4°C; the product was concentrated to 300 to 400 μL using an ultrafiltration tube (Amiconultra, Millipore).

[0232] Table 1 Raw material dosage of Example 8

[0233]

[0234] The Quant-it Ribogreen RNA quantitative detection kit (Thermo Fisher Scientific) was used to prepare RNA content standard working curve samples and test samples in a 96-well plate according to the manufacturer's instructions. The light absorption value of the samples at a specific wavelength was detected using an infinite 200 microplate reader (Tecan) to determine the ability of the nanomaterial to encapsulate mRNA. The particle size and shear surface potential (Zetapotential) of lipid nanoparticles were measured by light scattering and other methods using a ZetasizerPro (Marvern). The results are shown in Figure 2. Figures 11-12 shown.

[0235] according to Figures 11-12 It can be seen that the lipid nanoparticles of the present invention have higher uniformity (lower polydispersity coefficient) and more ideal surface electrical properties.

[0236] The apparent pKa of LNP formulations is related to the delivery efficiency of LNP to nucleic acids in vivo. The apparent pKa of each formulation was determined using a fluorescence assay based on 2-(p-tolyl)-6-naphthalenesulfonic acid (TNS). LNP formulations containing ionizable lipids / DSPC / cholesterol / DMG-PEG (50 / 10 / 38.5 / 1.5 mol %) were prepared as described above; TNS was made into a 300 μmol / L distilled water stock solution; the LNP formulation was diluted to 0.1 mg / mL of total lipid in 3 mL of buffer solution, which contained 50 mmol / L sodium citrate, 50 mmol / L sodium phosphate, 50 mmol / L sodium borate and 30 mmol / L sodium chloride, and its pH value was 3-9; TNS solution was added to a final concentration of 0.1 mg / mL, and after vortex mixing, the fluorescence intensity was measured at room temperature in a Molecular Devices Spectramax iD3 spectrometer using excitation wavelengths of 325 nm and 435 nm. The fluorescence data were subjected to a sigmoid best fit analysis and the pKa values ​​were measured at pH values ​​that reached half of the maximum fluorescence intensity. The results are shown in Table 2.

[0237] Table 2 Characterization data of lipid nanoparticles

[0238] mRNA-LNP samples Ionizable lipid structures Particle size (nm) Zeta potential (mV) PDI Encapsulation efficiency 1 Li-01 85.61 -2.794 0.0097 99.13 2 Li-29 109.6 -5.211 0.1015 99.10 3 Li-42 133.60 -7.253 0.0844 99.05 4 Li-46 88.44 -4.176 0.0742 99.82 5 Li-47 104.4 -4.118 0.0488 89.02

[0239] It can be seen from Table 2 that the lipid nanoparticles of the present invention have higher uniformity (lower polydispersity coefficient) and more ideal surface electrical properties, and at the same time have a higher encapsulation efficiency for the carrier.

[0240] Test animal studies

[0241] Lipid nanoparticles encapsulating firefly-luciferase mRNA were injected into 6-8 week old female C57BL / 6 mice (Beijing Huafukang Technology Co., Ltd.) via tail vein injection at a dose of 8 μg / mouse (100 μL). Luciferase substrate was injected at specific time points (6 h and 24 h) after administration, and the bioluminescence intensity in different tissues and organs was detected by in vivo imaging and in vitro imaging (IVIS Lumina, PerkinElmer). At the same time, the ionizable lipid in the lipid component of the nanomaterial was replaced with SM102 (Shanghai Aiweituo Pharmaceutical Technology Co., Ltd.) as a positive control. The results are shown in Figure 2. Figures 13 to 17 shown.

[0242] according to Figures 13 to 17 It can be seen that the lipid nanoparticles prepared with the ionizable lipids provided by the present invention have a higher in vivo liver transfection efficiency. Compared with the control group, obvious bioluminescence can be observed in both in vivo imaging and in vitro organ imaging obtained after dissecting the experimental animals.

[0243] As can be seen from the above examples, the lipid nanoparticles prepared by the ionizable lipids provided by the present invention have a higher in vivo mRNA transfection efficiency. The lipid nanoparticles prepared by the ionizable lipids of the present invention have a stable nanostructure and can be stored at low temperatures for a long time, thereby extending the shelf life of the drug and reducing the transportation conditions of the drug.

[0244] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An ionizable lipid, characterized in that Including compounds of formula A, stereoisomers of compounds of formula A, tautomers of compounds of formula A, solvates of compounds of formula A, N-oxides of compounds of formula A, pharmaceutically acceptable salts of compounds of formula A, deuterated compounds of compounds of formula A, prodrugs of compounds of formula A, compounds of formula B, stereoisomers of compounds of formula B, tautomers of compounds of formula B, solvates of compounds of formula B, N-oxides of compounds of formula B, pharmaceutically acceptable salts of compounds of formula B, deuterated compounds of compounds of formula B, prodrugs of compounds of formula B, compounds of formula C, stereoisomers of compounds of formula C, tautomers of compounds of formula C, solvates of compounds of formula C, N-oxides of compounds of formula C, pharmaceutically acceptable salts of compounds of formula C, deuterated compounds of compounds of formula C or prodrugs of compounds of formula C; In formula A, G is HO(CH2)2NCH3CH2CH(OH)CH2-; G1 is an alkylene group or R1-G3-O-CO-G1- is a methyl group as a whole; methyl group; G2 is an alkylene group; G3 is an alkylene group; R is -H, a straight-chain alkyl group, an acyl group or an ester group; R1 is a branched alkyl group; In formula B, G4 is an alkylene group or an ether group; G5 is an alkylene group; G6 is an alkylene group; R2 is a branched alkyl group; In formula C, R5 is hydroxyl, R6 is -O-, G7 is C4 alkylene, G8 is C5 alkylene, R3 is alkyl or alcoholic hydroxyl, and R4 is a branched alkyl; or, R5 is H, R6 is -CH2-, G7 is C5 alkylene, G8 is C5 alkylene, R3 is alcoholic hydroxyl, and R4 is a branched alkyl.

2. The ionizable lipid according to claim 1, characterized in that The ionizable lipid is any one of the compounds of formula Li-01 to formula Li-47:

3. The method for preparing the ionizable lipid according to any one of claims 1 to 2, characterized in that: The following steps are involved: Compound D and compound E are mixed and subjected to deoxygenation reaction to obtain compound F; the structure of compound D is shown in formula D; the structure of compound E is shown in formula E; the structure of compound F is shown in formula F; The compound F and the compound containing an R group are subjected to a substitution reaction to obtain a compound of formula A; the structure of the compound containing an R group is shown in formula G or formula H; Compound J and compound K are mixed and subjected to N-substitution reaction to obtain a compound of formula B; the structure of compound J is shown in formula J; the structure of compound K is shown in formula K; Compound L and compound M are mixed to perform N-substitution reaction to obtain a compound of formula C; the structure of compound L is shown in formula L; the structure of compound M is shown in formula M; Or, compound N and compound P are mixed to perform N substitution reaction to obtain a compound of formula C; the structure of compound N is shown in formula N; the structure of compound P is shown in formula P; In Formula D, Formula E, Formula F, Formula G, Formula H, Formula J, Formula K, Formula L, Formula M, Formula N or Formula P, G, G1, G2, G3, G4, G5, G6, G7, G8, R, R1, R2, R3, R4, R5 and R6 have the same meanings as in Formula A, Formula B or Formula C.

4. Use of the ionizable lipid according to any one of claims 1 to 2 or the ionizable lipid obtained by the preparation method according to claim 3 in the preparation of a drug carrier or a pharmaceutical composition.

5. A drug carrier, comprising an ionizable lipid; the ionizable lipid is the ionizable lipid according to any one of claims 1 to 2 or the ionizable lipid obtained by the preparation method according to claim 3.

6. The drug carrier according to claim 5, characterized in that The drug carrier may be in the form of lipid nanoparticles, polymer nanoparticles, lactic acid-glycolic acid copolymer nanoparticles, core-shell nanoparticles, liposomes or micelles.

7. The method for preparing the drug carrier according to any one of claims 5 to 6, characterized in that: The following steps are involved: The ionizable lipid and the alcohol solvent are mixed to obtain the drug carrier in the alcohol solvent.

8. A pharmaceutical composition, comprising a drug carrier and a carrier carried by the drug carrier; the drug carrier is the drug carrier according to any one of claims 5 to 6 or the drug carrier prepared by the method according to claim 7; the carrier comprises one or more of a drug reagent and a drug adjuvant.

9. The method for preparing the pharmaceutical composition according to claim 8, characterized in that: The following steps are involved: The drug carrier and the alcohol solvent are mixed to obtain an alcohol phase, the carrier and the buffer are mixed to obtain an aqueous phase, and the alcohol phase and the aqueous phase are mixed to perform a replacement reaction under dialysis conditions to obtain a drug composition.

10. Use of the ionizable lipid according to any one of claims 1 to 2, the drug carrier according to any one of claims 5 to 6, the pharmaceutical composition according to claim 8, or the ionizable lipid obtained by the preparation method according to claim 3, the drug carrier obtained by the preparation method according to claim 7, or the pharmaceutical composition obtained by the preparation method according to claim 9 in the preparation of targeted drugs.

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