Ionizable lipid compound as well as preparation method and application thereof

Abstract

The invention provides an ionizable lipid compound as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The ionizable lipid compound provided by the invention is a compound with a formula (1), or a salt, a stereoisomer and a tautomer thereof, belongs to a single-tail-chain multi-head lipid, has good surface activity and safety, and can be used for a lipid composition for delivering a therapeutic agent. The prepared lipid nanoparticles can show good effectiveness and safety in cells and in animal bodies. # imgabs0 #

Description

[0001] This is a divisional application of a Chinese patent with the application number 202410079369X, the application date of January 19, 2024, and the invention title of "Ionizable Lipid Compounds and Their Preparation Methods and Applications". The entire content is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biomedical technologies, and specifically relates to ionizable lipid compounds and their preparation methods and applications. Background Art

[0003] In recent years, nucleic acids have attracted much attention as multiple gene therapy products have been used in various applications. Gene therapy can be divided into three main approaches: 1) editing mutant genes using CRISPR Cas technology; 2) upregulating gene expression by inserting functional gene copies, using molecules including DNA plasmids (pDNA), minicircle DNA (mcDNA), synthetic mRNA, circular RNA, and self-amplifying RNA (saRNA); 3) downregulating gene expression using molecules such as small interfering RNA (siRNA), antisense oligonucleotides (ASO), short hairpin RNA (shRNA), and microRNA (miRNA).

[0004] Nucleic acids are macromolecular substances with a large number of negative charges, high molecular weights, and hydrophilicity. They have poor permeability across cell membranes and low stability in vivo, and are easily and rapidly cleared by the host. Therefore, delivering nucleic acids to the active sites within cells is the most challenging part of gene therapy. Especially when delivering RNA molecules, since RNA molecules usually have short-term activity and low retention rates within cells, more frequent drug administration is required. Thus, the toxicity of the payload and the vector are two very important metrics. The vector itself needs to overcome extracellular and intracellular barriers, provide protection against nuclease activity in the blood, enhance and assist cell uptake, and promote endosomal escape after entering the cell.

[0005] Currently, non-viral vectors for nucleic acid delivery cover a variety of types, such as lipids, polymers, peptides, proteins, and inorganic materials. Lipid nanoparticles are considered to be one of the most advanced non-viral vectors due to their safety, flexibility, and high efficiency. For example, the first approved siRNA drug, Patisiran, and the first mRNA vaccine both use lipid nanoparticles as their carriers. Traditional lipid nanoparticle formulations for nucleic acid drug delivery usually contain four main components, namely ionizable lipids (or cationic lipids), helper lipids, PEG lipids, and cholesterol. These components each play an important role in the delivery process. For example, the structure and function of ionizable lipids are particularly important. Their charge properties determine their ability to bind to negatively charged nucleic acid drugs. This binding not only helps protect the nucleic acid from degradation but also neutralizes the negative charge of the nucleic acid, thus enabling the successful delivery of the nucleic acid; phospholipids, as "helper lipids", help improve endosomal release and transfection efficiency; cholesterol helps enhance stability; while PEG lipids promote the formation of homogeneous lipid nanoparticles, improving stability and circulation time by preventing interactions with blood components and clearance. The ratio of these components is crucial for the delivery effect and can be adjusted and customized according to the needs of delivering different nucleic acids (such as siRNA and mRNA). It is worth mentioning that ionizable lipids are a new type of functional material with pH-responsive properties. In a low pH environment, protonation occurs, making the lipid more likely to interact with the negatively charged endosomal membrane, thus promoting the fusion process of endosomal disruption and nucleic acid release, and then achieving efficient delivery. Therefore, the structure and function of ionizable lipids play very important roles in both delivery and lysosomal escape, so their design and optimization are crucial.

[0006] Based on this, the present invention provides an ionizable lipid compound, its preparation method and application. The ionizable lipid compound provided by the present invention belongs to the single-tailed multi-headed lipid class and has good safety. It can be used to prepare a lipid composition for delivering a therapeutic agent, and the lipid nanoparticles prepared therefrom can exhibit good effectiveness and safety in cells and in animals. Summary of the Invention

[0007] Aiming at the problems existing in the prior art, the present invention provides an ionizable lipid compound, its preparation method and application. The ionizable lipid compound provided by the present invention belongs to the single-tailed multi-headed lipid class and has good safety. It can be used to prepare a lipid composition for delivering a therapeutic agent, and the lipid nanoparticles prepared therefrom can exhibit good effectiveness and safety in cells and in animals.

[0008] To achieve the above object, in the first aspect, the present invention provides an ionizable lipid compound, which is a compound of formula (1), or its salt, stereoisomer, tautomer:

[0009]

[0010] wherein A1 is NH or O;

[0011] R a is selected from C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 cycloalkyl;

[0012] The C6-C 24 alkyl and alkenyl are in straight-chain or branched-chain structures;

[0013] R b and R c are each independently selected from alkylamines;

[0014] The alkylamine is wherein, R a ’ is C1-C 12 alkyl, and the R b ’ and R b ” are each independently selected from H, C1-C6 alkylamines, R c ” is selected from C1-C6 alkyl which is unsubstituted or substituted with an amino group, and R c ”’ is H,

[0015] provided that when A1’ is -NH-CO- or -CO-O-, R c ’ is C1-C6 alkyl; when A1’ is -CO-, R c ’ does not exist.

[0016] In a second aspect, the present invention provides an ionizable lipid compound, and the ionizable lipid compound is a compound of formula (1), or a salt, stereoisomer, tautomer thereof:

[0017]

[0018] R a is selected from C6-C 24 alkyl alcohols; the C6-C 24 alkyl alcohols are in straight-chain or branched-chain structures; R b and R c are each independently selected from alkylamines;

[0019] The alkylamine is wherein, R a ’ is C1-C 12 alkyl, and the R b ’ and R b”each independently selected from H, C1-C6 alkylamines, R c ”selected from unsubstituted or amino-substituted C1-C6 alkyl, R c ”’ is H,

[0020] provided that when A1’ is -NH-CO- or -CO-O-, R c ’ is C1-C6 alkyl; when A1’ is -CO-, R c ’ does not exist.

[0021] In a preferred embodiment, provided that when R a is a straight-chain C6-C 24 alkyl, A1 is NH, and at the same time R b ’ and R b ”each independently selected from R c ”selected from unsubstituted or amino-substituted C1-C6 alkyl, R c ”’ is H, When R a is C6-C 24 alkyl alcohol, A1 is not O.

[0022] In a preferred embodiment, the condition is:

[0023] When R a is a branched C6-C 24 alkyl, A1 is NH, and R b and R c each independently selected from alkylamines.

[0024] In a preferred embodiment, R a is selected from the following compound structures:

[0025]

[0026] In a preferred embodiment, R b and R c are selected from the following compound structures:

[0027]

[0028] R d is selected from C1-C6 alkanes or cycloalkanes.

[0029] In a preferred embodiment, the compound of formula (1) is selected from at least one of the following compounds:

[0030]

[0031]

[0032]

[0033]

[0034] In a third aspect, the present invention provides a method for preparing the aforementioned ionizable lipid compound, including the following reaction steps:

[0035] R a ”-NH2 ① reacts with an α,β-unsaturated carbonyl compound ② to form an ionizable lipid compound ③:

[0036]

[0037] wherein, R a ”-NH2 is R a -NH2 or

[0038] R b ’ and R b ” are both H, or R b ’ and R b ” are both C1-C6 amines, or R b ’ and R b ” are both -R c ’-A1’-R c ”-NH2.

[0039] In a fourth aspect, the present invention provides a method for preparing the aforementioned ionizable lipid compound, including:

[0040] 1) R a ”-NH2 ① reacts with an α,β-unsaturated carbonyl compound ④ to form a compound ⑤;

[0041] 2) The compound ⑤ reacts with a nucleophile ⑥ to form an ionizable lipid compound ③;

[0042]

[0043] wherein, the nucleophile ⑥ is R b -NH2 or R b -OH; R a ”-NH2 is selected from R a -NH2 or

[0044]

[0045] R b ’ and R b ” are both H, or R b ’ and R b” are simultaneously C1-C6 amines, or R b ’ and R b ” are simultaneously -R c ’-A1’-R c ”-NH2; Z2 is a leaving group, and Z2 reacts with NH2 to obtain A1.

[0046] In a fifth aspect, the present invention provides a method for preparing the aforementioned ionizable lipid compound, including:

[0047] 1) R a ”-NH2 ① reacts with an α,β-unsaturated carbonyl compound ② to form a compound ⑦;

[0048] 2) The compound ⑦ reacts with an α,β-unsaturated carbonyl compound ⑧ to form a compound ⑨;

[0049] 3) The compound ⑨ reacts with a nucleophile ⑩ to form an ionizable lipid compound

[0050] (Formula 1c);

[0052] Wherein, R a ”-NH2 is R a -NH2, Z3 is a leaving group, and A3 reacts with Z3 to obtain A1.

[0053] The leaving group described above refers to the leaving part in a nucleophilic reaction or a condensation reaction, including but not limited to: H, OH, H2O, halogen (such as F, Cl, Br, and I), cyanate anion, inorganic acid (such as nitric acid, sulfuric acid, phosphoric acid), carboxylic acid (such as acetic acid, trifluoroacetic acid, and benzoic acid, etc.), sulfonic acid (such as methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and p-nitrobenzenesulfonic acid, etc.), carbon dioxide (CO2), nitrogen (N2), imidazole, alkoxy (R-O-), amino (-NHR, where R is an alkyl or aryl group from which H is removed), phenoxy, tertiary carbocation (such as tert-butyl cation), carbocation stabilized by an unsaturated system or a heteroatom, or various protecting groups described above.

[0054] In a preferred embodiment, in Formula 1a - Formula 1c, at least one nucleophile undergoes a Michael addition reaction with the β-carbon atom of at least one α,β-unsaturated carbonyl compound to form the bifurcated ionizable lipid compound containing a carbon-carbon bond, a carbon-oxygen bond, a carbon-nitrogen bond, a carbon-sulfur bond, or a carbon-selenium bond.

[0055] In a preferred embodiment, Formula 1a - Formula 1b further includes a step of iterative reaction using the compound of Formula (1) with a terminal amino group obtained by the reaction of Formula 1a - Formula 1b as a raw material according to Step 1 in Formula 1a or Steps 1 - 2 in Formula 1b;

[0056] The compound of formula (1) with a terminal amino group is

[0057] wherein R b ’ and R b ” are both H, or R b ’ and R b ” are both C1-C6 amines, or R b ’ and R b ” are both -R c ’-A1’-R c ”-NH2.

[0058] In a preferred embodiment, the reaction scheme of formula 1a-1c further includes using the compound of formula (1) with a terminal amino group obtained by the reaction scheme of formula 1a-1c as a raw material, and reacting with the step of reaction;

[0059] The compound of formula (1) with a terminal amino group is

[0060] wherein R b ’ and R b ” are both H, or R b ’ and R b ” are both C1-C6 amines, or R b ’ and R b ” are both -R c ’-A1’-R c ”-NH2.

[0061] In a preferred embodiment, the raw materials used in the reaction process further contain a protecting group, and the reaction steps include protecting and / or deprotecting steps.

[0062] In a sixth aspect, the present invention provides the use of the foregoing ionizable lipid compound in the following fields: (1) as an emulsifier, suspending agent, dispersant, solubilizer, lubricant, thickener, bacteriostatic agent or preservative; (2) preparing a cosmetic composition; (3) as a drug delivery carrier for preparing a lipid composition.

[0063] In a seventh aspect, the present invention provides a lipid composition comprising the foregoing ionizable lipid compound.

[0064] In a preferred embodiment, the lipid component of the lipid composition comprises other lipids, and the other lipids include phospholipids, cholesterol, and PEG-conjugated lipids.

[0065] In a preferred embodiment, the lipid component of the lipid composition further comprises other ionizable lipids, and the molar ratio of the ionizable lipid compound to the other ionizable lipids is 1:0.05 - 100.

[0066] In a preferred embodiment, the other ionizable lipids are selected from at least one of the following compounds:

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] In a preferred embodiment, the molar ratio of the ionizable lipid component to other lipids is 1:0.2 - 10; the active ingredient of the lipid composition includes a therapeutic agent and / or a prophylactic agent; the active ingredient accounts for 0.1 - 50% (w / w) of the total prescription amount.

[0073] In a preferred embodiment, the therapeutic agent and / or prophylactic agent is nucleic acid; the nucleic acid is DNA or RNA.

[0074] In a ninth aspect, the present invention provides the use of the foregoing lipid composition in the preparation of a drug for treating diseases in mammals.

[0075] In a preferred embodiment, the disease is characterized by a malfunctioning or abnormal protein or polypeptide activity; the diseases are selected from infectious diseases, cancers, proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases.

[0076] In a tenth aspect, the present invention provides a pharmaceutical composition comprising the foregoing lipid composition and a pharmaceutically acceptable carrier.

[0077] In a preferred embodiment, the pharmaceutical active compound is selected from anti-inflammatory compounds, steroids, statins, estradiol, BTK inhibitors, S1P1 agonists, glucocorticoid receptor modulators, and antihistamine drugs. Compared with the prior art, the present invention has the following beneficial effects:

[0078] 1. The ionizable lipid compound provided by the present invention has good surfactant properties and can be used to prepare lipid nanoparticles for use as a drug delivery carrier.

[0079] 2. The ionizable lipid compounds of the present invention are suitable for preparing lipid compositions. The Zeta potential of the lipid composition of mRNA prepared with the compounds in the examples is -20 mV to 20 mV, the dispersion coefficient is <0.5, the particle size is 10 - 200 nm, and the encapsulation efficiency is >60%.

[0080] 3. The lipid nanoparticles of mRNA and / or other nucleic acid substances (such as siRNA, microRNA, pDNA, etc.) prepared with the ionizable cationic lipid compounds of the present invention exhibit biological activity both in vitro cells and in animals, and have low toxicity. It shows that the lipid nanoparticles provided by the present invention have good safety and effectiveness as drug carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 Luciferase activity of luciferase mRNA lipid nanoparticles prepared with compound 6008 in HEK293T cells.

[0082] Figure 2 Whole body in vivo imaging at 6 h / 12 h after intravenous administration of luciferase mRNA lipid nanoparticles prepared with compound 6020 in mice. DETAILED DESCRIPTION OF THE INVENTION

[0083] The synthetic processes of the present invention can accommodate multiple functional groups, so various substituted starting materials can be used. These processes generally provide the desired final compounds at the end or near the end of the entire process. However, in some cases, it may be necessary to further convert the compound into its pharmaceutically acceptable salt. The compounds of the present invention can be prepared in various ways using commercially available starting materials, compounds known in the literature, or intermediates that are easily prepared, by adopting standard synthetic methods and procedures known to those skilled in the art or obvious to those skilled in the art based on the teachings herein. Standard synthetic methods and procedures for preparing organic molecules, as well as functional group transformations and operations, can be obtained from relevant scientific literature or from standard textbooks in the field. The following description of the synthetic methods is designed to illustrate rather than limit the general procedures for preparing the compounds of the present invention.

[0084] The compounds of the present invention having the various formulas described herein can be prepared from commercially available starting materials or starting materials that can be prepared using literature processes according to the processes described in the corresponding general synthetic routes. The variables (such as R1, R2, and R3, etc.) in each general synthetic route are defined as herein. Those of ordinary skill in the art should note that in the reaction procedures and synthetic schemes described herein, the order of some steps can be changed, such as the introduction and removal of protecting groups.

[0085] In the reaction schemes described herein, multiple stereoisomers can be produced. When no specific stereoisomer is indicated, this should be understood to include all possible stereoisomers produced by the reaction. Those of ordinary skill in the art should recognize that the reaction can be optimized to preferentially obtain one isomer, or new schemes can be designed to produce a single isomer. If a mixture is produced, techniques such as preparative thin-layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC can be used to separate the isomers.

[0086] Synthesis of Ionizable Lipid Compounds in Example 1

[0087] 1. Preparation of Compound 6001

[0088] Structural formula:

[0089] Molecular weight: 499.43

[0090] Add 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide to 6.4 mL of ethanol, stir and heat to 70 °C. After 3.5 h, add an additional 0.8 g of hydroxyethyl acrylamide and continue the reaction for 16 h. Then pass through a reverse column to obtain Compound 6002.

[0091] Add 0.7 g of Compound 6002, 0.4 g of palladium on carbon, and 28 mL of tetrahydrofuran to a reaction flask, stir, displace with hydrogen, and maintain pressure with a hydrogen balloon. After 16 h, filter and pass through a normal column (methanol - DCM system) to obtain 200 mg of Compound 6001. 1 H NMR (400 MHz, CDCl3) δ 7.54 (t, J = 5.5 Hz, 2H), 3.79 - 3.68 (m, 4H), 3.42 (dd, J = 10.0, 5.4 Hz, 4H), 2.89 (t, J = 6.0 Hz, 4H), 2.55 (dt, J = 11.8, 6.8 Hz, 6H), 1.54 (s, 2H), 1.27 (d, J = 11.2 Hz, 32H), 0.91 (t, J = 6.8 Hz, 3H).

[0092] 2. Preparation of Compound 6002

[0093] Structural formula:

[0094] Molecular weight: 497.42

[0095] Add 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide to 6.4 mL of ethanol, stir and heat to 70 °C. After 3.5 h, add an additional 0.8 g of hydroxyethyl acrylamide and continue the reaction for 16 h. Then concentrate the organic solvent, pass through a reverse column, and lyophilize to obtain a total of 400 mg of Compound 6002. 11H NMR (400 MHz, CDCl3) δ 7.31 (s, 2H), 5.38 (dd, J = 13.0, 7.4 Hz, 2H), 3.79 - 3.66 (m, 4H), 3.41 (dd, J = 10.1, 5.4 Hz, 4H), 2.82 - 2.72 (m, 4H), 2.44 (dd, J = 15.1, 9.2 Hz, 6H), 2.10 - 1.99 (m, 4H), 1.46 (s, 2H), 1.29 (s, 24H), 0.90 (t, J = 6.8 Hz, 3H).

[0096] Preparation of Compound 6003

[0097] Structural formula:

[0098] Molecular weight: 445.34

[0099] 4.00 g of tetradecylamine, 6.53 g of 2-hydroxyethyl acrylate and 80 mL of tert-butanol were added to a reaction flask, stirred and heated to 70 °C. After 29 h, the organic solvent was concentrated, passed through a reverse column, and freeze-dried to obtain 1.5 g of Compound 6003. 1 1H NMR (400 MHz, CDCl3) δ 4.29 (t, J = 7.5 Hz, 4H), 3.89 - 3.75 (m, 4H), 2.81 (t, J = 6.2 Hz, 4H), 2.50 (ddd, J = 23.2, 14.1, 7.1 Hz, 6H), 1.46 (s, 2H), 1.28 (s, 24H), 0.91 (t, J = 6.3 Hz, 3H).

[0100] Preparation of Compound 6004

[0101] Structural formula:

[0102] Molecular weight: 553.48

[0103] 0.8 g of oleylamine, 1.5 g of n-butyl acrylate and 5.6 mL of n-butanol were added to a reaction flask, stirred and heated to 100 °C. After 4 h, 1 mL of n-butyl acrylate was added, and the reaction continued for 1 h. Then it was passed through a normal-phase column (petroleum ether - ethyl acetate system), 0.4 g of sodium hydroxide, 1 mL of water and 10 mL of methanol were added, stirred and hydrolyzed for 30 min. 1 mL of concentrated hydrochloric acid and 10 mL of methanol were added to the mixed solution to adjust the pH to neutral, evaporated to dryness, dissolved in DCM, dried over anhydrous magnesium sulfate, filtered, the solvent was evaporated to dryness, 10 mL of DCM, 1.06 g of 4-amino-1-butanol, 0.81 g of HOBT and 2.3 g of EDCI were added, and the reaction was carried out at room temperature for 18 h. After concentrating the organic solvent, it was passed through a reverse column and freeze-dried to obtain 240 mg of Compound 6004. 1HNMR (400 MHz, CDCl3) δ 7.58 (d, J = 16.6 Hz, 2H), 5.44 - 5.31 (m, 2H), 3.72 (d, J = 16.5 Hz, 4H), 3.31 (d, J = 5.6 Hz, 4H), 3.17 (s, 4H), 2.84 (s, 2H), 2.69 (s, 4H), 2.09 - 2.01 (m, 4H), 1.67 (d, J = 2.7 Hz, 10H), 1.37 - 1.26 (m, 24H), 0.91 (t, J = 6.8 Hz, 3H).

[0104] 5. Preparation of Compound 6005

[0105] Structural formula:

[0106] Molecular weight: 597.54

[0107] Add 25.00 g of 11 - hentriacontanone, 62.05 g of ammonium acetate and 500 mL of methanol to a reaction flask and stir. Add 6.55 g of sodium cyanoborohydride. After reacting for 16 h, add 250 mL of water and 250 mL of DCM. Separate the layers. Extract the aqueous phase with 50 mL of DCM and combine the organic phases. Pass through a normal phase column (methanol - DCM system) to obtain 21.00 g of Compound 6005 - A.

[0108] Add 21.00 g of Compound 6005 - A, 58 mL of n - butyl acrylate and 100 mL of n - butanol latex to a reaction flask and stir. Heat up to 100 °C. Add an additional 10 mL of n - butyl acrylate. After 16 h, pass through a column (petroleum ether - ethyl acetate system) to obtain 31.00 g of Compound 6005 - B.

[0109] Prepare a solution by adding 31.00 g of Compound 6005 - B, 6.55 g of sodium hydroxide, 310 mL of methanol and 31 mL of water to a reaction flask and stir. After 20 min, add 150 mL of THF. Stir and heat up to 50 °C. After 30 min, add a solution prepared from 16.13 g of concentrated hydrochloric acid and 160 mL of methanol and stir. After 30 min, evaporate the solvent. Dissolve in 300 mL of DCM, dry with anhydrous magnesium sulfate, filter, and evaporate to obtain 27.00 g of an oily Compound 6005 - C.

[0110] Add 27.00 g of Compound 6005 - C, 25.00 g of 4 - amino - 1 - butanol, 41.85 g of EDCI, 14.74 g of HOBT and 310 mL of DCM to a reaction flask and stir. After 18 h, concentrate the organic solvents and pass through a column (methanol - DCM system) to remove the excess 4 - amino - 1 - butanol compound to obtain 6.80 g of Compound 6005. 1HNMR (400 MHz, CDCl3) δ 3.70 (t, J = 5.6 Hz, 4H), 3.28 (t, J = 5.6 Hz, 4H), 2.74 (t, J = 6.0 Hz, 4H), 2.40 - 2.45 (m, 1H), 2.34 (t, J = 6.4 Hz, 4H), 1.63 - 1.65 (m, 8H), 1.25 - 1.35 (m, 36H), 0.91 (t, J = 6.4 Hz, 6H).

[0111] Preparation of Compound 6006

[0112] Structural formula:

[0113] Molecular weight: 553.48

[0114] Add 1.00 g of oleylamine, 0.47 g of hydroxyethyl acrylamide and 8 mL of ethanol into a reaction flask, stir and heat to 70 °C. After 13 h, rotary evaporate the solvent and pass through a column (methanol - DCM system) to obtain 800 mg of intermediate 1. Add 1.5 mL of n - butyl acrylate and 5 mL of n - butanol, stir and heat to 100 °C. After 3 h, rotary evaporate the solvent and pass through a column (methanol - DCM system). Add a solution prepared from 0.4 g of sodium hydroxide, 10 mL of methanol and 1 mL of water, stir for 1 h, then add a solution prepared from 1 mL of concentrated hydrochloric acid and 8 mL of methanol, stir for 30 min, rotary evaporate the solvent, dissolve with 100 mL of DCM, dry with anhydrous magnesium sulfate, filter, rotary evaporate, add 0.45 g of 6 - amino - 1 - hexanol, 0.27 g of HOBT, 0.78 g of EDCI and 10 mL of DCM, stir. After 14 h, concentrate the organic solvent and pass through a column (methanol - DCM system) to obtain 300 mg of Compound 6006. 1 H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 6.95 (s, 1H), 5.43 - 5.28 (m, 2H), 3.74 - 3.67 (m, 2H), 3.64 (t, J = 6.3 Hz, 2H), 3.39 (dd, J = 10.0, 5.3 Hz, 2H), 3.24 (dd, J = 12.9, 6.7 Hz, 2H), 2.74 (t, J = 5.8 Hz, 4H), 2.48 - 2.42 (m, 2H), 2.41 - 2.33 (m, 4H), 2.01 (dd, J = 13.6, 6.8 Hz, 4H), 1.66 - 1.17 (m, 34H), 0.89 (t, J = 6.7 Hz, 3H).

[0115] 7. Compound 6007

[0116] Structural formula:

[0117] Molecular weight: 953.78

[0118]

[0119] At room temperature, add octadecylamine (50 g) to a three-necked flask. After adding MeOH (200 mL) and stirring to mix, cool the mixture to 5 °C in an ice-water bath. Then slowly add methyl acrylate (31.5 g) dropwise, stir to mix, and restore to room temperature. The reaction solution is reacted at room temperature for 4 h; concentrated under reduced pressure and purified by column chromatography (PE:EA = 20:1 - 10:1) to obtain product 2 (68 g). At room temperature, add product 2 (15.0 g), MeOH (100 mL) and ethylenediamine (1.0 g) to a single-necked flask, heat to 60 °C, and maintain this temperature to react overnight; concentrate under reduced pressure, add 30 mL of toluene, heat to 60 °C to dissolve, slowly cool to room temperature, and evaporate to dryness under reduced pressure to obtain compound 3 (15 g). At room temperature, add compound 3 (2 g) to a single-necked flask. After adding MeOH (20 mL) and stirring to dissolve, then slowly add methyl acrylate (2.5 g) and stir to mix. Heat to 60 °C and react overnight; stop the reaction, concentrate the reaction solution under reduced pressure, and purify by column chromatography (DCM:MeOH = 50:1 - 20:1) to obtain compound 4 (2.3 g). At room temperature, add compound 4 (2.3 g) and ethylenediamine (20 mL) to a single-necked flask, and react overnight at room temperature; stop the reaction and directly concentrate under reduced pressure to obtain compound 6007 (24 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.87 (t, J = 6.6 Hz, 2H), 3.55 - 3.36 (m, 8H), 3.33 (s, 8H), 3.27 - 3.16 (m, 2H), 3.06 - 2.90 (m, 4H), 2.77 (t, J = 6.5 Hz, 4H), 1.90 (dtdd, J = 21.2, 14.0, 9.5, 6.9 Hz, 4H), 1.73 (p, J = 7.7 Hz, 4H), 1.49 (tdd, J = 9.2, 7.9, 7.1, 4.0 Hz, 4H), 1.31 (s, 28H), 0.92 (t, J = 6.8 Hz, 3H).

[0120] 8. Compounds 6008 - 6018

[0121] Synthesis of Compound 6009

[0122] Structural formula:

[0123] At room temperature, a solution of freshly recrystallized hexadecylamine (0.03 mol) in methanol (20 mL) was added dropwise, under a nitrogen atmosphere, to a stirred solution of methyl methacrylate (6 mL) in methanol (20 mL). The reaction was carried out overnight. The reactants were evaporated to dryness at room temperature, and the residue was dissolved in chloroform and washed twice with 0.1 M NaOH solution. The chloroform solution was collected and dried over anhydrous calcium chloride. Then, a colorless oil was obtained by column chromatography. 1H-NMR (300 MHz, CDCl3): 0.78 (t, 3H), 1.16 (s, 30H), 2.38 (m, 6H), 2.71 (t, 4H), 3.57 (m, 6H).

[0124] Then, a solution of the above-mentioned colorless oil (11.05 g) in methanol (20 mL) was added to a vigorously stirred solution of 1,2-diaminoethane (75 g) in methanol (100 mL) at room temperature. After complete addition, the mixture was stirred at room temperature for another 24 hours. The solvent was removed under reduced pressure, keeping the temperature not higher than 40 °C. The excess 1,2-diaminoethane was removed using an azeotropic mixture of toluene and methanol (9:1). The remaining toluene was removed by azeotropic distillation with methanol. Finally, a white powder (10.5 g) was obtained, which was recrystallized repeatedly from chloroform and cyclohexane to finally obtain a white solid. 1H-NMR (300 MHz, CDCl3): 0.88 (t, 3H), 1.25 (s, 30H), 1.84 (s, 4H), 2.38 (m, 6H), 2.73 (m, 4H), 2.82 (m, 4H), 3.29 (m, 4H), 7.47 (s, 2H).

[0125] The preparation methods of 6008 and 6009 are only different in that: equimolar amounts of tetradecylamine are used instead of hexadecylamine. For 6010 - 6017, referring to the preparation method of 6009, they are prepared using equimolar amounts of the corresponding R a -NH2 instead of hexadecylamine.

[0126] For 6018, referring to the preparation method of 6007, it is prepared using equimolar amounts of the corresponding R a -NH2 instead of octadecylamine.

[0127] 9. Compound 6019

[0128] Structural formula:

[0129] Molecular weight: 753.66

[0130]

[0131] At room temperature, add compound 2483-46-7 (3 g) to a single-necked flask. After adding DCM (50 mL) and stirring to mix, then add 18807-71-1 (2.5 g), DCC (2.7 g), and DMAP (1.6 g), stir to mix, and react overnight at room temperature; concentrate under reduced pressure and purify by column chromatography (DCM:MeOH = 5:1) to obtain product 3 (4 g). At room temperature, add product 3 (5.0 g), MeOH (50 mL), and Pd / C (1.0 g) to a three-necked flask, displace hydrogen, heat to 50 °C, and stir for 1 h; stop the reaction, let the reaction solution cool to room temperature, filter, and rotary evaporate the filtrate to obtain product 4 (3.5 g). At room temperature, add raw material 5 (1.2 g) to a single-necked flask. After adding DMF (50 mL) and stirring to dissolve, then add product 4 (3.3 g), HATU (3.2 g), and TEA (0.9 g), stir to mix, and react overnight at room temperature; add water (500 mL) and EA (500 mL), perform liquid-liquid extraction, dry the organic phase with Na2SO4, concentrate under reduced pressure, and purify the crude product by column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain solid 6 (380 mg). At room temperature, add solid 6 (380 mg) and HCl / Dioxane (4 M) (1.6 mL) to a single-necked flask, and react for 2 h at room temperature; concentrate under reduced pressure and purify to obtain compound 6019 (220 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.87 (t, J = 6.6 Hz, 2H), 3.55 - 3.36 (m, 8H), 3.33 (s, 8H), 3.27 - 3.16 (m, 2H), 3.06 - 2.90 (m, 4H), 2.77 (t, J = 6.5 Hz, 4H), 1.90 (dtdd, J = 21.2, 14.0, 9.5, 6.9 Hz, 4H), 1.73 (p, J = 7.7 Hz, 4H), 1.49 (tdd, J = 9.2, 7.9, 7.1, 4.0 Hz, 4H), 1.31 (s, 28H), 0.92 (t, J = 6.8 Hz, 3H).

[0132] 10. Compound 6020

[0133] Structural formula:

[0134] Molecular weight: 810.19

[0135]

[0136] At room temperature, add octadecylamine (50 g) to a three-necked flask. After adding MeOH (200 mL) and stirring to mix, cool the mixture to 5 °C in an ice-water bath. Then, dropwise add methyl acrylate (31.5 g), stir to mix, and restore to room temperature. React for 4 h; stop the reaction, directly concentrate under reduced pressure, and purify by column chromatography (PE:EA = 20:1 - 10:1) to obtain product 2 (68 g). At room temperature, add product 2 (15.0 g), MeOH (100 mL), and ethylenediamine (1.0 g) to a single-necked flask. Heat to 60 °C and maintain this temperature to react overnight; stop the reaction, concentrate the reaction solution under reduced pressure, add 30 mL of toluene, heat to 60 °C to dissolve, slowly cool to room temperature, filter, and evaporate to dryness under reduced pressure to obtain compound 3 (15 g). At room temperature, add compound 3 (1.3 g) to a single-necked flask. After adding DMF (20 mL) and stirring to mix, add 35897 - 34 - 8 (3.6 g), EDCI (3.0 g), HOBt (2.1 g), and DIEA (2.0 g), stir to mix, and react overnight at room temperature; add water (200 mL) and directly lyophilize under reduced pressure to obtain compound 4 (4 g). At room temperature, add compound 4 (100 mg) and HCl / Dioxane (4 M) (2 mL) to a single-necked flask. React at room temperature for 1 h; stop the reaction, concentrate and purify the reaction solution to obtain compound 6020. 1H NMR (400 MHz, Methanol-d4) δ 3.90 (t, J = 6.4 Hz, 2H), 3.57 - 3.34 (m, 10H), 3.24 (dt, J = 16.1, 8.0 Hz, 8H), 2.77 (t, J = 6.5 Hz, 4H), 2.03 - 1.58 (m, 10H), 1.31 (s, 30H), 1.01 - 0.82 (m, 3H).

[0137] 11. Compound 6021

[0138] Structural formula:

[0139] Molecular weight: 1182.79

[0140]

[0141] At room temperature, into a single-necked flask, add compound 2 (3.5 g, the synthesis process is the same as product 2 in compound 6020), add MeOH (20 mL), stir and mix, then add TREN (23.0 g), stir and mix, heat up to 60 °C, stir overnight, concentrate under reduced pressure, and lyophilize the obtained crude product to obtain product 3 (25 g); at room temperature, into a single-necked flask, add product 3 (22.0 g) and DCM (200 mL), cool down to 0 °C, slowly dropwise add (Boc)2O (71.7 g), after dropping, restore to room temperature, react for 3 h, add water (200 mL) to quench, then add DCM (100 mL) for liquid-liquid extraction, dry the organic phase with sodium sulfate, concentrate under reduced pressure, and purify by column chromatography to obtain yellow oil 4 (5.8 g). At room temperature, into a single-necked flask, add yellow oil 4 (1.4 g), slowly add HCl / Dioxane (4 M) (15 mL) under an ice-water bath, stir and mix, after adding, restore to room temperature, react for 0.5 h, concentrate under reduced pressure to obtain compound 5 (800 mg). At room temperature, into a single-necked flask, add compound 5 (1.0 g), (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid (2.6 g), EDCI (1.4 g), HOBt (1.0 g), DIEA (1.0 g) and solvent DMF (10 mL), react overnight at room temperature, add 100 mL of water and 100 mL of EA, perform liquid-liquid extraction, wash the EA phase once with 100 mL of saturated brine, concentrate under reduced pressure, and purify by column chromatography (DCM:MeOH = 10:1) to obtain compound 6 (1.1 g). At room temperature, into a single-necked flask, add compound 6 (1.2 g), HCl / Dioxane (4 M) (10 mL) and solvent DCM (10 mL), react overnight at room temperature, concentrate under reduced pressure, and purify to obtain compound 6021 (220 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.96 (t, J = 6.6 Hz, 4H), 3.75 (dt, J = 14.0, 6.6 Hz, 4H), 3.62 - 3.45 (m, 12H), 3.36 - 3.33 (m, 14H), 3.25 - 3.17 (m, 2H), 3.04 - 2.90 (m, 8H), 2.83 (t, J = 6.9 Hz, 4H), 2.05 - 1.82 (m, 8H), 1.73 (p, J = 7.7 Hz, 10H), 1.53 (qd, J = 8.3, 7.7, 4.3 Hz, 8H), 1.30 (s, 28H), 0.97 - 0.87 (m, 3H).

[0142] 12. Compound 6023

[0143] Structural formula:

[0144] Molecular weight: 670

[0145]

[0146] At room temperature, add dodecylamine (10 g) to a three-necked flask. After adding MeOH (100 mL) and stirring to mix, cool the mixture to 5 °C in an ice-water bath. Slowly add methyl acrylate (10.2 g) dropwise, stir to mix, and then return to room temperature. Stop the reaction after the reaction solution has reacted at room temperature for 4 h. Concentrate under reduced pressure and purify by column chromatography (PE:EA = 20:1 - 10:1) to obtain product 2 (19 g). At room temperature, add product 2 (19.0 g), MeOH (200 mL), and ethylenediamine (127.7 g) to a single-necked flask. Heat the mixture to 60 °C and maintain this temperature to react overnight. Concentrate under reduced pressure to obtain compound 3 (20 g). Take 500 mg for purification and lyophilize to obtain compound 6023-1 (218 mg). At room temperature, add starting material 6023-1 (1.4 g) to a single-necked flask. After adding DCM (20 mL) and stirring to dissolve, add (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid (3.4 g), EDCI (1.9 g), and DMAP (1.2 g), stir to mix, and maintain at room temperature to react overnight. Separate and extract, dry, and concentrate under reduced pressure to obtain compound 4 (3.0 g). At room temperature, add compound 4 (2.8 g) and HCl / 1,4-Dioxane (4 M) (20 mL) to a single-necked flask and react at room temperature for 2 h; concentrate under reduced pressure, purify, and lyophilize to obtain compound 6023 (123 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.88 (t, J = 6.6 Hz, 2H), 3.55 - 3.36 (m, 8H), 3.36 - 3.33 (m, 2H), 3.31 - 3.14 (m, 4H), 3.04 - 2.93 (m, 4H), 2.77 (t, J = 6.5 Hz, 4H), 2.00 - 1.82 (m, 4H), 1.82 - 1.66 (m, 6H), 1.50 (qd, J = 8.2, 7.8, 4.2 Hz, 4H), 1.45 - 1.25 (m, 18H), 0.96 - 0.87 (m, 3H).

[0147] Compound 6024

[0148] Structural formula:

[0149] Molecular weight: 870.24

[0150]

[0151] At room temperature, add the product 6023-1 (1.4 g, synthesized in the same method as 6023-1 in compound 6023) into a single-necked flask. After adding MeOH (20 mL) and stirring until dissolved, slowly add methyl acrylate (2.8 g) and stir to mix. Heat the mixture to 60 °C and react overnight at this temperature. Then concentrate under reduced pressure to obtain compound 4 (2.0 g). At room temperature, add compound 4 (2.4 g) and ethylenediamine (20 mL) into a single-necked flask and react overnight at room temperature. Concentrate under reduced pressure and purify to obtain compound 6024 (191 mg). 1HNMR (400 MHz, Methanol-d4) δ 3.69 (t, J = 6.0 Hz, 4H), 3.60 - 3.48 (m, 20H), 3.42 (t, J = 6.1 Hz, 4H), 3.25 - 3.19 (m, 2H), 3.12 (t, J = 5.8 Hz, 8H), 2.83 (t, J = 6.5 Hz, 12H), 1.78 (tt, J = 11.0, 6.4 Hz, 2H), 1.51 - 1.18 (m, 20H), 0.91 (t, J = 6.7 Hz, 3H).

[0152] 14. Compound 6026

[0153] Structural formula:

[0154] Molecular weight: 443.67

[0155]

[0156] Compound 6026 was synthesized by referring to the synthetic route of compound 6019, with the only difference being that equimolar amounts of compound 1 were used to replace the raw material 5 of 6019, and equimolar amounts of N-(tert-butoxycarbonyl) ethanolamine were used to replace the intermediate 4 of compound 6019. 1HNMR (300 MHz, DMSO) δ: 4.51 (t, J = 7.3 Hz, 4H), 3.76 (t, J = 6.1 Hz, 4H), 3.18 (t, J = 6.7 Hz, 4H), 3.01 (t, J = 5.8 Hz, 2H), 2.49 (t, J = 7.1 Hz, 4H), 1.36 - 1.26 (m, 24H), 0.89 (t, J = 6.2 Hz, 3H).

[0157] Compounds 6025 - 6034 were obtained by referring to the synthetic routes of the aforementioned compounds, with the difference being that hydroxyethylamine was used to replace the corresponding ethylenediamine, and the amino group of hydroxyethylamine was first protected with protecting groups such as Fmoc or Boc, and the protecting group was removed by conventional methods after the reaction was completed.

[0158] Example 2 Preparation of Lipid Nanoparticles

[0159] Dilute luciferase mRNA in 50 mM sodium citrate buffer solution with pH 4.0, and the concentration of the mRNA solution is 135 μg / mL; prepare a lipid mixed ethanol solution according to the molar percentage ratio of ionizable lipid compound (ionizable lipid compound prepared in Example 1): other ionizable lipids: DSPC: cholesterol: 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethylacetamide (ALC-0159) of 15:35:10:38.5:1.5; after mixing the mRNA solution and the lipid mixed solution at a volume ratio of 3:1 in a nano-drug preparation device, obtain luciferase mRNA lipid nanoparticles by ultrafiltration, as shown in Table 1 specifically.

[0160] Table 1 Preparation of Lipid Nanoparticles

[0161]

[0162]

[0163] Comparative Example 1

[0164] Dilute luciferase mRNA in 50 mM sodium citrate buffer solution with pH 4.0, and the concentration of the mRNA solution is 135 μg / mL; prepare a lipid mixed ethanol solution according to the molar percentage ratio of SM102: other ionizable lipids (ALC-0315): DSPC: cholesterol: 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethylacetamide (ALC-0159) of 15:35:10:38.5:1.5; after mixing the mRNA solution and the lipid mixed solution at a volume ratio of 3:1 in a nano-drug preparation device, obtain luciferase mRNA lipid nanoparticles by ultrafiltration.

[0165] Comparative Example 2

[0166] Dilute luciferase mRNA in 50 mM sodium citrate buffer solution with pH 4.0, and the concentration of the mRNA solution is 135 μg / mL; prepare a lipid mixed ethanol solution according to the molar percentage ratio of SM102: DSPC: cholesterol: 2-[(polyethylene glycol)-2000]-N,N-ditetradecylethylacetamide (ALC-0159) of 50:10:38.5:1.5; after mixing the mRNA solution and the lipid mixed solution at a volume ratio of 3:1 in a nano-drug preparation device, obtain luciferase mRNA lipid nanoparticles by ultrafiltration.

[0167] Determination of Particle Size, Zeta Potential and Encapsulation Efficiency of Lipid Nanoparticles in Example 3

[0168] 1. Determination of particle size and polydispersity index (PDI): The average particle size and PDI of the sample solution of the lipid nanoparticles in Example 2 were determined by dynamic light scattering using a Malvern ZetaSizer Nano ZS90. The measurement angle was 90°, the refractive index of the dispersant was 1.330, and the test temperature was 25°C.

[0169] 2. Zeta potential: The Zeta potential of the sample solution of the lipid nanoparticles in Example 2 was determined using a Malvern ZetaSizer Nano ZS90 based on electrophoretic light scattering (ELS). The refractive index of the dispersant was 1.330, and the test temperature was 25°C.

[0170] 3. Encapsulation efficiency: According to the manufacturer's instructions, the encapsulation efficiency of luciferase mRNA in the lipid nanoparticles in Example 2 was determined using a Quant-it Ribogreen RNA quantification assay kit (ThermoFisher Scientific, UK). The average particle size, PDI, Zeta potential, and encapsulation efficiency data of each lipid nanoparticle are shown in Table 2.

[0171] Table 2 Summary of the apparent pKa, particle size, PDI, Zeta potential, and encapsulation efficiency of lipid nanoparticles

[0172]

[0173]

[0174] In vitro cell activity evaluation of the lipid nanoparticles in Example 4

[0175] The in vitro transfection efficiency of each lipid nanoparticle composition and the lipid nanoparticle composition of the comparative example was evaluated using HEK-293T cells. HEK293T cells were routinely cultured in DMEM + 10% FBS medium to ensure that the cells were in the logarithmic growth phase; one day before transfection, they were seeded onto a 96-well culture plate at an appropriate cell density and grown overnight. At the time of transfection, the cell confluence should reach 70 - 90%; the lipid nanoparticle compositions prepared from each exemplary compound were diluted into 4 different dose concentrations with DMEM and added to the 96-well cell culture plate so that the concentration in each well reached 400 ng, 200 ng, 100 ng, and 50 ng respectively, and Lipofectamine 2000 transfected with luciferase plasmid was used as a positive reference (PC). After incubation in a 37°C, 5% CO2 incubator for 24 h, the substrate was added to the wells, and the luciferase activity was measured using an enzyme-linked immunosorbent assay reader. The results are shown in Table 3. The luciferase activity of the luciferase mRNA lipid nanoparticles prepared from Compound 6006 in HEK293T cells is shown in the appendix Figure 1 .

[0176] Table 3 Luciferase Activity of Luciferase mRNA Lipid Nanoparticles in HEK293T Cells

[0177] Compound number HEK 293T cell luciferase activity (100 ng dose; RLU) 6001 2.1E+07 6002 4.7E+06 6003 9.8E+07 6004 5.8E+06 6005 2.9E+07 6006 2.3E+07 6007 6.6E+06 6015 5.1E+07 6019 8.4E+07 6020 1.0E+08 6023 2.3E+08 6026 6.5E+07 Comparative example 1 5.6E+05 Comparative example 2 2.6E+05

[0178] As can be seen from Table 2, the fluorescence intensity of luciferase expressed by the luciferase mRNA lipid nanoparticles prepared with the ionizable lipid compound of the example in HEK 293T cells (dose: 100 ng, 96-well plate) > 10 6 RLU, and it is superior to Comparative Examples 1-2, indicating that the luciferase mRNA lipid nanoparticles prepared with the ionizable lipid compound of the present invention exhibit good biological activity in vitro cells and have low cytotoxicity.

[0179] From the appendix Figure 1 It can be seen that at different doses (50 - 400 ng), the fluorescence intensity of luciferase expressed by the luciferase mRNA lipid nanoparticles prepared with Compound 6006 in HEK 293T cells > 10 7 RLU, and the fluorescence intensity increases with the increase of the dose, indicating that the expression level of luciferase increases correspondingly with the increase of the dose, and the prepared lipid nanoparticles have good mRNA delivery ability and safety.

[0180] In vivo Activity Evaluation of Lipid Nanoparticles in Example 5

[0181] Six- to eight-week-old female Balb / c mice were used to evaluate the in vivo transfection efficiency and safety of lipid nanoparticle combinations. Lipid nanoparticles prepared with Compound 6008, 6015, 6019 - 6021, 6023, 6024 and Comparative Example 1 were administered by tail vein injection at a single dose of 0.3 mpk. In vivo imaging of animals was performed using a PerkinElmer small animal imaging system at specific time points after administration (such as 6 h, 24 h), and bioluminescence signals were measured. The results are shown in Table 4. In vivo whole-body imaging of the luciferase mRNA lipid nanoparticles prepared with Compound 6020 at 6 h / 12 h after intravenous administration to mice is shown in the appendix Figure 2 .

[0182] Summary Table of In vitro / In vivo Activity Evaluation of Lipid Nanoparticles Prepared with Compounds

[0183]

[0184] As can be seen from Table 3, 6 h after administration of the lipid nanoparticles prepared with Compound 6008, 6015, 6019 - 6021, 6023, 6024, the mean fluorescence signal of the whole body of the mice is greater than 10 8p / s, and is superior to Comparative Example 1, indicating that the luciferase mRNA lipid nanoparticles containing the compound induced effective in vivo expression of luciferase. At the same time, after the mice were given each of the lipid nanoparticles prepared in Example 2, no toxic reactions such as abnormal activities were observed. It shows that the luciferase mRNA lipid nanoparticles prepared with the ionizable cationic lipid compound of the present invention exhibit biological activity in animals and have low toxicity.

[0185] The solution of the present invention is not limited to the technical means disclosed by the above technical means, but also includes technical solutions composed of any combination of the above technical features. The above is the specific implementation manner of the present invention. It should be noted that for those of ordinary skill in the art of the present invention, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

Claims

1. An ionizable lipid compound, characterized in that The ionizable lipid compound is a compound of formula (1), or a salt, stereoisomer, or tautomer thereof: Where A1 is NH or O; R a Selected from C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 Cycloalkyl; The C6-C 24 Alkyl and alkenyl groups are straight chain or branched chain structures; R b and R c Each independently selected from alkylamines; The alkylamine is Among them, R a ' is C1-C 12 Alkyl, the R b ' and R b " are each independently selected from H, C1-C6 alkylamine, R c " is selected from C1-C6 alkyl which is unsubstituted or substituted with amino, R c ”' is H, Provided that, when A1' is -NH-CO- or -CO-O-, R c ' is a C1-C6 alkyl group; when A1' is -CO-, R c 'Does not exist.

2. An ionizable lipid compound, characterized in that The ionizable lipid compound is a compound of formula (1), or a salt, stereoisomer, or tautomer thereof: R a Selected from C6-C 24 Alkyl alcohol; the C6-C 24 Alkyl alcohol is a straight chain or branched chain structure; R b and R c Each independently selected from alkylamines; The alkylamine is Among them, R a ' is C1-C 12 Alkyl, the R b ' and R b " are each independently selected from H, C1-C6 alkylamine, R c " is selected from C1-C6 alkyl which is unsubstituted or substituted with amino, R c ”' is H, Provided that, when A1' is -NH-CO- or -CO-O-, R c ' is a C1-C6 alkyl group; when A1' is -CO-, R c 'Does not exist.

3. The ionizable lipid compound according to any one of claims 1 to 2, characterized in that The condition is that when R a For a straight chain C6-C 24 When the alkyl group is alkyl, A1 is NH, and R b ' and R b "Each independently selected R c " is selected from C1-C6 alkyl which is unsubstituted or substituted with amino, R c ”' is H, When R a C6-C 24 When it is an alkyl alcohol, A1 is not O.

4. The ionizable lipid compound according to any one of claims 1 to 2, characterized in that The conditions are: When R a For branched C6-C 24 When it is alkyl, A1 is NH, R b and R c Each is independently selected from alkylamines.

5. The ionizable lipid compound according to any one of claims 1 to 2, characterized in that The R a Select from the following compound structures:

6. The ionizable lipid compound according to any one of claims 1 to 2, characterized in that The R b and R c Select from the following compound structures: R d Selected from C1-C6 alkanes or cycloalkanes.

7. The ionizable lipid compound according to any one of claims 1 to 2, characterized in that The compound of formula (1) is selected from at least one of the following compounds:

8. The method for preparing an ionizable lipid compound according to any one of claims 1 to 7, characterized in that: The process comprises the following reaction steps: R a ” -NH2① reacts with α,β-unsaturated carbonyl compound② to generate ionizable lipid compound③: Among them, R a ” -NH2 is R a -NH2 or R b ' and R b " is H, or R b ' and R b " is a C1-C6 amine, or R b ' and R b "At the same time 9. The method for preparing an ionizable lipid compound according to any one of claims 1 to 7, characterized in that: include: 1) R a ” -NH2① reacts with α,β-unsaturated carbonyl compound④ to form compound⑤; 2) Compound ⑤ reacts with a nucleophile ⑥ to generate an ionizable lipid compound ③; Among them, the nucleophile ⑥ is R b -NH2 or R b -OH; R a ” -NH2 is selected from R a -NH2 or R b ' and R b " is H, or R b ' and R b " is a C1-C6 amine, or R b ' and R b "At the same time Z2 is a leaving group, and Z2 reacts with NH2 to obtain A1.

10. The method for preparing an ionizable lipid compound according to any one of claims 1 to 7, characterized in that: include: 1) R a -NH2① reacts with α,β-unsaturated carbonyl compound② to produce compound ⑦; 2) Compound ⑦ reacts with an α,β-unsaturated carbonyl compound ⑧ to form compound ⑨; 3) Compound ⑨ reacts with nucleophile ⑩ to generate ionizable lipid compound Among them, Z3 is a leaving group, and A3 reacts with Z3 to obtain A1.

11. The preparation method according to any one of claims 8 to 10, characterized in that: In Formula 1a to Formula 1c, at least one nucleophilic reagent undergoes a Michael addition reaction with the β-carbon atom of at least one α,β-unsaturated carbonyl compound to generate the ionizable lipid compound with two forks containing a carbon-carbon bond, a carbon-oxygen bond, a carbon-nitrogen bond, a carbon-sulfur bond or a carbon-selenium bond; Alternatively, the reaction formula 1a-1c also includes using the compound of formula (1) with a terminal amino group obtained by the reaction formula 1a-1c as a raw material, and The steps of the reaction; The compound of formula (1) having a terminal amino group is Among them, R b ' and R b " is H, or R b ' and R b " is a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2; Alternatively, Formula 1a-Formula 1b further comprises the step of using the compound of Formula (1) with a terminal amino group obtained by the reaction of Formula 1a-Formula 1b as a raw material, and performing an iterative reaction according to Step 1 in Formula 1a or Steps 1-2 in Formula 1b; The compound of formula (1) having a terminal amino group is Among them, R b ' and R b " is H, or R b ' and R b " is a C1-C6 amine, or R b ' and R b "At the same time -R c '-A1'-R c ”-NH2; Alternatively, the raw materials used in the reaction process also contain a protecting group, and the reaction steps include protection and / or deprotection steps.

12. Use of the ionizable lipid compound according to any one of claims 1 to 7 in the following fields: (1) as an emulsifier, suspending agent, dispersant, solubilizer, lubricant, thickener, antibacterial agent or preservative; (2) for preparing cosmetic compositions; (3) as a drug delivery carrier for preparing lipid compositions.

13. A lipid composition, characterized in that The invention comprises the ionizable lipid compound according to any one of claims 1 to 7.

14. The lipid composition of claim 13, wherein The lipid component of the lipid composition comprises other lipids including phospholipids, cholesterol and PEG-conjugated lipids.

15. The lipid composition of claim 14, wherein The lipid component of the lipid composition further comprises other ionizable lipids, and the molar ratio of the ionizable lipid compound to the other ionizable lipids is 1:0.05-100.

16. The lipid composition of claim 15, wherein The other ionizable lipids are selected from at least one of the following compounds:

17. The lipid composition of claim 14, wherein The molar ratio of the ionizable lipid component to other lipids is 1:0.2-10; the active ingredients of the lipid composition include therapeutic agents and / or preventive agents; and the active ingredients account for 0.1-50% (w / w) of the total prescription.

18. The lipid composition of claim 17, wherein The therapeutic and / or preventive agent is a nucleic acid; the nucleic acid is DNA or RNA.

19. Use of the lipid composition according to any one of claims 13 to 18 in the preparation of a medicament for treating a disease in a mammal, characterized in that: The disease is characterized by dysfunctional or abnormal protein or polypeptide activity; the disease is selected from infectious diseases, cancer, proliferative diseases, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases.

20. A pharmaceutical composition, characterized in that Comprising the lipid composition according to any one of claims 13 to 18 and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition according to claim 20, characterized in that Also included are pharmaceutically active compounds selected from the group consisting of anti-inflammatory compounds, steroids, statins, estradiol, BTK inhibitors, S1P1 agonists, glucocorticoid receptor modulators, and antihistamines.

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