Ionizable lipid compound and preparation method and application thereof

Abstract

The present invention provides an ionizable lipid compound and a preparation method and application thereof, belonging to the field of biomedical technology. The ionizable lipid compound provided by the present invention is a compound having formula (1), or a salt, stereoisomer, or tautomer thereof, which 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 lipid nanoparticles prepared by the lipid nanoparticles can show good effectiveness and safety in cells and animals.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an ionizable lipid compound and a preparation method and application thereof. Background Art

[0002] In recent years, nucleic acids have attracted attention as multiple gene therapy products have been used for 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 such as DNA plasmids (pDNA), minicircular 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).

[0003] Nucleic acids are macromolecules with a large amount of negative charge, high molecular weight and hydrophilicity. They have poor permeability on cell membranes and low stability in the body, and are easily cleared by the host. Therefore, delivering nucleic acids to active sites in cells is the most challenging part of gene therapy. Especially when delivering RNA molecules, because RNA molecules usually have short-term activity, low retention rate in cells, and need to be administered more frequently, 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 from nuclease activity in the blood, enhance and assist cellular absorption, and promote endosomal escape after entering the cell.

[0004] 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 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 parts, namely ionizable lipids (or cationic lipids), auxiliary 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 to protect nucleic acids from degradation, but also neutralizes the negative charge of nucleic acids, thereby achieving successful delivery of nucleic acids; phospholipids as "auxiliary lipids" help improve endosomal release and transfection efficiency; cholesterol helps to enhance stability; and PEG lipids promote the formation of homogeneous lipid nanoparticles, which improve stability and circulation time by preventing interactions with blood components and being cleared. The ratio of these components is crucial to 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, a protonation reaction occurs, making it easier for lipids to interact with the negatively charged endosomal membrane, thereby promoting the fusion process of endosomal destruction and nucleic acid release, thereby achieving efficient delivery. Therefore, the structure and function of ionizable lipids play a very important role in both delivery and lysosomal escape, so their design and optimization are crucial.

[0005] Based on this, the present invention provides an ionizable lipid compound and a preparation method and application thereof. The ionizable lipid compound provided by the present invention belongs to a single-tail chain multi-head lipid class with good safety, and can be used to prepare a lipid composition for delivering therapeutic agents. The lipid nanoparticles prepared therefrom can show good effectiveness and safety in cells and animals. Summary of the invention

[0006] The present invention aims at the problems existing in the prior art and provides an ionizable lipid compound and a preparation method and application thereof. The ionizable lipid compound provided by the present invention belongs to a single-tail chain multi-head lipid with good safety, can be used to prepare a lipid composition for delivering a therapeutic agent, and the lipid nanoparticles prepared therefrom can show good effectiveness and safety in cells and animals.

[0007] To achieve the above object, in a first aspect, the present invention provides an ionizable lipid compound, wherein the ionizable lipid compound is a compound of formula (1), or a salt, stereoisomer, or tautomer thereof:

[0008]

[0009] Where A1 is NH or O;

[0010] R a Selected from C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 The cycloalkyl group of C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 The cycloalkyl group of R is a straight chain or branched chain structure; b and R c Each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkynyl, C3-C 12 Cycloalkane, C6-C 12 Aromatic hydrocarbon groups, C1-C 12 Alkyl alcohol, C1-C 12 Heterocyclic groups, alkylamines;

[0011] 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, or -R c '-A1'-R c ”-NH2;

[0012] Provided that, when A1' is -CO-NH-, -NH-CO- or -CO-O-, R c ' is a C1-C6 alkyl group; when A1' is -CO-, R c 'Does not exist.

[0013] In a second aspect, the present invention further provides an ionizable lipid compound, characterized in that the ionizable lipid compound is a compound of formula (1), or a salt, stereoisomer, or tautomer thereof:

[0014]

[0015] R a Selected from C6-C 24 Alkyl alcohol, C6-C24 Short chain polyethylene oxide; the C6-C 24 Alkyl alcohol, C6-C 24 Short-chain polyoxyethylene is a straight chain or branched chain structure; R b and R c Each independently selected from C1-C 12 Alkyl, C2-C 12 Alkenyl, C1-C 12 Alkynyl, C3-C 12 Cycloalkane, C6-C 12 Aromatic hydrocarbon groups, C1-C 12 Alkyl alcohol, C1-C 12 Heterocyclic groups, alkylamines;

[0016] 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, or -R c '-A1'-R c ”-NH2;

[0017] Provided that, when A1' is -CO-NH-, -NH-CO- or -CO-O-, R c ' is a C1-C6 alkyl group; when A1' is -CO-, R c 'Does not exist.

[0018] In a preferred embodiment, R b and R c Each independently selected from C1-C 12 Alkyl alcohols, alkyl amines.

[0019] In a preferred embodiment, provided that when R a For a straight chain C6-C 24 When A1 is not NH, or R b and R c Not C1-C 12 Alkyl alcohol; when R a C6-C 24 When alkyl alcohol, A1 is not O, or R b and R c Not for C6-C 24 alkyl.

[0020] In a preferred embodiment, the condition is that when R a C6-C 24 In the case of alkenyl, A1 is NH, R b and R c Each independently selected from C1-C 12 Alkyl alcohols;

[0021] When R a For branched C6-C 24 When it is alkyl, A1 is NH, R b and R c Each independently selected from C1-C 12 Alkyl alcohols, alkyl amines;

[0022] R a For a straight chain C6-C 24 When it is alkyl, A1 is NH or O, R b and R c Each independently selected from alkylamines;

[0023] When R a For a straight chain C6-C 24 When it is alkyl, A1 is O, R b and R c Each independently selected from C1-C 12 Alkyl alcohols;

[0024] When R a C6-C 24 In the case of short-chain polyoxyethylene, A1 is NH or O, R b and R c Each independently selected from alkylamines;

[0025] Or when R a C6-C 24 In the case of alkyl alcohol, A1 is NH or O, R b and R c Each is independently selected from alkylamines.

[0026] In a preferred embodiment, R a Select from the following compound structures:

[0027]

[0028] In a preferred embodiment, R b and R c Select from the following compound structures:

[0029]

[0030] R d Selected from C1-C6 alkanes or cycloalkanes.

[0031] In a preferred embodiment, the compound of formula (1) is selected from at least one of the following compounds:

[0032]

[0033]

[0034]

[0035] In a third aspect, the present invention provides a method for preparing the aforementioned ionizable lipid compound, comprising the following reaction steps:

[0036] R a ”-NH2① reacts with α,β-unsaturated carbonyl compound② to generate ionizable lipid compound③:

[0037]

[0038] Among them, R a -NH2 is R a -NH2 or

[0039] 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.

[0040] In a fourth aspect, the present invention provides a method for preparing the aforementioned ionizable lipid compound, comprising:

[0041] 1) R a ”-NH2① reacts with α,β-unsaturated carbonyl compound④ to form compound⑤;

[0042] 2) Compound ⑤ reacts with a nucleophile ⑥ to generate an ionizable lipid compound ③;

[0043]

[0044] Among them, the nucleophile ⑥ is R b -NH2 or R b -OH; R a -NH2 is selected from R a -NH2 or

[0045]

[0046] 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; Z2 is a leaving group, and Z2 reacts with NH2 to obtain A1.

[0047] In a fifth aspect, the present invention provides a method for preparing the aforementioned ionizable lipid compound, comprising:

[0048] 1) R a ”-NH2① reacts with α,β-unsaturated carbonyl compound② to form compound⑦;

[0049] 2) Compound ⑦ reacts with an α,β-unsaturated carbonyl compound ⑧ to form compound ⑨;

[0050] 3) Compound ⑨ reacts with nucleophile ⑩ to generate ionizable lipid compound

[0051]

[0052] Among them, R a -NH2 is R a -NH2, Z3 is a leaving group, A3 reacts with Z3 to obtain A1.

[0053] The leaving group mentioned above refers to the leaving part in the nucleophilic reaction or condensation reaction, including but not limited to: H,

[0054] 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 (RO-), amino (-NHR, wherein R is an alkyl or aryl group from which H is removed), phenoxy, tertiary carbon cation (such as tert-butyl cation), carbon cation stabilized by an unsaturated system or a heteroatom, or the various protecting groups mentioned above.

[0055] In a preferred embodiment, in Formula 1a-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 a bifurcated carbon-carbon bond, a carbon-oxygen bond, a carbon-nitrogen bond, a carbon-sulfur bond or a carbon-selenium bond.

[0056] In a preferred embodiment, 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;

[0057] The compound of formula (1) having a terminal amino group is

[0058] 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.

[0059] In a preferred embodiment, the reaction formula 1a-1c further comprises 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;

[0060] The compound of formula (1) having a terminal amino group is

[0061] 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.

[0062] In a preferred embodiment, the raw materials used in the reaction process also contain a protecting group, and the reaction steps include protection and / or deprotection steps.

[0063] In a fourth aspect, the present invention provides the use of the aforementioned ionizable lipid compound in an emulsifier, suspending agent, dispersant, solubilizer, lubricant, thickener, antibacterial agent or preservative.

[0064] In a fifth aspect, the present invention provides use of the aforementioned ionizable lipid compound in the preparation of a cosmetic composition.

[0065] In a sixth aspect, the present invention provides the use of the aforementioned ionizable lipid compound in the preparation of a lipid composition, characterized in that the ionizable lipid compound is used as a drug delivery carrier.

[0066] In a seventh aspect, the present invention provides a lipid composition comprising the aforementioned ionizable lipid compound.

[0067] In a preferred embodiment, the lipid component of the lipid composition comprises other lipids, including phospholipids, cholesterol and PEG-conjugated lipids.

[0068] 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.

[0069] In a preferred embodiment, the other ionizable lipids are selected from at least one of the following compounds:

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] 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 preventive agent; and the active ingredient accounts for 0.1-50% (w / w) of the total prescription.

[0076] In a preferred embodiment, the therapeutic and / or prophylactic agent is a nucleic acid; the nucleic acid is DNA or RNA.

[0077] In an eighth aspect, the present invention provides use of the aforementioned lipid composition in the preparation of a medicament for treating a disease in a mammal.

[0078] In a preferred embodiment, 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.

[0079] In a ninth aspect, the present invention provides a pharmaceutical composition comprising the aforementioned lipid composition and a pharmaceutically acceptable carrier.

[0080] In a preferred embodiment, the pharmaceutically active compound is selected from anti-inflammatory compounds, steroids, statins, estradiol, BTK inhibitors, S1P1 agonists, glucocorticoid receptor modulators and antihistamines. Compared with the prior art, the present invention has the following beneficial effects:

[0081] 1. The ionizable lipid compound provided by the present invention has a good surfactant and can be used to prepare lipid nanoparticles for use as a drug delivery carrier.

[0082] 2. The ionizable lipid compound of the present invention is suitable for preparing lipid compositions. The mRNA lipid composition prepared using the compound of the embodiment has a Zeta potential of -20mV-20mV, a dispersion coefficient of <0.5, a particle size of 10-200nm, and an encapsulation rate of >60%.

[0083] 3. The lipid nanoparticles of mRNA and / or other nucleic acid substances (such as siRNA, microRNA, pDNA, etc.) prepared by the ionizable cationic lipid compound of the present invention exhibit biological activity in vitro and in vivo in animals and have low toxicity. This shows that the lipid nanoparticles provided by the present invention have good safety and effectiveness as drug carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 Luciferase activity of luciferase mRNA lipid nanoparticles prepared for compound 6008 in HEK293T cells.

[0085] Figure 2 Whole-body in vivo imaging of luciferase mRNA lipid nanoparticles prepared for compound 6020 6h / 12h after intravenous administration to mice. DETAILED DESCRIPTION

[0086] The synthesis process of the present invention can tolerate multiple functional groups, so various substituted starting materials can be used. These processes generally provide the desired final compound at the end or near the end of the entire process, but 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 a variety of ways using commercially available starting materials, compounds known in the literature, or by easily prepared intermediates, by using standard synthetic methods and procedures known to those skilled in the art or obvious to the technician based on the teachings of this article. Standard synthetic methods and procedures for preparing organic molecules and 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 method is designed to illustrate but not limit the general procedures for preparing the compounds of the present invention.

[0087] 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 (e.g., R1, R2, and R3, etc.) in each general synthetic route are as defined herein. One of ordinary skill in the art should note that in the reaction procedures and synthetic schemes described herein, the order of certain steps may vary, such as the introduction and removal of protecting groups.

[0088] In the reaction scheme described herein, multiple stereoisomers can be produced. When a specific stereoisomer is not indicated, this should be understood to include all possible stereoisomers produced by the reaction. It will be appreciated by those of ordinary skill in the art that the reaction can be optimized to preferentially obtain a type of isomer, or a new scheme can be designed to produce a single isomer. If a mixture is produced, isomers can be separated using techniques such as preparative thin layer chromatography, preparative HPLC, preparative chiral HPLC or preparative SFC.

[0089] Example 1 Synthesis of ionizable lipid compounds

[0090] 1. Preparation of Compound 6001

[0091] Structural formula:

[0092] Molecular weight: 499.43

[0093] 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide were added to 6.4 mL of ethanol, and the mixture was heated to 70°C with stirring. After 3.5 h, 0.8 g of hydroxyethyl acrylamide was added, and the reaction was continued for 16 h. The mixture was passed through a reverse column to obtain compound 6002.

[0094] 0.7 g of compound 6002, 0.4 g of palladium on carbon and 28 mL of tetrahydrofuran were added to a reaction bottle and stirred. The gas was replaced with hydrogen and the pressure was maintained with a hydrogen balloon. After 16 h, the mixture was filtered and passed through a column (methanol-DCM system) in the forward direction to obtain 200 mg of compound 6001. 1 H NMR (400MHz, CDCl3) δ7.54(t,J=5.5Hz,2H),3.79-3.68(m,4H),3.42(dd,J=10.0,5.4Hz,4H),2.89(t,J =6.0Hz, 4H), 2.55 (dt, J = 11.8, 6.8Hz, 6H), 1.54 (s, 2H), 1.27 (d, J = 11.2Hz, 32H), 0.91 (t, J = 6.8Hz, 3H).

[0095] 2. Preparation of Compound 6002

[0096] Structural formula:

[0097] Molecular weight: 497.42

[0098] 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide were added to 6.4 mL of ethanol, stirred and heated to 70°C. After 3.5 h, 0.8 g of hydroxyethyl acrylamide was added. After the reaction was continued for 16 h, the organic solvent was concentrated, the mixture was passed through a reverse column and freeze-dried to obtain 400 mg of compound 6002. 1 H NMR(400MHz, CDCl3) δ7.31(s,2H),5.38(dd,J=13.0,7.4Hz,2H),3.79-3.66(m,4H),3.41(dd,J=10.1,5.4Hz,4H),2. 82-2.72(m,4H),2.44(dd,J=15.1,9.2Hz,6H),2.10-1.99(m,4H),1.46(s,2H),1.29(s,24H),0.90(t,J=6.8Hz,3H).

[0099] 3. Preparation of Compound 6003

[0100] Structural formula:

[0101] Molecular weight: 445.34

[0102] 4.00 g of tetradecylamine, 6.53 g of 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 H NMR(400MHz, CDCl3)δ4.29(t,J=7.5Hz,4H),3.89-3.75(m,4H),2.81(t,J=6.2Hz,4H), 2.50(ddd,J=23.2,14.1,7.1Hz,6H),1.46(s,2H),1.28(s,24H),0.91(t,J=6.3Hz,3H).

[0103] 4. Preparation of Compound 6004

[0104] Structural formula:

[0105] Molecular weight: 553.48

[0106] 0.8 g of oleylamine, 1.5 g of n-butyl acrylate and 5.6 mL of n-butanol were added to the reaction flask, stirred and heated to 100°C. After 4 hours, 1 mL of n-butyl acrylate was added, and the reaction was continued for 1 hour. Then, the mixture was passed through a normal phase column (petroleum ether-ethyl acetate system), and 0.4 g of sodium hydroxide, 1 mL of water, and 10 mL of methanol were added, and stirred for hydrolysis for 30 minutes. 1 mL of concentrated hydrochloric acid and 10 mL of methanol were added to obtain a mixed solution, and the pH was adjusted to neutral. The solution was spin-dried, dissolved in DCM, dried over anhydrous magnesium sulfate, filtered, and the solvent was spin-dried. 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 hours. After concentrating the organic solvent, the mixture was passed through a reverse phase column and freeze-dried to obtain 240 mg of compound 6004. 1 HNMR (400MHz, CDCl3) δ7.58(d,J=16.6Hz,2H),5.44-5.31(m,2H),3.72(d,J=16.5Hz,4H),3.31(d,J=5.6Hz,4H),3.17( s,4H),2.84(s,2H),2.69(s,4H),2.09-2.01(m,4H),1.67(d,J=2.7Hz,10H),1.37-1.26(m,24H),0.91(t,J=6.8Hz,3H).

[0107] 5. Preparation of Compound 6005

[0108] Structural formula:

[0109] Molecular weight: 597.54

[0110] 25.00 g of 11-henicosone, 62.05 g of ammonium acetate and 500 mL of methanol were added to a reaction flask and stirred. 6.55 g of sodium cyanoborohydride was added. After reacting for 16 h, 250 mL of water and 250 mL of DCM were added, the liquids were separated, the aqueous phase was extracted with 50 mL of DCM, and the organic phases were combined and passed through a normal phase column (methanol-DCM system) to obtain 21.00 g of compound 6005-A.

[0111] 21.00 g of compound 6005-A, 58 mL of n-butyl acrylate and 100 mL of n-butanol latex were stirred in a reaction bottle and heated to 100° C. 10 mL of n-butyl acrylate was added and after 16 hours, the mixture was passed through a column (petroleum ether-ethyl acetate system) to obtain 31.00 g of compound 6005-B.

[0112] A solution prepared by mixing 31.00 g of compound 6005-B and 6.55 g of sodium hydroxide, 310 mL of methanol and 31 mL of water was added to a reaction flask and stirred. After 20 minutes, 150 mL of THF was added, and the temperature was raised to 50°C with stirring. After 30 minutes, 16.13 g of concentrated hydrochloric acid and 160 mL of methanol were added and stirred. After 30 minutes, the solvent was dried by spin drying, 300 mL of DCM was added to dissolve, the mixture was dried over anhydrous magnesium sulfate, filtered and dried to obtain 27.00 g of oily compound 6005-C.

[0113] 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 were added to a reaction flask and stirred. After 18 hours, the organic solvent was concentrated and passed through a column (methanol-DCM system) to remove excess 4-amino-1-butanol compound to obtain 6.80 g of compound 6005. 1 HNMR (400MHz, CDCl3) δ3.70(t,J=5.6Hz,4H),3.28(t,J=5.6Hz,4H),2.74(t,J=6.0Hz,4H),2.40- 2.45(m,1H),2.34(t,J=6.4Hz,4H),1.63-1.65(m,8H),1.25-1.35(m,36H),0.91(t,J=6.4Hz,6H).

[0114] 6. Preparation of Compound 6006

[0115] Structural formula:

[0116] Molecular weight: 553.48

[0117] Add 1.00g oleylamine, 0.47g hydroxyethyl acrylamide and 8mL ethanol to the reaction bottle, stir and heat to 70°C. After 13h, spin dry the solvent and pass through a column (methanol-DCM system) to obtain 800mg of intermediate 1. Add 1.5mL n-butyl acrylate and 5mL n-butanol, stir and heat to 100°C. After 3h, spin dry the solvent and pass through a column (methanol-DCM system). Add 0.4g sodium hydroxide and 10mL methanol, 1mL water to make a solution, stir for 1h, add 1mL concentrated hydrochloric acid and 8mL methanol to make a solution, stir for 30min, spin dry the solvent and add 100mL DCM to dissolve, dry over anhydrous magnesium sulfate, filter, spin dry, add 0.45g 6-amino-1-hexanol, 0.27g HOBT, 0.78g EDCI and 10mL DCM to stir, concentrate the organic solvent after 14h, pass through a column (methanol-DCM system), and obtain 300mg of compound 6006. 1HNMR(400MHz, CDCl3)δ7.83(s,1H),6.95(s,1H),5.43-5.28(m,2H),3.74-3 .67(m,2H),3.64(t,J=6.3Hz,2H),3.39(dd,J=10.0,5.3Hz,2H),3.24(dd,J= 12.9,6.7Hz,2H),2.74(t,J=5.8Hz,4H),2.48-2.42(m,2H),2.41-2.33(m,4 H), 2.01 (dd, J = 13.6, 6.8 Hz, 4H), 1.66-1.17 (m, 34H), 0.89 (t, J = 6.7 Hz, 3H).

[0118] 7. Compound 6007

[0119] Structural formula:

[0120] Molecular weight: 953.78

[0121]

[0122] At room temperature, add compound octadecylamine (50 g) to a three-necked flask, add MeOH (200 mL) and stir to mix, cool to 5 degrees Celsius in an ice-water bath, slowly drop methyl acrylate (31.5 g), stir to mix, return to room temperature, and react at room temperature for 4 hours; 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 keep the 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, compound 3 (2 g) was added to a single-mouth bottle, MeOH (20 mL) was added and stirred to dissolve, and then methyl acrylate (2.5 g) was slowly added and stirred to mix, and the temperature was raised to 60°C and left overnight; the reaction was stopped, and the reaction solution was concentrated under reduced pressure, and purified by column chromatography (DCM: MeOH = 50: 1-20: 1) to obtain compound 4 (2.5 g). At room temperature, compound 4 (2.3 g) and ethylenediamine (20 mL) were added to a single-mouth bottle, and the reaction was left overnight at room temperature; the reaction was stopped, and the reaction was directly concentrated under reduced pressure to obtain compound 6007 (24 mg). 1H NMR (400MHz, Methanol-d4) δ3.87(t,J=6.6Hz,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=6.5Hz,4H) ,1.90(dtdd,J=21.2,14.0,9.5,6.9Hz,4H),1.73(p,J=7.7Hz,4H),1.49(tdd,J=9.2,7.9,7.1,4.0Hz,4H),1.31(s,28H),0.92(t,J=6.8Hz,3H).

[0123] 8. Compounds 6008-6018

[0124] Synthesis of compound 6009

[0125] Structural formula:

[0126] At room temperature, the methanol (20mL) solution of freshly recrystallized hexadecylamine (0.03mol) was added dropwise to a solution in the methanol (20mL) of stirred methyl methacrylate (6mL) under a nitrogen atmosphere, and the reaction was overnight. The reactant was spin-dried at room temperature, and the residue was dissolved in chloroform and washed twice with 0.1M NaOH solution. The chloroform solution was collected and dried over anhydrous calcium chloride. Then a colorless oil was obtained by column chromatography. 1H-NMR (300MHz, CDCl3): 0.78 (t, 3H), 1.16 (s, 30H), 2.38 (m, 6H), 2.71 (t, 4H), 3.57 (m, 6H).

[0127] Then, a solution of the above colorless oil (11.05 g) in methanol (20 ml) was added to a solution of 1,2-diaminoethane (75 g) in methanol (100 ml) which was stirred vigorously 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. Excess 1,2-diaminoethane was removed with 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 repeatedly recrystallized with chloroform and cyclohexane to obtain a white solid. 1H-NMR (300MHz, 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).

[0128] The preparation methods of 6008 and 6009 differ only in that an equimolar amount of tetradecylamine is used to replace hexadecylamine. 6010-6017 refer to the preparation method of 6009, using an equimolar amount of the corresponding R a It is prepared by replacing hexadecylamine with -NH2.

[0129] 6018 was prepared by referring to the method of 6007, using an equimolar amount of the corresponding R a -NH2 is used to replace octadecylamine.

[0130] 9. Compound 6019

[0131] Structural formula:

[0132] Molecular weight: 753.66

[0133]

[0134] At room temperature, compound 2483-46-7 (3 g) was added to a single-necked bottle, DCM (50 mL) was added and stirred, and then 18807-71-1 (2.5 g), DCC (2.7 g), and DMAP (1.6 g) were added, stirred, and reacted overnight at room temperature; the mixture was concentrated under reduced pressure and purified by column chromatography (DCM: MeOH = 5:1) to obtain product 3 (4 g). At room temperature, product 3 (5.0 g), MeOH (50 mL) and Pd / C (1.0 g) were added to a three-necked bottle, hydrogen was replaced, the temperature was raised to 50°C, and stirred for 1 h; the reaction was stopped, the reaction solution was cooled to room temperature, filtered, and the filtrate was dried to obtain product 4 (3.5 g). At room temperature, raw material 5 (1.2 g) was added to a single-mouth bottle, DMF (50 mL) was added and stirred to dissolve, and then product 4 (3.3 g), HATU (3.2 g), and TEA (0.9 g) were added and stirred to mix, and the mixture was reacted overnight at room temperature; water (500 mL) and EA (500 mL) were added, and the mixture was separated and extracted, and the organic phase was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (DCM: MeOH = 20: 1-10: 1) to obtain solid 6 (380 mg). At room temperature, solid 6 (380 mg) and HCl / Dioxane (4M) (1.6 mL) were added to a single-mouth bottle, and the mixture was reacted for 2 h at room temperature; the mixture was concentrated under reduced pressure and purified to obtain compound 6019 (220 mg). 1HNMR (400MHz, Methanol-d4) δ3.87(t,J=6.6Hz,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=6.5Hz,4H ), 1.90 (dtdd, J = 21.2, 14.0, 9.5, 6.9Hz, 4H), 1.73 ( p, J = 7.7Hz, 4H), 1.49 ( tdd, J = 9.2, 7.9, 7.1, 4.0Hz, 4H), 1.31 ( s, 28H), 0.92 ( t, J = 6.8Hz, 3H).

[0135] 10. Compound 6020

[0136] Structural formula:

[0137] Molecular weight: 810.19

[0138]

[0139] At room temperature, add compound octadecylamine (50 g) to a three-necked flask, add MeOH (200 mL) and stir to mix, cool to 5 degrees Celsius in an ice-water bath, then drop methyl acrylate (31.5 g), stir to mix, return to room temperature, and react for 4 hours; stop the reaction, directly concentrate under reduced pressure, purify by column chromatography (PE: EA = 20: 1-10: 1), and 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 keep the 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, compound 3 (1.3 g) was added to a single-necked bottle, DMF (20 mL) was added and stirred, and then 35897-34-8 (3.6 g), EDCI (3.0 g), HOBt (2.1 g), and DIEA (2.0 g) were added, stirred, and reacted at room temperature overnight; water (200 mL) was added and the mixture was directly freeze-dried under reduced pressure to obtain compound 4 (4 g). Compound 4 (100 mg) and HCl / Dioxane (4 M) (2 mL) were added to a single-necked bottle at room temperature and reacted for 1 h. The reaction was stopped and the reaction solution was concentrated under reduced pressure 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).

[0140] 11. Compound 6021

[0141] Structural formula:

[0142] Molecular weight: 1182.79

[0143]

[0144] At room temperature, compound 2 (3.5 g, the synthesis process is the same as that of product 2 in compound 6020) was added to a single-necked bottle, MeOH (20 mL) was added and stirred, TREN (23.0 g) was added, the mixture was stirred, the temperature was raised to 60°C, the mixture was stirred overnight, and the mixture was concentrated under reduced pressure. The crude product was lyophilized to obtain product 3 (25 g). At room temperature, product 3 (22.0 g) and DCM (200 mL) were added to a single-necked bottle, the temperature was lowered to 0°C, (Boc)2O (71.7 g) was slowly added dropwise, the temperature was restored to room temperature after the addition was completed, the reaction was carried out for 3 h, water (200 mL) was added for extraction, and DCM (100 mL) was added for separation and extraction. The organic phase was dried over sodium sulfate, concentrated under reduced pressure, and purified by column to obtain yellow oil 4 (5.8 g). At room temperature, yellow oil 4 (1.4 g) was added to a single-mouth bottle, HCl / Dioxane (4M) (15 mL) was slowly added under an ice-water bath and stirred and mixed, the temperature was restored to room temperature after addition, the reaction was continued for 0.5 h, and the mixture was concentrated under reduced pressure to obtain compound 5 (800 mg). At room temperature, compound 5 (1.0 g), (S)-2,6-di-tert-butyloxycarbonylaminohexanoic acid (2.6 g), EDCI (1.4 g), HOBt (1.0 g), DIEA (1.0 g) and solvent DMF (10 mL) were added to a single-mouth bottle, the reaction was continued overnight at room temperature, 100 mL of water and 100 mL of EA were added, the mixture was separated and extracted, the EA phase was washed once with 100 mL of saturated brine, concentrated under reduced pressure, and purified by column chromatography (DCM: MeOH = 10: 1) to obtain compound 6 (1.1 g). Into a single-necked bottle at room temperature, compound 6 (1.2 g), HCl / Dioxane (4M) (10 mL) and solvent DCM (10 mL) were added. The reaction was allowed to react overnight at room temperature. The mixture was concentrated under reduced pressure and purified to obtain compound 6021 (220 mg). 1H NMR(400MHz, Methanol-d4)δ3.96(t,J=6.6Hz,4H),3.75(dt,J=14.0,6.6Hz,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=6.9Hz,4H),2.05-1.82(m,8H),1.73(p,J=7.7Hz ,10H),1.53(qd,J=8.3,7.7,4.3Hz,8H),1.30(s,28H),0.97-0.87(m,3H).

[0145] 12. Compound 6023

[0146] Structural formula:

[0147] Molecular weight: 670

[0148]

[0149] At room temperature, compound dodecylamine (10 g) was added to a three-necked flask, MeOH (100 mL) was added and stirred, then cooled to 5°C in an ice-water bath, methyl acrylate (10.2 g) was slowly added dropwise, stirred, and returned to room temperature. The reaction solution was allowed to react at room temperature for 4 hours, then the reaction was stopped, concentrated under reduced pressure, and purified by column chromatography (PE: EA = 20: 1-10: 1) to obtain product 2 (19 g). At room temperature, product 2 (19.0 g), MeOH (200 mL) and ethylenediamine (127.7 g) were added to a single-necked flask, the temperature was raised to 60°C, the temperature was maintained for overnight reaction, and the compound 3 (20 g) was obtained after concentration under reduced pressure. 500 mg was purified and lyophilized to obtain compound 6023-1 (218 mg). At room temperature, add raw material 6023-1 (1.4 g) to a single-mouth bottle, add DCM (20 mL) and stir to dissolve, then add (S)-2,6-di-tert-butyloxycarbonylaminocaproic acid (3.4 g), EDCI (1.9 g), DMAP (1.2 g) and stir to mix, keep at room temperature, react overnight, separate and extract, dry, and concentrate under reduced pressure. Compound 4 (3.0 g) is obtained. At room temperature, add compound 4 (2.8 g) and HCl / 1,4-Dioxane (4M) (20 mL) to a single-mouth bottle, react for 2 h at room temperature; concentrate under reduced pressure, purify, and freeze-dry to obtain compound 6023 (123 mg). 1H NMR(400MHz, Methanol-d4)δ3.88(t,J=6.6Hz,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=6.5Hz,4H),2.00-1.82(m,4H),1.82-1.66(m,6H),1.50(qd,J=8.2,7.8,4.2Hz,4H),1.45-1.25(m,18H),0.96-0.87(m,3H).

[0150] 13. Compound 6024

[0151] Structural formula:

[0152] Molecular weight: 870.24

[0153]

[0154] At room temperature, product 6023-1 (1.4 g, the synthesis method is the same as 6023-1 in compound 6023) was added to a single-mouth bottle, MeOH (20 mL) was added and stirred to dissolve, and then methyl acrylate (2.8 g) was slowly added and stirred to mix. The temperature was raised to 60°C, and the reaction was carried out overnight at this temperature. The mixture was concentrated under reduced pressure to obtain compound 4 (2.0 g). At room temperature, compound 4 (2.4 g) and ethylenediamine (20 mL) were added to a single-mouth bottle, and the reaction was carried out overnight at room temperature. The mixture was concentrated under reduced pressure and purified to obtain compound 6024 (191 mg). 1HNMR (400MHz, Methanol-d4) δ3.69(t,J=6.0Hz,4H),3.60-3.48(m,20H),3.42(t,J=6.1Hz,4H),3.25-3.19(m,2H),3 .12(t,J=5.8Hz,8H),2.83(t,J=6.5Hz,12H),1.78(tt,J=11.0,6.4Hz,2H),1.51-1.18(m,20H),0.91(t,J=6.7Hz,3H).

[0155] 14. Compound 6026

[0156] Structural formula:

[0157] Molecular weight: 443.67

[0158]

[0159] Compound 6026 was synthesized by referring to the synthetic route of compound 6019, except that an equal molar amount of compound 1 was used to replace the raw material 5 of compound 6019, and an equal molar amount of N-(tert-butyloxycarbonyl)ethanolamine was used to replace the intermediate 4 of compound 6019. 1H NMR (300MHz, 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).

[0160] Compounds 6025-6034 were obtained by referring to the synthetic route of the aforementioned compounds, except that hydroxyethylamine was used instead of the corresponding ethylenediamine, and the amino group of the hydroxyethylamine was first protected with a protecting group such as Fmoc or Boc, and the protecting group was removed by conventional methods after the reaction was completed.

[0161] Example 2 Preparation of lipid nanoparticles

[0162] Luciferase mRNA was diluted in 50 mM sodium citrate buffer solution at pH 4.0, and the concentration of the mRNA solution was 135 μg / ml; a lipid mixed ethanol solution was prepared according to the molar percentage ratio of 15:35:10:38.5:1.5 of ionizable lipid compound (ionizable lipid compound prepared in Example 1): other ionizable lipids: DSPC: cholesterol: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159); the mRNA solution and the lipid mixed solution were mixed in a nano drug preparation equipment at a volume ratio of 3:1, and then ultrafiltration was performed to obtain luciferase mRNA lipid nanoparticles, as shown in Table 1.

[0163] Table 1 Preparation of lipid nanoparticles

[0164]

[0165]

[0166] Comparative Example 1

[0167] Luciferase mRNA was diluted in 50mM sodium citrate buffer solution at pH 4.0, and the concentration of the mRNA solution was 135μg / ml; a lipid mixed ethanol solution was prepared in a molar ratio of 15:35:10:38.5:1.5 of SM102: other ionizable lipids (ALC-0315): DSPC: cholesterol: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159); the mRNA solution and the lipid mixed solution were mixed in a nano drug preparation device at a volume ratio of 3:1, and then ultrafiltration was performed to obtain luciferase mRNA lipid nanoparticles.

[0168] Comparative Example 2

[0169] Luciferase mRNA was diluted in 50mM sodium citrate buffer solution at pH 4.0, and the concentration of the mRNA solution was 135μg / ml; a lipid mixed ethanol solution was prepared in a molar ratio of 50:10:38.5:1.5 of SM102:DSPC:cholesterol:2-[(polyethylene glycol)-2000]-N,N-dioctyl acetamide (ALC-0159); the mRNA solution and the lipid mixed solution were mixed in a nano drug preparation device at a volume ratio of 3:1, and then ultrafiltration was performed to obtain luciferase mRNA lipid nanoparticles.

[0170] Example 3 Determination of particle size, zeta potential and encapsulation efficiency of lipid nanoparticles

[0171] 1. Particle size and polydispersity index (PDI) determination: Malvern ZetaSizer Nano ZS90 was used to determine the average particle size and PDI of the sample solution of lipid nanoparticles in Example 2 by dynamic light scattering. The measurement angle was 90°, the refractive index of the dispersant was 1.330, and the test temperature was 25°C.

[0172] 2. Zeta potential: The zeta potential of the sample solution of lipid nanoparticles in Example 2 was measured using Malvern ZetaSizerNano ZS90 based on electrophoretic light scattering (ELS) technology, with a dispersant refractive index of 1.330 and a test temperature of 25°C.

[0173] 3. Encapsulation efficiency: According to the manufacturer's instructions, the Quant-it Ribogreen RNA Quantification Assay Kit (ThermoFisher Scientific, UK) was used to determine the encapsulation efficiency of luciferase mRNA in the lipid nanoparticles in Example 2. The average particle size, PDI, Zeta potential and encapsulation efficiency data of each lipid nanoparticle are shown in Table 2.

[0174] Table 2 Summary of apparent pKa, particle size, PDI, Zeta potential and encapsulation efficiency of lipid nanoparticles

[0175] Compound No. Particle size nm PDI Potential mV Encapsulation rate% 6001 107.2 0.140 -3.06 100 6002 52.81 0.108 3.47 97.8 6003 65.54 0.112 -10.5 89.1 6004 48.72 0.07 3.34 96.5 6005 58.67 0.190 2.93 97.7 6006 74.43 0.067 -9.82 80.6 6007 62.94 0.122 2.82 89.4 6015 54.43 0.036 10.1 80.7 6019 79.50 0.146 0.76 80.3 6020 90.01 0.098 -10.0 85.6 6023 42.84 0.071 9.42 90.2 6024 42.44 0.088 10.8 84.5 6026 51.34 0.110 5.41 86.4 Comparative Example 1 90.45 0.201 5.60 79.5 Comparative Example 2 80.02 0.178 4.82 80.5

[0176] Example 4 Evaluation of in vitro cell activity of lipid nanoparticles

[0177] HEK-293T cells were used to evaluate the in vitro transfection efficiency of each lipid nanoparticle composition and the comparative lipid nanoparticle composition. HEK293T cells were routinely cultured with DMEM + 10% FBS medium to ensure that the cells were in the logarithmic growth phase; one day before transfection, the cells were seeded on a 96-well culture plate at an appropriate cell density and grown overnight. During transfection, the cell fusion degree should reach 70-90%; the lipid nanoparticle composition prepared by each exemplary compound was diluted into 4 different dose concentrations with DMEM, and added to a 96-well cell culture plate, so that the concentrations in each well reached 400ng, 200ng, 100ng and 50ng, respectively, and the luciferase plasmid was transfected with Lipofectamine 2000 as a positive reference (PC). After incubation for 24 h in a 37°C, 5% CO2 incubator, substrate was added to the wells and luciferase activity was measured using a microplate reader. The results are shown in Table 3. The luciferase activity of luciferase mRNA lipid nanoparticles prepared by compound 6006 in HEK293T cells is shown in Appendix Figure 1 .

[0178] Table 3 Luciferase activity of luciferase mRNA lipid nanoparticles in HEK293T cells

[0179]

[0180]

[0181] As can be seen from Table 2, the fluorescence intensity of luciferase expressed by the luciferase mRNA lipid nanoparticles prepared by using the ionizable lipid compound of the embodiment in HEK 293T cells (dose: 100 ng, 96-well plate) is >10 6 RLU, and is better than Comparative Examples 1-2, indicating that the luciferase mRNA lipid nanoparticles prepared using the ionizable lipid compound of the present invention exhibit good biological activity in in vitro cells and have low cytotoxicity.

[0182] From the attached Figure 1 It can be seen that at different doses (50-400 ng), the fluorescence intensity of luciferase expressed by luciferase mRNA lipid nanoparticles prepared by compound 6006 in HEK 293T cells was >10 7 RLU, and the fluorescence intensity increased with the increase of dose, indicating that the expression of luciferase increased accordingly with the increase of dose, and the prepared lipid nanoparticles had good mRNA delivery ability and safety.

[0183] Example 5 Evaluation of in vivo activity of lipid nanoparticles

[0184] The in vivo transfection efficiency and safety of lipid nanoparticle compositions were evaluated using female Balb / c mice aged 6-8 weeks. Lipid nanoparticles prepared by compounds 6008, 6015, 6019-6021, 6023, 6024 and Comparative Example 1 were administered by tail vein injection, with a single dose of 0.3 mpk. The animals were imaged in vivo using a PerkinElmer small animal imaging system at specific time points (e.g., 6h, 24h) after administration, and the bioluminescent signals were measured. The results are shown in Table 4. Whole-body live imaging of luciferase mRNA lipid nanoparticles prepared by compound 6020 at 6h / 12h after intravenous administration of mice is shown in the attached Figure 2 .

[0185] Table 4 Summary of in vivo / in vitro activity evaluation of lipid nanoparticles prepared by compounds

[0186]

[0187] As can be seen from Table 3, after 6 h of administration of lipid nanoparticles prepared by compounds 6008, 6015, 6019-6021, 6023, and 6024, the average fluorescence signal of the whole body of mice was greater than 10 8p / s, and is better than Comparative Example 1, indicating that the luciferase mRNA lipid nanoparticles containing the compound induce effective expression of luciferase in vivo. At the same time, after the mice were given the lipid nanoparticles prepared in Example 2, no toxic reactions such as abnormal activity were observed. This shows that the luciferase mRNA lipid nanoparticles prepared using the ionizable cationic lipid compound of the present invention exhibit biological activity in animals and have low toxicity.

[0188] The solutions of the present invention are not limited to the technical means disclosed by the above technical means, but also include technical solutions composed of any combination of the above technical features. The above is a specific implementation method of the present invention. It should be pointed out that for ordinary technicians in the field of the present invention, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also considered 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; R a Selected from C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 Alkyl alcohol, C6-C 24 The cycloalkyl group of C6-C 24 The alkyl group is a branched structure; the C6-C 24 Alkenyl, C6-C 24 Alkyl alcohol is a straight chain or branched chain structure; R b and R c Each independently selected from C1-C 12 Alkyl alcohol.

2. The ionizable lipid compound according to claim 1, characterized in that R a Select from the following compound structures:

3. The ionizable lipid compound according to claim 1, characterized in that R b and R c Select from the following compound structures: R d Selected from C1-C6 alkyl or cycloalkyl.

4. An ionizable lipid compound selected from at least one of the following compounds:

5. The method for preparing an ionizable lipid compound according to any one of claims 1 to 4, 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, A3 reacts with Z3 to obtain A1, A3 is a nucleophilic reagent, R a , R b , R c It has the same definition as any one of claims 1 to 4.

6. The preparation method according to claim 5, characterized in that: In 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.

7. The preparation method according to any one of claims 5 to 6, characterized in that: The raw materials used in the reaction process also contain protecting groups, and the reaction steps include protecting and / or deprotecting steps.

8. Use of the ionizable lipid compound according to any one of claims 1 to 4 in an emulsifier, suspending agent, dispersant, solubilizer, lubricant, thickener, antibacterial agent or preservative.

9. Use of the ionizable lipid compound according to any one of claims 1 to 4 in the preparation of a cosmetic composition.

10. Use of the ionizable lipid compound according to any one of claims 1 to 4 in the preparation of a lipid composition, characterized in that: The ionizable lipid compound is used as a drug delivery vehicle.

11. A lipid composition, characterized in that The invention comprises the ionizable lipid compound according to any one of claims 1 to 4.

12. The lipid composition of claim 11, wherein The lipid component of the lipid composition comprises other lipids including phospholipids, cholesterol and PEG-conjugated lipids.

13. The lipid composition of claim 11, 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.

14. The lipid composition of claim 13, wherein The other ionizable lipids are selected from at least one of the following compounds:

15. 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.

16. The lipid composition of claim 15, wherein The therapeutic and / or preventive agent is a nucleic acid; the nucleic acid is DNA or RNA.

17. Use of the lipid composition according to any one of claims 11 to 16 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.

18. A pharmaceutical composition, characterized in that Comprising the lipid composition according to any one of claims 11 to 16 and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to claim 18, 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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