Amino lipid compound, lipid nanoparticles constructed by amino lipid compound and pharmaceutical composition

Abstract

The invention discloses an amino lipid compound, lipid nanoparticles constructed by the amino lipid compound and a pharmaceutical composition, and the amino lipid compound has a structure as shown in a formula I. The amino lipid compound has the advantages of strong delivery capacity, good stability and high safety in vivo. When being used as a vaccine or a nucleic acid drug carrier, the lipid nano-particles constructed by the lipid nano-particles show excellent biological activity, high protein expression level and remarkable immunocompetence, have low adverse reaction occurrence rate and good stability, and can be stored, transported and used at normal temperature.

Description

TECHNICAL FIELD

[0001] The present application relates to an amino lipid compound, a lipid nanoparticle and a pharmaceutical composition constructed therefrom, and in particular to an amino lipid compound, a lipid nanoparticle and a pharmaceutical composition constructed therefrom with excellent stability, safety, metabolic properties and delivery performance. BACKGROUND

[0002] Gene therapy involves delivering a gene with specific genetic information into target cells by artificial methods to produce proteins that can regulate, treat or cure diseases caused by genetic defects, or achieve clinical therapeutic effects by regulating the expression of related genes. However, since both nucleic acids and cell membranes are negatively charged, unmodified nucleic acids are difficult to directly enter cells and are easily destroyed by nucleic acid degradation enzymes in cells, so effective gene delivery and treatment cannot be achieved. In order to solve this problem, it is usually necessary to use viral vectors or non-viral vectors to assist the delivery of genes.

[0003] Viral vectors are favored due to their high transfection efficiency in vitro and in vivo, but they also have some disadvantages, such as high toxicity, strong immune response, limited gene carrying capacity, poor targeting, and complex preparation process. In contrast, non-viral vectors are increasingly attracting attention due to their ease of preparation, transportation and storage, as well as high safety, effectiveness, and non-immunogenicity.

[0004] Lipid nanoparticles (LNPs) are a widely used non-viral vector, especially suitable for vaccine development and nucleic acid drug delivery. Ionizable lipids are the core component of LNPs, although a variety of compounds have been reported for the preparation of lipid nanoparticles, but in practical applications, the efficiency of these compounds in vaccine and nucleic acid drug delivery is not high, the drug stability is poor, and there may be problems such as long elimination half-life, high toxicity, insufficient safety, etc., which limit their application in clinical.

[0005] DLin-MC3-DMA (MC3) is the first ionizable amino lipid used in the approved small nucleic acid drug Onpattro, but MC3 is slowly degraded and eliminated in vivo, and multiple doses can accumulate in the liver to produce toxic side effects, usually requiring the use of corticosteroid drugs (such as dexamethasone), H1 and H2 blockers before administration to reduce and avoid related adverse reactions caused by infusion, and its application effect in vaccine delivery is not ideal, with low protein expression and antibody production levels.

[0006] SM102, as an important component of mRNA vaccines, especially for the COVID-19 vaccine of Moderna, plays a key role in mRNA delivery. Although SM102 can improve the stability and delivery efficiency of mRNA, the delivery efficiency may still be affected by various factors, such as the nature of the lipid component, the nature of the drug, and the characteristics of the target cells or tissues, etc., which may not be able to effectively deliver mRNA to the target cells or tissues, resulting in poor therapeutic effect. Therefore, there is an urgent need to develop new lipid compounds with high delivery efficiency, good stability, moderate elimination half-life, and high safety to meet the needs of intracellular therapeutic agent delivery. SUMMARY

[0007] The first object of the present application is to provide an amino lipid compound that is beneficial for drug delivery, the second object is to provide a lipid nanoparticle constructed from the amino lipid compound, and the third object is to provide a pharmaceutical composition constructed from the lipid nanoparticle.

[0008] Technical solution: The amino lipid compound or its pharmaceutically acceptable salt, stereoisomer according to the present application, wherein the amino lipid compound has the structure shown in formula I:

[0009]

[0010] L 1 , L 2 are each independently selected from O or C, and L 1 , L 2 are different from O;

[0011] R 1 , R 2 are each independently selected from H, -CH2OH, -O(CH2)2OH, -CH2O(CH2)2OH, or R 1 is absent;

[0012] R 3 , R 4 are each independently selected from H or -OH.

[0013] The term "pharmaceutically acceptable salt" means a salt of a compound of this application which is found to be suitable for use in pharmaceutical applications, due to the presence of specific substituents on the compound discovered in this application, with a relatively non-toxic acid or base. When the compound of this application contains relatively acidic functionalities, base addition salts can be obtained by contacting the free acid form of such compounds with a sufficient amount of the appropriate base in a pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When the compound of this application contains relatively basic functionalities, acid addition salts can be obtained by contacting the free base form of such compounds with a sufficient amount of the appropriate acid in a pure solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include mineral acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming the carbonate or bicarbonate salt), phosphoric acid (forming phosphates, monohydrogenphosphates, dihydrogenphosphates), sulfuric acid (forming sulfates or bisulfates), hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like; also salts with amino acids such as arginine, lysine and the like, and salts with organic acids like glucuronic acid. Certain specific compounds of this application contain both basic and acidic functionalities, allowing the compounds to be converted into either base or acid addition salts. Preferred are the base addition salts. The free form of the compounds and the various salt forms can be interconverted by standard techniques known in the art. The free form of the compounds can be regenerated from the various salt forms by contacting with a suitable acid or base, as appropriate. The different salt forms can be converted into each other by standard techniques known in the art.

[0014] The "pharmaceutically acceptable salts" of the present application can be synthesized from the parent compound that contains a sufficiently basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by contacting the free acid or base forms of these compounds with a sufficient amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both. Generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, among others, are preferred.

[0015] The term "stereoisomer" refers to compounds of the present application having different stereochemical configurations at chiral atoms in the structure of the compounds. For example, the carbon atom attached to the nitrogen atom in the structure of compound 2 is a chiral carbon atom, which will form stereoisomers of compound 2 having R-configuration and S-configuration.

[0016] Preferably, in the structure, when L 1 , L 2 is O, R 3 , R 4 is H.

[0017] Preferably, in the structure, when L1 , L 2 are both C, R 1 is selected from -CH2OH, -O(CH2)2OH.

[0018] Preferably, the amino lipid compound is selected from any one of the following compounds:

[0019]

[0020]

[0021] The lipid nanoparticle of the present application is constructed from the amino lipid compound of the present application or a pharmaceutically acceptable salt thereof or a stereoisomer thereof and one or more of a helper lipid, a structural lipid, a polymer-lipid conjugate.

[0022] Preferably, the lipid nanoparticle has a particle size of 1-1000 nm.

[0023] Further preferably, the lipid nanoparticle has a particle size of 1-200 nm.

[0024] Preferably, in the lipid nanoparticle, the molar percentage content of the amino lipid compound of the present application or a pharmaceutically acceptable salt thereof or a stereoisomer thereof is 25-75%.

[0025] Further preferably, in the lipid nanoparticle, the molar percentage content of the amino lipid compound of the present application or a pharmaceutically acceptable salt thereof or a stereoisomer thereof is selected from 32-35%, 35-40%, 40-42%, 42-45%, 45-48%, 48-55%, 55-65%.

[0026] Preferably, the lipid nanoparticle contains 25-75 parts by weight of the amino lipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 5-45 parts by weight of the helper lipid, 0-55 parts by weight of the structural lipid, and 0-5 parts by weight of the polymer-lipid conjugate.

[0027] Further preferably, the lipid nanoparticle contains any one of the following combinations:

[0028] 25-65 parts by weight of the amino lipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 5-42 parts by weight of the helper lipid, 10-55 parts by weight of the structural lipid, and 0.5-4 parts by weight of the polymer-lipid conjugate.

[0029] or 28 to 60 parts of an aminolipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 5 to 42 parts of a helper lipid, 15.5 to 53.5 parts of a structural lipid, 0.5 to 3.5 parts of a polymer-lipid conjugate;

[0030] or 35 to 60 parts of an aminolipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 5 to 40 parts of a helper lipid, 18.5 to 53.5 parts of a structural lipid, 1.5 to 3 parts of a polymer-lipid conjugate;

[0031] Further more preferably, the lipid nanoparticle contains, in parts by weight, any one of the following combinations:

[0032] 45 parts of an aminolipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 11 parts of a helper lipid, 41.5 parts of a structural lipid, 2.5 parts of a polymer-lipid conjugate;

[0033] or 42 parts of an aminolipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 10.5 parts of a helper lipid, 45 parts of a structural lipid, 2.5 parts of a polymer-lipid conjugate;

[0034] or 42 parts of an aminolipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 16 parts of a helper lipid, 39.5 parts of a structural lipid, 2.5 parts of a polymer-lipid conjugate;

[0035] or 40 parts of an aminolipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 16 parts of a helper lipid, 41.5 parts of a structural lipid, 2.5 parts of a polymer-lipid conjugate;

[0036] or 40 parts of an aminolipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 18 parts of a helper lipid, 39.5 parts of a structural lipid, 2.5 parts of a polymer-lipid conjugate;

[0037] or 35 parts of an aminolipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 16 parts of a helper lipid, 46.5 parts of a structural lipid, 2.5 parts of a polymer-lipid conjugate;

[0038] or 35 parts of an aminolipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 25 parts of a helper lipid, 36.5 parts of a structural lipid, 3.5 parts of a polymer-lipid conjugate;

[0039] or 28 parts of an aminolipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 33.5 parts of a helper lipid, 35 parts of a structural lipid, 3.5 parts of a polymer-lipid conjugate;

[0040] or 32 parts of an aminolipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 37 parts of a helper lipid, 40.5 parts of a structural lipid, 0.5 parts of a polymer-lipid conjugate;

[0041] or 35 parts of an aminolipid compound or a pharmaceutically acceptable salt thereof or a stereoisomer thereof, 40 parts of a helper lipid, 22.5 parts of a structural lipid, 2.3 parts of a polymer-lipid conjugate.

[0042] Preferably, the helper lipid is selected from the group consisting of phospholipids, which can be semi-synthetic or of natural origin, or chemically modified.

[0043] Further preferably, the helper lipid is selected from the group consisting of one or more of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), 1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-0-4'-(N,N,N-trimethyl)homoserine (DGTS), lysophospholipids.

[0044] More preferably, the helper lipid is selected from the group consisting of one or more of DSPC, DOPE, DOPC, DOPS.

[0045] More preferably, the helper lipid is selected from the group consisting of one or more of DSPC, DOPE.

[0046] Preferably, the structural lipid is selected from the group consisting of sterols.

[0047] Further preferably, the structural lipid is selected from the group consisting of one or more of 20a-hydroxycholesterol, cholesterol, cholesterol ester, steroid hormones, steroid vitamins, bile acids, cholestanol, ergosterol, beta-sitosterol, oxidized cholesterol derivatives.

[0048] More preferably, the structural lipid is selected from the group consisting of one or more of cholesterol, cholesterol ester, steroid hormones, steroid vitamins, bile acids.

[0049] More preferably, the structural lipid is selected from the group consisting of high purity cholesterol, in particular injection grade high purity cholesterol, such as CHO-HP.

[0050] More preferably, the structural lipid is selected from the group consisting of 20a-hydroxycholesterol.

[0051] Preferably, the polymer-lipid conjugate is constructed from a polymer component and a lipid component, which can improve the stability of the lipid nanoparticle in vivo. The polymer component is selected from one or more of a hydrophilic polymer component, an amphiphilic polymer component.

[0052] Further preferably, the polymer-lipid conjugate is constructed from a hydrophilic polymer component and a lipid component.

[0053] Further preferably, the polymer-lipid conjugate is constructed from an amphiphilic polymer component and a lipid component.

[0054] More preferably, the hydrophilic polymer component is selected from one or more of polyethylene glycol (PEG), poly(oxazolines) (POX), poly(glycerols) (PGs), poly(hydroxypropylmethacrylate) (PHPMA), poly(2-hydroxyethylmethacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethylacrylamide) (PDMA), poly(N-acryloyl morpholine) (PAcM), polyaminoacids, glycosaminoglycans (GAGs), heparin, hyaluronic acid (HA), polysialic acid (PSA), elastin-like polypeptide (ELPs), serum albumin, CD47.

[0055] More preferably, the hydrophilic polymer component is selected from polyethylene glycol.

[0056] In particular, the polymer-lipid conjugate is selected from one or more of the following: polyethylene glycol-lipid (PEG-lipid), polyoxazoline-lipid, polyglycerol-lipid, polyhydroxypropyl methacrylate-lipid, poly(2-hydroxyethyl methacrylate)-lipid, poly(N-(2-hydroxypropyl)methacrylamide)-lipid, polyvinylpyrrolidone-lipid, poly(N,N-dimethylacrylamide)-lipid, poly(N-acryloyl morpholine)-lipid, glycosaminoglycan-lipid, heparin-lipid, hyaluronic acid-lipid, polysialic acid-lipid, elastin-like polypeptide-lipid, serum albumin-lipid, CD47-lipid.

[0057] In some embodiments, the polymer-lipid is selected from a PEG-lipid. In an alternative particular example, the polymer-lipid is a PEG-lipid. In some embodiments, the PEG-lipid is selected from one or more of the following: PEG-myristoyl glycerol diester (PEG-DMG), PEG-distearoyl phosphatidyl ethanolamine (PEG-DSPE), PEG-diacylglycerol (PEG-DAG), PEG-dialkyloxypropyl (PEG-DAA), PEG-phospholipid, PEG-ceramide (PEG-Cer), PEG-1,2-distearoyl-rac-glycerol (PEG-DSG), PEG-1,2-dipalmitoyl-rac-glycerol (PEG-DPG). More preferably, the PEG-lipid is one or more of the following: PEG-DMG, PEG-DSG, PEG-DPG. PEG-DMG is a polyethylene glycol derivative of 1,2-dimyristate glycerol. In some embodiments, the average molecular weight of the PEG in the PEG-lipid is between 2000 and 5000. In an alternative particular example, the average molecular weight of the PEG in the PEG-lipid is 2000. In some embodiments, the PEG-lipid is PEG2000-DMG.

[0058] More preferably still, the amphoteric polymer component is selected from one or more of poly(carboxybetaine) (pCB), poly(sulfobetaine) (pSB), phosphobetaine-base polymers, phosphorylcholine polymers, poly(carboxybetaine acrylamide) (pCBAA), poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyl phosphorylcholine), poly(vinyl-pyridiniopropanesulfonate), poly(carboxybetaine) based on vinylimidazole, poly(sulfobetaine) based on vinylimidazole, poly(sulfobetaine) based on vinylpyridine.

[0059] In particular, the polymer-lipid is selected from one or more of polyhydroxybetaine-lipid, poly(sulfobetaine)-lipid, phosphobetaine-base polymer-lipid, phosphorylcholine polymer-lipid. In some embodiments, the polymer-lipid is selected from one or more of poly(carboxybetaine acrylamide)-lipid, poly(carboxybetaine methacrylate)-lipid, poly(sulfobetaine methacrylate)-lipid, poly(methacryloyloxyethyl phosphorylcholine) lipid, poly(vinyl-pyridiniopropanesulfonate)-lipid, poly(carboxybetaine) based on vinylimidazole-lipid, poly(sulfobetaine) based on vinylimidazole-lipid, poly(sulfobetaine) based on vinylpyridine-lipid.

[0060] Preferably, the lipid component is selected from one or more of myristoyl glycerol (1,2-dimyristoyl-sn-glycerol, DMG), distearoyl-phosphatidyl-ethanolamine (DSPE), diacylglycerol (DAG), dialkyloxypropyl (DAA), phospholipid, ceramide (Cer), 1,2-distearoyl-rac-glycerol (DSG), and dipalmitoyl-rac-glycero (DPG).

[0061] In some embodiments, the lipid nanoparticle of the present application comprises DOPE, cholesterol (preferably injection grade high purity cholesterol, such as CHO-HP), PEG-lipid (e.g. DMG-PEG2000), and the amino lipid compound of the present application or a pharmaceutically acceptable salt thereof or a stereoisomer thereof; wherein the molar ratio between the amino lipid compound of the present application or a pharmaceutically acceptable salt thereof or a stereoisomer thereof and DOPE, cholesterol, PEG-lipid is (25-60):(5-42):(15.5-53.5):(0.5-3.5), more preferably 50:11:43.5:2.5, 50:10:38.5:1.5.

[0062] In some embodiments, the lipid nanoparticle of the present application comprises DSPC, cholesterol (preferably injection grade high purity cholesterol, such as CHO-HP), PEG-lipid (e.g. DMG-PEG2000), and the amino lipid compound of the present application or a pharmaceutically acceptable salt thereof or a stereoisomer thereof; wherein the molar ratio between the amino lipid compound of the present application or a pharmaceutically acceptable salt thereof or a stereoisomer thereof and DSPC, cholesterol, PEG-lipid is (25-60):(5-42):(15.5-53.5):(0.5-3.5), more preferably 50:11:43.5:2.5, 50:10:38.5:1.5.

[0063] In particular, the lipid nanoparticle of the present application is constructed by the following method:

[0064] Step one: mixing the amino lipid compound of the present application, the helper lipid, the structural lipid, and the polymer-lipid conjugate in proportion and dissolving them in an organic solvent to obtain an organic mixture;

[0065] Step two: using a micro-injection pump, mix the organic mixture prepared in step one with the salt solution in a micro-channel chip according to a ratio to obtain a crude solution;

[0066] Step three: dialyze and filter the crude solution prepared in step two to obtain the lipid nanoparticle.

[0067] In step one, the organic solvent is preferably anhydrous ethanol; the amount ratio of the amino lipid compound, the auxiliary lipid, the structural lipid, and the polymer-lipid conjugate is as previously described.

[0068] In step two, the salt solution is preferably a sodium acetate aqueous solution, more preferably a 50 mM, pH = 4.0 sodium acetate aqueous solution; the organic mixture and the salt solution are preferably mixed at a volume ratio of 1:3; the micro-channel chip is preferably a T-shaped, Y-shaped, fluid dynamic focusing type, or staggered chevron type micro-channel chip.

[0069] The pharmaceutical composition described in the present application comprises a nucleic acid drug and the lipid nanoparticle described in the present application.

[0070] Since the amino lipid compound or its pharmaceutically acceptable salt or its stereoisomer described in the present application contains a long non-polar residue, the compound has a hydrophobic characteristic, and since the structure contains an amino group, it also has a hydrophilic characteristic. This amphiphilic characteristic can be used to form lipid nanoparticles, such as lipid bilayers, micelles, liposomes, etc.

[0071] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, such as a diluent or excipient.

[0072] Specifically, in the construction process of the pharmaceutical composition described in the present application, the salt solution dissolving the nucleic acid drug is replaced with a blank salt solution in step two of the lipid nanoparticle construction method, and the construction is completed.

[0073] The lipid nanoparticle constructed by the amino lipid compound or its pharmaceutically acceptable salt or its stereoisomer described in the present application has excellent performance in encapsulating bioactive ingredients, and can be used to deliver one or more of a variety of therapeutic agents to cells, tissues or organs. The lipid nanoparticle described in the present application is applied to deliver bioactive ingredients (such as DNA, RNA, etc.) to cells, tissues or organs.

[0074] In addition, the lipid nanoparticle or pharmaceutical composition described in the present application containing bioactive ingredients (such as DNA, RNA, etc.) is also applied to produce polypeptides and / or proteins of interest, prepare drugs, and prepare drugs for nucleic acid transfer.

[0075] The amino lipid compound, or the pharmaceutically acceptable salt or stereoisomer thereof, or the lipid nanoparticle constructed therefrom, can be applied to deliver a bioactive ingredient (such as DNA, RNA, etc.) into a cell, a tissue or an organ.

[0076] In some embodiments, the application comprises a step of contacting a cell, a tissue or an organ with a lipid nanoparticle comprising a bioactive ingredient (such as a nucleic acid (e.g., mRNA) encoding a polypeptide and / or protein of interest). In some embodiments, the tissue or organ is selected from any one of the following: spleen, liver, kidney, lung, femur, ocular tissue, vascular endothelium in blood vessels, lymph, tumor tissue.

[0077] In some embodiments, the cell is a mammalian cell, which can be a cell of any mammal. In some embodiments, the mammal is selected from any one of the following: human, mouse, rat, pig, cat, dog, horse, goat, cow, monkey.

[0078] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages:

[0079] The amino lipid compound designed in the present application has good stability during in vivo circulation and rapid degradation, and the delivery efficiency is significantly improved, and has the advantages of strong delivery capacity, good stability and high safety. When the lipid nanoparticle constructed therefrom is used as a vaccine or a nucleic acid drug carrier, it exhibits excellent biological activity, high protein expression level and significant immune activity, and has a low incidence of adverse reactions, and in addition, it has good stability and can be stored, transported and used at room temperature. BRIEF DESCRIPTION OF DRAWINGS

[0080] Figure 1 Results of in vivo immune performance evaluation of the compound of the present application. DETAILED DESCRIPTION

[0081] The technical solutions of the present application are further described below in conjunction with examples.

[0082] Example 1: Synthesis of Compound 1

[0083]

[0084] In a 250 mL reaction flask, the starting material A (22.3 g, 100 mmol) and the starting material B (25.6 g, 100 mmol) were sequentially added, 100 mL of dichloromethane, after stirring and dissolving, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23.0 g, 120 mmol), 4-dimethylaminopyridine (0.61 g, 5 mmol), N,N-diisopropylethylamine (25.8 g, 200 mmol) were added, and the reaction was allowed to proceed at room temperature for 2 h, washed with water 3 times, dried using anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (petroleum ether: ethyl acetate = 10:1 to 2:1) to obtain the intermediate I-1 (41.5 g, yield 90%).

[0085] 1 H NMR (500 MHz, Chloroform-d) δ 4.66, 3.45, 2.29, 1.85, 1.56, 1.54, 1.52, 1.42, 1.42, 1.39, 1.31, 1.31, 1.29, 1.29, 1.28, 1.27, 1.27, 1.27, 1.26, 1.25, 1.25, 0.89.

[0086]

[0087] In a 100 mL reaction flask, the starting material D (3.50 g, 10 mmol) and the starting material C (8.7 g, 100 mmol) were sequentially added, 30 mL of ethanol, after stirring and dissolving, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h, 100 mL of dichloromethane was added, washed with water 3 times, dried using anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain the intermediate I-2 (2.38 g, yield 67%).

[0088] 1 H NMR (500 MHz, Chloroform-d) δ 4.66, 3.45, 2.29, 1.85, 1.56, 1.54, 1.52, 1.42, 1.42, 1.39, 1.31, 1.31, 1.29, 1.29, 1.28, 1.27, 1.27, 1.27, 1.26, 1.25, 1.25, 0.89.

[0089]

[0090] In a 100 mL reaction flask, intermediate I-1 (355 mg, 1 mmol), intermediate I-2 (554 mg, 1.2 mmol), acetonitrile 20 mL, after stirring and dissolving, potassium carbonate (276 mg, 2 mmol), potassium iodide (166 mg, 1 mmol) were added, and the reaction was allowed to proceed at room temperature for 24 h. After adding dichloromethane 100 mL, washing with water 3 times, drying with anhydrous sodium sulfate, and concentrating, compound 1 (501 mg, yield 68%) was obtained by purification using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1).

[0091] 1 H NMR (500 MHz, Chloroform-d) δ 4.83 (p, J = 7.0 Hz, 1H), 4.12 - 4.00 (m, 1H), 3.59 (dt, J = 12.3, 6.8 Hz, 1H), 3.51 (dt, J = 12.4, 6.8 Hz, 1H), 3.28 (t, J = 6.7 Hz, 1H), 2.61 - 2.49 (m, 5H), 2.35 - 2.18 (m, 5H), 1.74 - 1.65 (m, 5H), 1.68 - 1.58 (m, 5H), 1.61 - 1.53 (m, 5H), 1.57 - 1.50 (m, 5H), 1.53 - 1.43 (m, 5H), 1.46 - 1.18 (m, 45H), 0.94 - 0.83 (m, 10H).

[0092] Example 2: Synthesis of compound 2

[0093] Compound intermediate II-1 was synthesized in the same manner as example 1.

[0094]

[0095] In a 100 mL reaction flask, intermediate I-1 (355 mg, 1 mmol), intermediate I-2 (554 mg, 1.2 mmol), acetonitrile 20 mL, after stirring and dissolving, potassium carbonate (276 mg, 2 mmol), potassium iodide (166 mg, 1 mmol) were added, and the reaction was allowed to proceed at room temperature for 24 h. After adding dichloromethane 100 mL, washing with water 3 times, drying with anhydrous sodium sulfate, and concentrating, compound 1 (501 mg, yield 68%) was obtained by purification using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1).

[0096] 1H NMR (500 MHz, Chloroform-d) δ 4.07 (t, J = 7.0 Hz, 2H), 3.72 (dd, J = 12.5, 7.0 Hz, 1H), 3.71 - 3.63 (m, 2H), 3.60 (dt, J = 12.5, 6.8 Hz, 1H), 3.53 (d, J = 6.8 Hz, 2H), 3.19 (t, J = 6.7 Hz, 1H), 3.19 - 3.07 (m, 1H), 3.05 - 2.95 (m, 1H), 2.98 - 2.89 (m, 2H), 2.31 (td, J = 7.1, 0.8 Hz, 2H), 1.96 (hept, J = 7.0 Hz, 1H), 1.88 - 1.74 (m, 2H), 1.67 - 1.41 (m, 7H), 1.43 - 1.22 (m, 17H), 0.94 - 0.84 (m, 3H).

[0097]

[0098] In a 100 mL reaction flask, sequentially added intermediate I-1 (355 mg, 1 mmol), intermediate II-1 (556 mg, 1.2 mmol), acetonitrile 20 mL, after stirring and dissolving, added potassium carbonate (276 mg, 2 mmol), potassium iodide (166 mg, 1 mmol), reacted at room temperature for 24 h, added dichloromethane 100 mL, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain compound 2 (509 mg, yield 68%).

[0099] 1 H NMR (500 MHz, Chloroform-d) δ 4.14 - 3.99 (m, 0H), 3.74 - 3.58 (m, 1H), 2.63 - 2.50 (m, 1H), 2.35 - 2.18 (m, 1H), 1.81 - 1.66 (m, 1H), 1.66 (td, J = 7.0, 1.7 Hz, 1H), 1.65 - 1.53 (m, 1H), 1.53 (ddd, J = 7.0, 5.0, 3.6 Hz, 1H), 1.53 - 1.44 (m, 1H), 1.47 - 1.35 (m, 2H), 1.37 - 1.32 (m, 1H), 1.35 - 1.28 (m, 1H), 1.31 - 1.18 (m, 8H), 0.94 - 0.83 (m, 2H).

[0100] Example 3: Synthesis of compound 3

[0101] Compound intermediate III-1 was synthesized in the same manner as example 1.

[0102]

[0103] In a 100 mL reaction flask, intermediate I-1 (355 mg, 1 mmol) and intermediate III-1 (556 mg, 1.2 mmol) were sequentially added, acetonitrile 20 mL, stirred and dissolved, then potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added, and the reaction was allowed to proceed at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain compound 3 (510 mg, yield 67%).

[0104] 1 H NMR (500 MHz, Chloroform-d) δ 4.14 - 4.00 (m, 2H), 3.77 - 3.63 (m, 2H), 3.63 - 3.54 (m, 3H), 3.20 - 3.10 (m, 1H), 3.03 - 2.91 (m, 3H), 2.85 (dt, J = 9.0, 6.0 Hz, 1H), 2.31 (td, J = 7.1, 0.8 Hz, 2H), 1.85 - 1.74 (m, 1H), 1.77 - 1.66 (m, 3H), 1.66 - 1.41 (m, 7H), 1.43 - 1.22 (m, 17H), 0.94 - 0.84 (m, 3H).

[0105]

[0106] In a 100 mL reaction flask, intermediate I-1 (355 mg, 1 mmol) and intermediate III-1 (556 mg, 1.2 mmol) were sequentially added, acetonitrile 20 mL, stirred and dissolved, then potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added, and the reaction was allowed to proceed at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain compound 3 (510 mg, yield 67%).

[0107] 1H NMR (500 MHz, Chloroform-d) δ 4.14 - 3.99 (m, 0 H), 3.74 - 3.53 (m, 1 H), 2.67 - 2.51 (m, 1 H), 2.35 - 2.18 (m, 1 H), 1.85 - 1.66 (m, 1 H), 1.70 - 1.59 (m, 1 H), 1.63 - 1.38 (m, 3 H), 1.42 - 1.35 (m, 1 H), 1.35 (ddd, J=5.5, 4.0, 1.1 Hz, 1 H), 1.35 - 1.28 (m, 1 H), 1.31 - 1.24 (m, 3 H), 1.28 - 1.18 (m, 4 H), 0.94 - 0.83 (m, 2 H).

[0108] Example 4: Synthesis of compound 4

[0109] Compound intermediate IV-1 was synthesized in the same method as example 1.

[0110]

[0111] In a 100 mL reaction bottle, raw material D (3.50 g, 10 mmol) and raw material G (17.3 g, 100 mmol) were sequentially added, 30 mL of ethanol was stirred and dissolved, then 2.58 g of N, N-diisopropyl ethylamine (20 mmol) was added, and the reaction was carried out at room temperature for 24 h. 100 mL of dichloromethane was added, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and then purified using a flash column chromatography system (dichloromethane:methanol=20:1 to 5:1) to obtain intermediate IV-1 (5.76 g, yield 70%).

[0112] 1H NMR (500 MHz, Chloroform-d) δ 4.15 (t, J = 7.4 Hz, 1H), 4.12-4.01 (m, 2H), 3.72 (dq, J = 12.1, 7.1 Hz, 1H), 3-59 (dq, J = 12.1, 7.1 Hz, 1H), 3.56-3.42 (m, 2H), 3.35 (dd, J = 12.3, 7.0 Hz, 1H), 3.24 (dd, J = 12.3, 7.0 Hz, 1H), 3.11 (dtd, J = 12.3, 7.0, 5.9 Hz, 1H), 3.01-2.91 (m, 1H), 2.70 (dp, J = 8.6, 7.0 Hz, 1H), 2.55 (dt, J = 8.6, 5.9 Hz, 1H), 2.35-2.27 (m, 2H), 1.84 (hept, J = 7.0 Hz, 1H), 1.65-1.57 (m, 2H), 1.60-1.54 (m, 6H), 1.57-1.41 (m, 6H), 1.45-1.35 (m, 3H), 1.38-1.31 (m, 4H), 1.34-1.28 (m, 3H), 1.31-1.22 (m, 8H), 0.94-0.84 (m, 3H).

[0113]

[0114] In a 100 mL reaction flask, sequentially added intermediate I-1 (355 mg, 1 mmol), intermediate IV-1 (560 mg, 1.2 mmol), acetonitrile 20 mL, after stirring and dissolving, added potassium carbonate (276 mg, 2 mmol), potassium iodide (166 mg, 1 mmol), reacted at room temperature for 24 h, added dichloromethane 100 mL, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain compound 4 (530 mg, yield 72%).

[0115] 1 H NMR (500 MHz, Chloroform-d) δ 3.65-3.42 (m, 1H), 2.64-2.47 (m, 1H), 2.35-2.18 (m, 1H), 1.73-1.59 (m, 2H), 1.63-1.53 (m, 1H), 1.56-1.50 (m, 1H), 1.50-1.37 (m, 1H), 1.40-1.28 (m, 2H), 1.31-1.26 (m, 1H), 1.28-1.18 (m, 5H), 0.94-0.84 (m, 2H).

[0116] Example 5: Synthesis of compound 5

[0117] Compound intermediate V-1 was synthesized in the same manner as example 1.

[0118]

[0119] In a 100 mL reaction flask, the starting material D (3.50 g, 10 mmol) and the starting material H (15.9 g, 100 mmol) were sequentially added, 30 mL of ethanol was added, and after stirring and dissolving, 2.58 g (20 mmol) of N,N-diisopropyl ethylamine was added, and the reaction was allowed to proceed at room temperature for 24 hours. 100 mL of dichloromethane was added, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain intermediate V-1 (3.4 g, yield 68%).

[0120] 1 H NMR (500 MHz, Chloroform-d) δ 4.14-4.00 (m, 2H), 3.82 (t, J=7.5 Hz, 1H), 3.76-3.65 (m, 2H), 3.68-3.60 (m, 1H), 3.58-3.43 (m, 2H), 3.00-2.88 (m, 1H), 2.76 (dp, J=8.8, 7.0 Hz, 1H), 2.66-2.59 (m, 1H), 2.62-2.56 (m, 1H), 2.35-2.27 (m, 2H), 1.80-1.72 (m, 1H), 1.74-1.63 (m, 3H), 1.66-1.59 (m, 1H), 1.63-1.56 (m, 3H), 1.59-1.53 (m, 2H), 1.56-1.45 (m, 3H), 1.48-1.39 (m, 2H), 1.43-1.35 (m, 3H), 1.39-1.33 (m, 4H), 1.35-1.31 (m, 1H), 1.31 (d, J=3.3 Hz, 1H), 1.30 (d, J=1.4 Hz, 1H), 1.31-1.21 (m, 10H), 0.94-0.84 (m, 3H).

[0121]

[0122] In a 100 mL reaction flask, intermediate I-1 (355 mg, 1 mmol), intermediate V-1 (556 mg, 1.2 mmol), acetonitrile 20 mL, after stirring and dissolving, potassium carbonate (276 mg, 2 mmol), potassium iodide (166 mg, 1 mmol) were added, and the reaction was allowed to proceed at room temperature for 24 h. After adding dichloromethane 100 mL, washing with water 3 times, drying with anhydrous sodium sulfate, concentrating, and purifying with a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1), compound 5 (521 mg, yield 70%) was obtained.

[0123] 1 H NMR (500 MHz, Chloroform-d) δ 3.77-3.59 (m, 1H), 2.57 (dt, J = 37.5, 7.0 Hz, 1H), 2.35-2.18 (m, 1H), 1.80-1.68 (m, 1H), 1.71-1.61 (m, 1H), 1.64-1.42 (m, 3H), 1.45-1.18 (m, 9H), 0.94-0.84 (m, 2H).

[0124] Example 6: Synthesis of compound 6

[0125]

[0126] In a 250 mL reaction flask, raw material I (23.8 g, 100 mmol) and raw material B (25.6 g, 100 mmol) were sequentially added, dichloromethane 100 mL, after stirring and dissolving, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23.0 g, 120 mmol), 4-dimethylaminopyridine (0.61 g, 5 mmol), N,N-diisopropylethylamine (25.8 g, 200 mmol) were added, and the reaction was allowed to proceed at room temperature for 2 h. After washing with water 3 times, drying with anhydrous sodium sulfate, concentrating, and purifying with a flash column chromatography system (petroleum ether: ethyl acetate = 10:1 to 2:1), intermediate VI-1 (40.1 g, yield 90%) was obtained.

[0127] 1 H NMR (500 MHz, Chloroform-d) δ 3.54-3.44 (m, 1H), 2.30 (td, J = 7.1, 1.0 Hz, 1H), 1.76-1.66 (m, 1H), 1.69-1.59 (m, 1H), 1.62-1.49 (m, 1H), 1.47-1.22 (m, 11H), 0.94-0.84 (m, 2H).

[0128]

[0129] In a 100 mL reaction flask, intermediate VI-1 (476 mg, 1 mmol) and intermediate VI-2 (41 mg, 1.2 mmol) were sequentially added, acetonitrile 20 mL, stirred and dissolved, then potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added, and the reaction was allowed to proceed at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain compound 6 (510 mg, yield 68%).

[0130] 1 H NMR (500 MHz, Chloroform-d) δ 4.17-4.10 (m, 1H), 4.12-4.00 (m, 2H), 3.81 (h, J=7.0 Hz, 1H), 3.62-3.48 (m, 2H), 3.28 (t, J=6.7 Hz, 1H), 3.00 (dt, J=8.8, 7.4 Hz, 1H), 2.92 (dt, J=12.3, 7.1 Hz, 1H), 2.82 (dt, J=12.2, 7.2 Hz, 1H), 2.70 (dp, J=9.0, 7.0 Hz, 1H), 2.31 (td, J=7.1, 1.7 Hz, 2H), 1.75 (hept, J=7.0 Hz, 1H), 1.66-1.20 (m, 31H), 0.94-0.84 (m, 3H).

[0131]

[0132] In a 100 mL reaction flask, intermediate VI-1 (476 mg, 1 mmol) and intermediate VI-2 (41 mg, 1.2 mmol) were sequentially added, acetonitrile 20 mL, stirred and dissolved, then potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added, and the reaction was allowed to proceed at room temperature for 24 h. Dichloromethane 100 mL was added, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain compound 6 (510 mg, yield 68%).

[0133] 1H NMR (500 MHz, Chloroform-d) δ 4.83 (1H), 4.14 - 3.99 (m, 2H), 3.77 (d, J = 8.2 Hz, 2H), 3.71 (dq, J = 8.3, 6.8 Hz, 2H), 3.59 (dt, J = 12.3, 6.8 Hz, 1H), 3.51 (dt, J = 12.4, 6.8 Hz, 1H), 3.28 (t, J = 6.7 Hz, 1H), 2.93 (dd, J = 12.4, 6.8 Hz, 2H), 2.82 (dd, J = 12.4, 6.9 Hz, 2H), 2.58 (1H), 2.30 (td, J = 7.1, 1.7 Hz, 2H), 2.28 - 2.16 (m, 2H), 1.80 (hept, J = 6.9 Hz, 1H), 1.75 - 1.45 (m, 25H), 1.45 - 1.31 (m, 11H), 1.32 (s, 1H), 1.31 (d, J = 2.9 Hz, 1H), 1.31 - 1.27 (m, 3H), 1.30 - 1.23 (m, 23H), 1.24 (d, J = 0.9 Hz, 2H), 1.25 - 1.19 (m, 1H), 0.94 - 0.84 (m, 9H).

[0134] Example 7: Synthesis of compound 7

[0135] Compound intermediate VII-1 was synthesized in the same manner as example 6.

[0136]

[0137] In a 100 mL reaction flask, raw material L (3.50 g, 10 mmol) and raw material J (12.9 g, 100 mmol) were sequentially added, 30 mL of ethanol was added, and after stirring and dissolving, N, N-diisopropyl ethylamine (2.58 g, 20 mmol) was added, and the reaction was allowed to proceed at room temperature for 24 h. 100 mL of dichloromethane was added, washed with water 3 times, dried with anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain intermediate VII-1 (3.26 g, yield 68%).

[0138] 1H NMR (500 MHz, Chloroform-d) δ 4.09 (dt, J = 12.5, 7.1 Hz, 1H), 4.02 (dt, J = 12.5, 7.1 Hz, 1H), 3.63 - 3.49 (m, 2H), 3.28 (t, J = 6.7 Hz, 1H), 3.16 - 3.06 (m, 1H), 3.01 - 2.91 (m, 1H), 2.68 (dp, J = 8.6, 6.9 Hz, 1H), 2.55 (dt, J = 8.8, 6.0 Hz, 1H), 2.31 (td, J = 7.1, 0.8 Hz, 2H), 1.76 (hept, J = 7.0 Hz, 1H), 1.66 - 1.22 (m, 35H), 0.94 - 0.84 (m, 3H).

[0139]

[0140] In a 100 mL reaction flask, intermediate VII-1 (397 mg, 1 mmol), intermediate VI-1 (476 mg, 1.2 mmol), acetonitrile 20 mL, after stirring and dissolving, add potassium carbonate (276 mg, 2 mmol), potassium iodide (166 mg, 1 mmol), react at room temperature for 24 h, add dichloromethane 100 mL, wash with water 3 times, dry with anhydrous sodium sulfate, concentrate, and purify with flash column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain compound 7 (521 mg, yield 72%).

[0141] 1 H NMR (500 MHz, Chloroform-d) δ 4.83 (p, J = 7.0 Hz, 1H), 4.14 - 3.99 (m, 2H), 3.86 (d, J = 8.4 Hz, 1H), 3.69 (dt, J = 8.3, 7.0 Hz, 1H), 3.62 - 3.57 (m, 1H), 3.54 - 3.49 (m, 1H), 3.28 (t, J = 6.7 Hz, 1H), 2.91 (dd, J = 12.3, 7.0 Hz, 1H), 2.81 - 2.76 (m, 1H), 2.63 - 2.54 (m, 5H), 2.35 - 2.18 (m, 5H), 1.70 - 1.60 (m, 5H), 1.57 - 1.53 (m, 5H), 1.52 - 1.42 (m, 10H), 1.38 - 1.33 (m, 5H), 1.32 - 1.18 (m, 40H), 0.94 - 0.84 (m, 10H).

[0142] Example 8: Preparation of Lipid Nanoparticles

[0143] The lipids MC3 and SM102 used are shown below. MC3 and SM102 are commercially available or can be prepared according to techniques known in the art.

[0144]

[0145] Preparation of the lipid nanoparticles:

[0146] Preparation method one: the amino lipid compounds prepared in Examples 1-7 were mixed with DOPE, cholesterol, DMG-PEG2000 in a molar ratio of 50:11:43.5:2.5, respectively, and dissolved in anhydrous ethanol. Using a microsyringe pump, the ratio of ethanol solution to sodium acetate solution (50 mM, pH = 4.0) was controlled at 1:3, and the crude solution of the lipid nanoparticles was prepared in a microfluidic chip; then the dialysis box (Fisher, MWCO 20K) was used to dialyze in 1xPBS at 4°C for 6 h, and filtered with a 0.22 μιη microporous filter before use.

[0147] Preparation method two: the above amino lipid compounds were mixed with DSPC, cholesterol, DMG-PEG2000 in a molar ratio of 50:10:38.5:1.5, and the preparation method was the same as method one.

[0148] Example 9: Evaluation of the in vivo delivery performance of the lipid nanoparticles for luciferase mRNA

[0149] The lipid nanoparticles prepared in Example 8 were used for the biological experiment of evaluating the in vivo delivery performance for luciferase mRNA (Fluc mRNA, GenBank No. X84847).

[0150] Animal preparation: 6-week-old female BALB / c mice were selected, and the body weight was about 20 g. The feeding environment was a SPF level feeding room, and the animal experiment was strictly performed according to the guidelines of the national health organization and the requirements of animal ethics.

[0151] In vivo delivery: 3 mice were randomly selected in each group, and the lipid nanoparticles were injected using the tail vein administration method at a dosage of 0.5 mg / kg. After 12 h, D-luciferin potassium salt (200 μί, 10 mg / mL) was injected into each mouse through the tail vein. After 10 min, the mice were placed under the in vivo imaging system (IVIS-200, Xenogen), the total fluorescence intensity of each mouse was observed, and the representative amino lipid compound administration method for delivering Fluc mRNA expression intensity is shown in Table 1. MC3 was used as a control, and SM102 was a comparative compound.

[0152] Table 1: Expression intensity of the representative amino lipid compound for delivering Fluc mRNA through tail vein administration

[0153]

[0154]

[0155] Example 10: Delivery of vectors in vivo for ovalbumin mRNA delivery and immune performance evaluation

[0156] Preparation of lipid nanoparticles:

[0157] Preparation method: The amino lipid compounds prepared in Examples 1-7 were mixed and dissolved in anhydrous ethanol in a molar ratio of 50:10:38.5:1.5 of DOPE, cholesterol, PEG2000-DMG, respectively. Ovalbumin mRNA (OVA mRNA, Gene ID: V00383) was dissolved in sodium acetate solution (50 mM, pH = 4.0). Using a microsyringe pump, the volume ratio of ethanol solution to sodium acetate solution (50 mM, pH = 4.0) was controlled to be 1:3, and the crude solution of lipid nanoparticles was prepared in a microfluidic chip. Then, the crude solution was dialyzed in a dialysis box (Fisher, MWCO 20K) in 1xPBS at 4°C for 6h. Before use, the obtained lipid nanoparticles were filtered through a 0.22μm microporous filter. The mass ratio of amino lipid compound to ovalbumin mRNA (OVA mRNA) in the obtained lipid nanoparticles was about 10:1.

[0158] Animal preparation: 6-week-old female BALB / c mice weighing about 20g were selected and raised in a SPF-level feeding room. Animal experiments were strictly conducted in accordance with the guidelines and animal ethics requirements of the national health organization.

[0159] In vivo delivery: 3 mice were randomly selected from each group, and the lipid nanoparticles were injected into the leg muscles at a dose of 0.5mg / kg (Day 0). Seven days later, the same amount was used for reinforcement (Day 7). On day 21, the tail vein blood was taken for serological analysis. MC3 was used as a control.

[0160] Enzyme-linked immunosorbent assay (ELISA): a flat-bottom 96-well plate was pre-coated with 50mM carbonate buffer, and the concentration of OVA protein was 0.5μg protein per well (pH = 9.6) at 4°C overnight, then blocked with 5% glycine. The antiserum obtained from the immunized animals was diluted to 10 -2 to 10 -6and incubated at room temperature for 1 h at 37 °C. Horseradish peroxidase (HRP)-coupled goat anti-mouse IgG was labeled at a dilution of 1 : 10000 in PBS-T-1% BSA. After addition of the HRP substrate, the absorbance at 450 nm was detected in an ELISA reader (Bio-Rad).

[0161] As shown in Figure 6, MC3 control IgG antibody titer was the lowest, the IgG antibody titer produced by compound 6 and SM102 was comparable, and the IgG antibody titer of other compounds was significantly better than the control, which also showed that the amino lipid compounds designed in the application had excellent immunological activity and also had strong adjuvant effect. Figure 1

[0162] Example 11: Evaluation of the stability of the lipid nanoparticles prepared from the amino lipid compounds

[0163] Preparation of the lipid nanoparticles: The lipid nanoparticles were prepared according to Formulation Method 1 and Method 2, respectively, and were administered subcutaneously.

[0164] Characterization of the lipid nanoparticles: The particle size and PDI of the prepared lipid nanoparticles were determined by MicroCal iTC200 (Malvern). 40 μL of LNP solution was taken for particle size measurement, and the cycle was repeated three times, with each cycle lasting 30 s. Particle size detection was performed on the day of preparation (week 0), one week (week 1) and four weeks (week 4) of storage at 25 °C, respectively.

[0165] Animal preparation: 6-week-old male BALB / c mice weighing about 20 g were selected, and the feeding environment was a SPF-level feeding room. Animal experiments were strictly conducted in accordance with the guidelines of the national health organization and the requirements of animal ethics.

[0166] In vivo delivery: 3 mice were randomly selected from each group, and the amount of use was 0.5 mg / kg, and the lipid nanoparticles were injected subcutaneously (Day 0). After 7 days, the same amount was used for reinforcement again (Day 7). The expression intensity of subcutaneously administered Fluc mRNA was detected on the same day (week 0) and four weeks (week 4), and the experimental results are shown in Table 2.

[0167] Table 2 DLS characterization of LNP prepared from amino lipid compounds and Fluc-mRNA subcutaneous expression intensity

[0168]

[0169]

[0170] ​As shown in Table 2, the lipid nanoparticles constructed by the amino lipid compound designed in the application have excellent stability.

[0171] In summary, the amino lipid compound designed in the application and the lipid nanoparticles constructed by the amino lipid compound not only have excellent immunocompetence, but also have significant immune enhancement effect, and also exhibit excellent stability.

Claims

1. An aminolipid compound or a pharmaceutically acceptable salt, stereoisomer thereof, characterized by, The amino lipid compound has a structure shown in formula I: L 1 , L 2 are each independently selected from O or C, and L 1 , L 2 are different from O; R 1 , R 2 each independently is selected from H, -CH2OH, -0(CH2)2OH, -CH2O(CH2)2OH, or R 1 is absent; R 3 , R 4 are each independently selected from H or -OH.

2. The aminolipid compound or pharmaceutically acceptable salt, stereoisomer thereof according to claim 1, characterized in that, In the structure, when L 1 , L 2 is O, R 3 , R 4 is H.

3. The aminolipid compound or pharmaceutically acceptable salt, stereoisomer thereof according to claim 1, characterized in that, In the structure, when L 1 , L 2 are all C, R 1 is selected from -CH2OH, -O(CH2)2OH.

4. The aminolipid compound or pharmaceutically acceptable salt, stereoisomer thereof according to claim 1, characterized by, The amino lipid compound is selected from any one of the following compounds:

5. A lipid nanoparticle characterized in that, It is constructed from the amino lipid compound or its pharmaceutically acceptable salt or its stereoisomer of claim 1 and one or more of auxiliary lipids, structural lipids, polymer-lipid conjugate.

6. The lipid nanoparticle of claim 5, wherein, In parts by weight, wherein it contains 25-75 parts of the amino lipid compound or its pharmaceutically acceptable salt or its stereoisomer, 5-45 parts of auxiliary lipids, 0-55 parts of structural lipids, 0-5 parts of polymer-lipid conjugate.

7. The lipid nanoparticle of claim 5, wherein, The auxiliary lipids are selected from one or more of distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, dioleoylphosphatidylserine, 1,2-dioctadecanoyl-sn-glycero-3-phosphate-(1'-rac-glycerol), dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-0-4'-(N,N,N-trimethyl)homoserine, lysophospholipid.

8. The lipid nanoparticle of claim 5, wherein, The structural lipids are selected from one or more of 20α-hydroxycholesterol, cholesterol, cholesteryl ester, sterol hormones, sterol vitamins, bile acids, cholestanol, ergosterol, β-sitosterol, oxidized cholesterol derivatives.

9. The lipid nanoparticle of claim 5, wherein, The polymer-lipid conjugate is constructed from a polymer component and a lipid component; wherein the polymer component is selected from one or more of polyethylene glycol, polyoxazoline, polyglycerol, polyhydroxypropyl methacrylate, poly(2-hydroxyethyl methacrylate), poly(N-(2-hydroxypropyl)methacrylamide), polyvinylpyrrolidone, poly(N,N-dimethylacrylamide), poly(N-acryloyl morpholide), polyamino acid, glycosaminoglycan, heparin, hyaluronic acid, polysialic acid, elastin-like protein, serum albumin, CD47, polycarboxybetaine, polysulfobetaine, phosphobetaine-based polymer and phosphocholine-based polymer, poly(carboxybetaine acrylamide), poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyl phosphorylcholine), poly(vinylpyridyl propyl sulfonate), polyvinylimidazolyl betaine, polyvinylimidazolyl sulfobetaine, polyvinylpyridyl sulfobetaine, and the lipid component is selected from one or more of myristoylglycerol diester, distearoylphosphatidylethanolamine, diacylglycerol, dialkyloxypropyl, phospholipid, ceramide, 1,2-distearoyl-rac-glycerol, 1,2-dipalmitoyl-rac-glycerol.

10. A pharmaceutical composition, characterized by, It comprises a nucleic acid drug and the lipid nanoparticle of claim 5. The amino lipid compound has a structure shown in formula I: The amino lipid compound is selected from any one of the following compounds: It is constructed from the amino lipid compound or its pharmaceutically acceptable salt or its stereoisomer of claim 1 and one or more of auxiliary lipids, structural lipids, polymer-lipid conjugate. In parts by weight, wherein it contains 25-75 parts of the amino lipid compound or its pharmaceutically acceptable salt or its stereoisomer, 5-45 parts of auxiliary lipids, 0-55 parts of structural lipids, 0-5 parts of polymer-lipid conjugate. The auxiliary lipids are selected from one or more of distearoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, dioleoylphosphatidylserine, 1,2-dioctadecanoyl-sn-glycero-3-phosphate-(1'-rac-glycerol), dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-0-4'-(N,N,N-trimethyl)homoserine, lysophospholipid. The structural lipids are selected from one or more of 20α-hydroxycholesterol, cholesterol, cholesteryl ester, sterol hormones, sterol vitamins, bile acids, cholestanol, ergosterol, β-sitosterol, oxidized cholesterol derivatives. The polymer-lipid conjugate is constructed from a polymer component and a lipid component; wherein the polymer component is selected from one or more of polyethylene glycol, polyoxazoline, polyglycerol, polyhydroxypropyl methacrylate, poly(2-hydroxyethyl methacrylate), poly(N-(2-hydroxypropyl)methacrylamide), polyvinylpyrrolidone, poly(N,N-dimethylacrylamide), poly(N-acryloyl morpholide), polyamino acid, glycosaminoglycan, heparin, hyaluronic acid, polysialic acid, elastin-like protein, serum albumin, CD47, polycarboxybetaine, polysulfobetaine, phosphobetaine-based polymer and phosphocholine-based polymer, poly(carboxybetaine acrylamide), poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyl phosphorylcholine), poly(vinylpyridyl propyl sulfonate), polyvinylimidazolyl betaine, polyvinylimidazolyl sulfobetaine, polyvinylpyridyl sulfobetaine, and the lipid component is selected from one or more of myristoylglycerol diester, distearoylphosphatidylethanolamine, diacylglycerol, dialkyloxypropyl, phospholipid, ceramide, 1,2-distearoyl-rac-glycerol, 1,2-dipalmitoyl-rac-glycerol. It comprises a nucleic acid drug and the lipid nanoparticle of claim 5.

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