A lipid compound and its use

Abstract

The present invention relates to a compound, a lipid compound nanoparticle, a nucleic acid nanoparticle complex, a pharmaceutical composition and their application in the field of drug delivery. The compound, lipid compound nanoparticle or nucleic acid nanoparticle complex provided by the present invention has good biocompatibility, high transfection efficiency, low toxicity and excellent technical effects.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and biotechnology, and specifically relates to a lipid compound and a use thereof. Background Art

[0002] Gene transfection is a technology that transfers or delivers nucleic acids with biological functions into cells and allows the nucleic acids to maintain their biological functions in the cells. Gene vectors refer to a tool that introduces exogenous therapeutic genes into biological cells. Currently, the gene vectors with potential for industrial transformation in the world are mainly viral vectors and non-viral vectors.

[0003] Viral vectors are gene delivery tools that use the ability of viruses to transmit their genomes into other cells for infection. Currently, lentivirus, adenovirus, retrovirus vectors, and adeno-associated virus vectors have good application prospects. However, due to their inherent physical and chemical properties and biological activity, viral vectors have serious disadvantages, such as high production costs, limited loading capacity, poor targeting, insertion integration, teratogenicity, carcinogenicity, and mutagenesis, which are not conducive to the development of universal and general therapies.

[0004] Non-viral vectors mainly include: liposome nanoparticles, composite nanoparticles, cationic polymer nanoparticles, polypeptide nanoparticles, etc. Among them, liposome nanoparticles are the main non-viral vectors currently used in the development of RNA drugs. At present, the first RNAi drug (Patisiran) and the first mRNA drug (BNT162b2, Comirnaty) have been launched one after another, which fully verifies the clinical application value of liposome nanoparticles (LNP). Compared with viral vectors, liposome nanoparticles have the advantages of low production cost, clear chemical structure, easy quality control, targeted drug delivery through targeted modification, and theoretically unlimited encapsulation capacity. However, most liposome lipid materials are non-degradable and highly toxic, so it is difficult to meet the clinical needs of repeated administration. In addition, there are problems such as poor in vivo transfection effect, nucleic acid metabolism or clearance in serum, and low bioavailability.

[0005] Therefore, there is still an urgent need for nanoparticles with good biocompatibility and high transfection efficiency. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides a compound.

[0008] A compound, the structure of which is shown in formula A,

[0009]

[0010] in,

[0011] a is an integer from 0 to 10 (i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10);

[0012] b is an integer from 1 to 9 (i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9);

[0013] c is an integer from 1 to 9 (i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9);

[0014] R1 is C1-C20 alkyl (i.e., C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl or C20 alkyl);

[0015] R2 is hydrogen or -C(=O)Ra;

[0016] Ra is a C1-C20 alkyl group (i.e., a C1 alkyl group, a C2 alkyl group, a C3 alkyl group, a C4 alkyl group, a C5 alkyl group, a C6 alkyl group, a C7 alkyl group, a C8 alkyl group, a C9 alkyl group, a C10 alkyl group, a C11 alkyl group, a C12 alkyl group, a C13 alkyl group, a C14 alkyl group, a C15 alkyl group, a C16 alkyl group, a C17 alkyl group, a C18 alkyl group, a C19 alkyl group or a C20 alkyl group);

[0017] R3 is a hydroxyl group,

[0018] R4 is

[0019] Rb is C1-C20 alkyl (i.e., C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, C6 alkyl, C7 alkyl, C8 alkyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl, C19 alkyl or C20 alkyl);

[0020] w is an integer from 1 to 9 (i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9);

[0021] z is an integer from 1 to 9 (i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9);

[0022] X1 is O or NH;

[0023] Each X2 is independently selected from O or NH;

[0024] Y1 is selected from O or NH;

[0025] Y2 is selected from O or NH.

[0026] In some embodiments, the compound represented by formula A is selected from the compound represented by formula I, the compound represented by formula II, the compound represented by formula III or the compound represented by formula IV.

[0027]

[0028]

[0029] Wherein, in the compound represented by formula II, w is an integer of 1-9 (i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9); z is an integer of 1-9 (i.e., 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9);

[0030] In the compound represented by formula I, R3 is hydroxyl,

[0031] In some embodiments, in the compound represented by formula A or the compound represented by formula I, X1 is O; each X2 is independently selected from O or NH; Y1 is selected from O or NH; Y2 is selected from O or NH.

[0032] In some embodiments, in the compound represented by formula A or the compound represented by formula I, X1 is O; each X2 is O; Y1 is selected from O; Y2 is selected from O.

[0033] In some embodiments, in the compound represented by formula A or the compound represented by formula I, X1 is NH; each X2 is independently selected from O or NH; Y1 is selected from O or NH; Y2 is selected from O or NH.

[0034] In some embodiments, in the compound of formula A or formula I, X1 is NH; each X2 is NH; Y1 is selected from NH; Y2 is selected from NH. In some embodiments, the compound of formula A is selected from the compound of formula I, wherein R1 is an unsubstituted straight-chain alkyl group of C1 to C15 (i.e., an unsubstituted straight-chain alkyl group of C1, an unsubstituted straight-chain alkyl group of C2, an unsubstituted straight-chain alkyl group of C3, an unsubstituted straight-chain alkyl group of C4, an unsubstituted straight-chain alkyl group of C5, an unsubstituted straight-chain alkyl group of C6, an unsubstituted straight-chain alkyl group of C7, an unsubstituted straight-chain alkyl group of C8, an unsubstituted straight-chain alkyl group of C9, an unsubstituted straight-chain alkyl group of C10, an unsubstituted straight-chain alkyl group of C11, an unsubstituted straight-chain alkyl group of C12, an unsubstituted straight-chain alkyl group of C13, an unsubstituted straight-chain alkyl group of C14, or an unsubstituted straight-chain alkyl group of C15). alkyl), R2 is hydrogen or -C(=O)Ra, Ra is an unsubstituted straight-chain alkyl group of C5 to C15 (i.e., an unsubstituted straight-chain alkyl group of C5, an unsubstituted straight-chain alkyl group of C6, an unsubstituted straight-chain alkyl group of C7, an unsubstituted straight-chain alkyl group of C8, an unsubstituted straight-chain alkyl group of C9, an unsubstituted straight-chain alkyl group of C10, an unsubstituted straight-chain alkyl group of C11, an unsubstituted straight-chain alkyl group of C12, an unsubstituted straight-chain alkyl group of C13, an unsubstituted straight-chain alkyl group of C14 or an unsubstituted straight-chain alkyl group of C15), a is an integer of 0 to 6 (i.e., 0, 1, 2, 3, 4, 5 or 6), b is an integer of 3 to 5 (i.e., 3, 4 or 5), and c is an integer of 3 to 5 (i.e., 3, 4 or 5).

[0035] In some embodiments, the compound represented by formula A is selected from the compound represented by formula I, wherein R1 is an unsubstituted straight-chain alkyl group of C5 to C11 (i.e., an unsubstituted straight-chain alkyl group of C5, an unsubstituted straight-chain alkyl group of C6, an unsubstituted straight-chain alkyl group of C7, an unsubstituted straight-chain alkyl group of C8, an unsubstituted straight-chain alkyl group of C9, an unsubstituted straight-chain alkyl group of C10, or an unsubstituted straight-chain alkyl group of C11), R2 is hydrogen or -C(=O)Ra, and Ra is an unsubstituted straight-chain alkyl group of C5 to C13 (i.e., unsubstituted C5 straight-chain alkyl, unsubstituted C6 straight-chain alkyl, unsubstituted C7 straight-chain alkyl, unsubstituted C8 straight-chain alkyl, unsubstituted C9 straight-chain alkyl, unsubstituted C10 straight-chain alkyl, unsubstituted C11 straight-chain alkyl, unsubstituted C12 straight-chain alkyl or unsubstituted C13 straight-chain alkyl), a is an integer from 0 to 6 (i.e., 0, 1, 2, 3, 4, 5 or 6), b is an integer from 3 to 5 (i.e., 3, 4 or 5), and c is an integer from 3 to 5 (i.e., 3, 4 or 5).

[0036] In some embodiments, the compound represented by formula A is selected from the compound represented by formula I, and the compound represented by formula I is selected from compound L0111, compound L0112, compound L0113, compound L0114, compound L0115, compound L0116, compound L0117, compound L0118, compound L0119, compound L0120, compound L0121, compound L0122, compound L0123, compound L0124, compound L0125, compound L0132, compound L0133, compound L0134, compound L0135 or compound L0136,

[0037]

[0038]

[0039]

[0040]

[0041] In some embodiments, the compound represented by formula A is selected from the compound represented by formula II, wherein a is an integer of 0 to 5 (i.e., 0, 1, 2, 3, 4 or 5), b is an integer of 3 to 10 (i.e., 3, 4, 5, 6, 7, 8, 9 or 10), c is an integer of 1 to 5 (i.e., 1, 2, 3, 4 or 5), w is an integer of 1 to 5 (i.e., 1, 2, 3, 4 or 5), z is an integer of 5-10 (i.e., 5, 6, 7, 8, 9 or 10), and Ra is an unsubstituted C5 to C15 straight-chain alkyl group (i.e., an unsubstituted C5 straight-chain alkyl group, an unsubstituted C6 straight-chain alkyl group, an unsubstituted C7 straight-chain alkyl group, an unsubstituted C8 straight-chain alkyl group, an unsubstituted C9 straight-chain alkyl group, an unsubstituted Rb is an unsubstituted C5-C15 straight chain alkyl group (i.e., an unsubstituted C5 straight chain alkyl group, an unsubstituted C6 straight chain alkyl group, an unsubstituted C7 straight chain alkyl group, an unsubstituted C8 straight chain alkyl group, an unsubstituted C9 straight chain alkyl group, an unsubstituted C10 straight chain alkyl group, an unsubstituted C11 straight chain alkyl group, an unsubstituted C12 straight chain alkyl group, an unsubstituted C13 straight chain alkyl group, an unsubstituted C14 straight chain alkyl group or an unsubstituted C15 straight chain alkyl group).

[0042] In some embodiments, the compound represented by formula A is selected from the compound represented by formula II, wherein a is an integer of 0 to 3 (i.e., 0, 1, 2 or 3), b is an integer of 4 to 6 (i.e., 4, 5 or 6), c is an integer of 1 to 3 (i.e., 1, 2 or 3), w is an integer of 1 to 3 (i.e., 1, 2 or 3), z is an integer of 5 to 8 (i.e., 5, 6, 7 or 8), Ra is an unsubstituted C5 to C13 straight-chain alkyl group (i.e., an unsubstituted C5 straight-chain alkyl group, an unsubstituted C6 straight-chain alkyl group, an unsubstituted C R is an unsubstituted straight-chain alkyl group of C7, an unsubstituted straight-chain alkyl group of C8, an unsubstituted straight-chain alkyl group of C9, an unsubstituted straight-chain alkyl group of C10, an unsubstituted straight-chain alkyl group of C11, an unsubstituted straight-chain alkyl group of C12 or an unsubstituted straight-chain alkyl group of C13), and Rb is an unsubstituted straight-chain alkyl group of C9-C13 (i.e., an unsubstituted straight-chain alkyl group of C9, an unsubstituted straight-chain alkyl group of C10, an unsubstituted straight-chain alkyl group of C11, an unsubstituted straight-chain alkyl group of C12 or an unsubstituted straight-chain alkyl group of C13).

[0043] In some embodiments, the compound represented by formula A is selected from the compound represented by formula II, wherein a is 1, b is 5, c is 1, w is 1, z is 6, Ra is an unsubstituted C5-C13 straight-chain alkyl group (i.e., an unsubstituted C5 straight-chain alkyl group, an unsubstituted C6 straight-chain alkyl group, an unsubstituted C7 straight-chain alkyl group, an unsubstituted C8 straight-chain alkyl group, an unsubstituted C9 straight-chain alkyl group, an unsubstituted C10 straight-chain alkyl group, an unsubstituted C11 straight-chain alkyl group, an unsubstituted C12 straight-chain alkyl group or an unsubstituted C13 straight-chain alkyl group), and Rb is an unsubstituted C10 straight-chain alkyl group, an unsubstituted C11 straight-chain alkyl group or an unsubstituted C12 straight-chain alkyl group.

[0044] In some embodiments, the compound represented by formula A is selected from the compound represented by formula II, and the compound represented by formula II is selected from compound L0126, compound L0127 or compound L0128.

[0045]

[0046] In some embodiments, the compound represented by formula A is selected from the compound represented by formula III, wherein a, b and c are the same and are integers from 1 to 9 (i.e., 1, 2, 3, 4, 5, 6, 7, 8 or 9).

[0047] In some embodiments, the compound represented by formula A is selected from the compound represented by formula III, wherein a, b and c are the same and are integers of 1 to 5 (ie, 1, 2, 3, 4 or 5).

[0048] In some embodiments, the compound represented by formula A is selected from the compound represented by formula III, wherein a, b and c are the same and are all integers of 1 to 5 (i.e., 1, 2, 3, 4 or 5); Ra is an unsubstituted C5 to C15 straight-chain alkyl group (i.e., an unsubstituted C5 straight-chain alkyl group, an unsubstituted C6 straight-chain alkyl group, an unsubstituted C7 straight-chain alkyl group, an unsubstituted C8 straight-chain alkyl group, an unsubstituted C9 straight-chain alkyl group, an unsubstituted C10 straight-chain alkyl group, an unsubstituted C11 straight-chain alkyl group, an unsubstituted C12 straight-chain alkyl group, an unsubstituted C13 straight-chain alkyl group); R is an unsubstituted C5-C15 straight chain alkyl group (i.e., an unsubstituted C5 straight chain alkyl group, an unsubstituted C6 straight chain alkyl group, an unsubstituted C7 straight chain alkyl group, an unsubstituted C8 straight chain alkyl group, an unsubstituted C9 straight chain alkyl group, an unsubstituted C10 straight chain alkyl group, an unsubstituted C11 straight chain alkyl group, an unsubstituted C12 straight chain alkyl group, an unsubstituted C13 straight chain alkyl group, an unsubstituted C14 straight chain alkyl group or an unsubstituted C15 straight chain alkyl group).

[0049] In some embodiments, the compound represented by formula A is selected from the compound represented by formula III, wherein a, b and c are the same and are all integers of 3 to 5 (i.e., 3, 4 or 5); Ra is an unsubstituted C5-C11 straight-chain alkyl group (i.e., an unsubstituted C5 straight-chain alkyl group, an unsubstituted C6 straight-chain alkyl group, an unsubstituted C7 straight-chain alkyl group, an unsubstituted C8 straight-chain alkyl group, an unsubstituted C9 straight-chain alkyl group, an unsubstituted C10 straight-chain alkyl group or an unsubstituted C11 straight-chain alkyl group), and Rb is an unsubstituted C8-C13 straight-chain alkyl group (i.e., an unsubstituted C8 straight-chain alkyl group, an unsubstituted C9 straight-chain alkyl group, an unsubstituted C10 straight-chain alkyl group, an unsubstituted C11 straight-chain alkyl group, an unsubstituted C12 straight-chain alkyl group or an unsubstituted C13 straight-chain alkyl group).

[0050] In some embodiments, the compound represented by formula A is selected from the compound represented by formula III, wherein a, b and c are the same and are all integers of 3 to 5 (i.e., 3, 4 or 5); Ra is an unsubstituted C5 to C11 straight chain alkyl group (i.e., an unsubstituted C5 straight chain alkyl group, an unsubstituted C6 straight chain alkyl group, an unsubstituted C7 straight chain alkyl group, an unsubstituted C8 straight chain alkyl group, an unsubstituted C9 straight chain alkyl group, an unsubstituted C10 straight chain alkyl group or an unsubstituted C11 straight chain alkyl group), and Rb is an unsubstituted C11 straight chain alkyl group.

[0051] In some embodiments, the compound represented by formula A is selected from the compound represented by formula III, and the compound represented by formula III is selected from compound L0129 and compound L0130.

[0052]

[0053] In some embodiments, the compound represented by formula A is selected from the compound represented by formula IV, wherein a is an integer of 0 to 5, b is an integer of 3 to 10, c is an integer of 3 to 10, z is an integer of 5 to 10, Ra is an unsubstituted C5 to C15 straight chain alkyl, and Rb is an unsubstituted C5 to C15 straight chain alkyl.

[0054] In some embodiments, the compound represented by formula A is selected from the compound represented by formula IV, wherein a is an integer of 0 to 3, b is an integer of 4 to 6, c is an integer of 4 to 6, z is an integer of 5 to 8, Ra is an unsubstituted C5 to C13 straight chain alkyl, and Rb is an unsubstituted C9 to C13 straight chain alkyl.

[0055] In some embodiments, the compound represented by formula A is selected from the compound represented by formula IV, wherein a is 1, b is 5, c is 5, z is 6, Ra is an unsubstituted C5-C13 straight-chain alkyl, and Rb is an unsubstituted C10-C15 straight-chain alkyl.

[0056] In some embodiments, the compound represented by formula A is selected from the compound represented by formula IV, wherein the compound represented by formula IV is selected from compound L0131,

[0057]

[0058] In a second aspect, the present invention provides a lipid compound nanoparticle.

[0059] In some embodiments, a lipid compound nanoparticle comprises the following components: the compound described in the first aspect and auxiliary materials.

[0060] In some embodiments, a lipid compound nanoparticle comprises the following components: the compound described in the first aspect, a nucleic acid and auxiliary materials.

[0061] In some embodiments, the auxiliary material is selected from at least one of: PEG derivatives, lipids, lipidoids, alcohols, sugars or inorganic salts.

[0062] In some embodiments, the PEG derivative is selected from at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, PEG-modified stearic acid, and PEG-modified phosphatidylserine.

[0063] In some embodiments, the PEG derivative includes 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)], dilauroylphosphatidylethanolamine-polyethylene glycol, dimyristoylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylcholine polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol, PEG-distearylglycerol, PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide, PEG-dipalmitoylphosphatidylethanolamine or PEG-1,2-dimyristyloxypropyl-3-amine.

[0064] In some embodiments, the PEG derivative includes at least one of DMG-PEG2000, mPEG-DSPE, mPEG-STA, mPEG-PS, mPEG-DMPE, and mPEG-DPPE.

[0065] In some embodiments, the lipid comprises a lipid selected from a phospholipid or a sterol.

[0066] In some embodiments, the phospholipid includes at least one selected from phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-phosphocholine, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

[0067] In some embodiments, the sterol comprises at least one of cholesterol, lanosterol, 5α-cholestane-3β-ol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, or α-tocopherol.

[0068] In some embodiments, the lipid compound nanoparticles include the compound described in the first aspect, a PEG derivative and a lipid, wherein the lipid is selected from at least one of a phospholipid and a sterol.

[0069] In some embodiments, based on the total molar amount of each component in the lipid compound nanoparticles, the content of the compound described in the first aspect is 14.8 mol%-70.0 mol%.

[0070] In some embodiments, the content of the PEG derivative is 0.4 mol%-10 mol%, calculated based on the total molar amount of each component in the lipid compound nanoparticles.

[0071] In some embodiments, the content of the phospholipid is 5.0 mol%-30.0 mol%, calculated based on the total molar amount of each component in the lipid compound nanoparticles.

[0072] In some embodiments, the content of the sterol is 10.0 mol%-75.0 mol% based on the total molar amount of each component in the lipid compound nanoparticle. In some embodiments, the content of the sterol is 15.0 mol%-75.0 mol% based on the total molar amount of each component in the lipid compound nanoparticle.

[0073] In some embodiments, the molar ratio of PEG derivative: phospholipid: sterol: the compound described in the first aspect in the lipid compound nanoparticles is (0.4-10.0): (5.0-30.0): (10.0-75.0): (14.8-70.0). In some embodiments, the molar ratio of PEG derivative: phospholipid: sterol: the compound described in the first aspect in the lipid compound nanoparticles is (0.4-10.0): (5.0-30.0): (15.0-75.0): (14.8-70.0).

[0074] In some embodiments, the molar ratio of PEG derivative: phospholipid: sterol: compound of any one of claims 1 to 8 in the lipid compound nanoparticles is 2.50:16.00:16.50:65.00, 1.00:5.00:64.00:30.00, 0.40:8.00:56.60:35.00, 1.00:8.00:61.00:30.00, 1.20:12.00:38.30:48.50, 1.70:9.00:40.80:48.50, 2.50:16.00:21.50:60.00, 1.20:9.00:4 7.80:42.00,1.00:16.00:34.50:48.50,2.50:8.00:41.00:48.50,1.50:8.00:25.50:65.00,2.50:11.50:51.00:35.00,1.50:8.00:30.50:60.00,1.00:5.00:62.00:32.00,0.60:5.00:54.40:40.00,1.50:16.00:47.50:35.00,1.63:12.99:45.38:40.00,1.20:8.00 :55.8:35.00,1.30:8.00:55.70:35.00,0.40:5.00:59.60:35.00,1.00:20.00:37.00:42.00,1.00:25.00:34.00:40.00,3.20:12.00:29.80:55.00,1.00:8.00:53.00:38.00,2.50:9.50:33.00:55.00,10.00:16.00:54.00:20.00,3.00:30.00:42.00:25.00,3.20: 16.80:10.00:70.00, 3.00:17.00:25.00:55.00, 3.20:16.80:15.00:65.00, 4.20:11.00:70.00:14.80, 6.20:6.80:75.00:15.00, 1.50:11.50:38.50:48.50, 7.50:9.61:35.56:47.33, 3.00:9.50:32.50:55.00, 0.95:7.58:26.47:65.00 or 1.40:11.15:38.95:48.50.

[0075] In some embodiments, the molar ratio of PEG derivative: phospholipid: sterol: the compound described in the first aspect in the lipid compound nanoparticles is (0.4-1.5): (8.0-11.5): (38.5-56.6): (35.0-48.5).

[0076] In some embodiments, the molar ratio of PEG derivative: phospholipid: sterol: compound described in the first aspect in the lipid compound nanoparticles is 0.40:8.00:56.60:35.00, 1.50:11.50:38.50:48.50 or 1.40:11.15:38.95:48.50.

[0077] In a third aspect, the present invention provides a nucleic acid-nanoparticle complex.

[0078] A nucleic acid nanoparticle complex comprises: a nucleic acid and at least one of the lipid compound nanoparticles described in the second aspect.

[0079] In some embodiments, the ratio of the molar amount of ionizable nitrogen atoms in the compound of any one of claims 1 to 10 to the molar amount of phosphorus atoms in the nucleic acid in the nucleic acid-nanoparticle complex is 5 to 50. In some embodiments, the ratio of the molar amount of ionizable nitrogen atoms in the compound of any one of claims 1 to 10 to the molar amount of phosphorus atoms in the nucleic acid in the nucleic acid-nanoparticle complex is 5, 6, 7, 8, 9, 10, 15, 19, 20, 25, 30, 35, 40, 45 or 50.

[0080] In some embodiments, the nucleic acid includes: deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). RNA includes but is not limited to small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), self-replicating RNA (saRNA), small hairpin RNA (shRNA), circular RNA (circRNA), messenger RNA (mRNA) and combinations thereof. mRNA can be synthesized according to various known methods. For example, mRNA can be synthesized by in vitro transcription (IVT). The specific conditions vary depending on the specific application. In a fourth aspect, the present invention provides a pharmaceutical composition.

[0081] A pharmaceutical composition comprises the lipid compound nanoparticles according to the second aspect or the nucleic acid nanoparticle complexes according to the third aspect, and pharmaceutically acceptable excipients.

[0082] The pharmaceutical composition may be in the form of an injection, suppository, eye drops, tablets, capsules, suspensions or inhalants.

[0083] In some embodiments, the pharmaceutical composition contains at least one RNA for treating or preventing a disease. The composition containing RNA contains at least a portion of a coding RNA and a non-coding RNA; the coding RNA includes at least one coding region encoding at least one therapeutic protein or polypeptide and an immunogenic protein or peptide; the coding RNA is mRNA.

[0084] The therapeutic protein or polypeptide may be a cytokine, a chemokine, a suicide gene product, an immunogenic protein or peptide, an apoptosis inducer, an angiogenesis inhibitor, a heat shock protein, a tumor antigen, a β-catenin inhibitor, a STING pathway activator, a checkpoint regulator, an innate immune activator, an antibody, a dominant negative receptor and a decoy receptor, a myeloid-derived suppressor cell (MDSCs) inhibitor, an IDO pathway inhibitor, and a protein or peptide that binds to an apoptosis inhibitor;

[0085] The immunogenic protein or peptide may be a full-length or partial sequence of at least one protein or peptide from one of the following viruses or bacteria: SARS-CoV-2, human papillomavirus (HPV), influenza virus A or B, or any other orthomyxovirus (influenza virus C); picornaviruses, such as rhinoviruses or hepatitis A virus; togaviruses, such as alphaviruses or rubella viruses, e.g., Sindbis virus, Semliki Forest virus, or measles virus; rubella virus; coronavirus, especially It is a SARS-CoV-2, HCV-229E or HCV-OC43 subtype; a rhabdovirus, such as rabies virus; a paramyxovirus, such as mumps virus; a reovirus, such as rotavirus of group A, B or C; a hepadnavirus, such as hepatitis B virus; a papovavirus, such as human papillomavirus of any serotype; an adenovirus, especially types 1 to 47; a herpesvirus, such as herpes simplex virus 1, 2 or 3; a cytomegalovirus, preferably CMVpp65; an Epstein-Barr virus; a vaccinia virus; a bacterium, Chlamydophila pneumoniae pneumoniae); flaviviruses, such as dengue virus types 1 to 4, yellow fever virus, West Nile virus, Japanese encephalitis virus; hepatitis C virus; caliciviruses; filoviruses, such as Ebola virus; Borna virus; bunyaviruses, such as Rift Valley fever virus; arenaviruses, such as lymphocytic choriomeningitis virus or hemorrhagic fever virus; retroviruses, such as HIV; parvoviruses.

[0086] In a fifth aspect, the present invention provides a use of the compound described in the first aspect, the lipid compound nanoparticles described in the second aspect, the nucleic acid nanoparticle complex described in the third aspect, or the pharmaceutical composition described in the fourth aspect.

[0087] Use of the compound of the first aspect, the lipid compound nanoparticle of the second aspect, the nucleic acid nanoparticle complex of the third aspect or the pharmaceutical composition of the fourth aspect in the preparation of a product for in vivo delivery of nucleic acids.

[0088] The present invention provides a ribonucleic acid vaccine with RNA (such as messenger RNA (mRNA)) as the core and the nanoparticles described in the first aspect as the delivery carrier, which can safely induce the body's naturally existing specific immune system to produce almost any target protein or fragment thereof, including vaccines for infectious pathogens such as bacteria and viruses and tumor vaccines. In some embodiments, the RNA is modified. The RNA vaccine disclosed in the present invention can be used to induce immune responses to infectious pathogens or cancers, including cellular immune responses and humoral immune responses, without the risk of, for example, insertion mutagenesis. Depending on the incidence of infectious pathogens and cancers, the RNA vaccine with the nanoparticles described in the first aspect as the delivery carrier can be used in various environments. The RNA vaccine can be used to prevent and / or treat infectious pathogens or cancers of various metastatic stages or degrees. The RNA vaccine with the nanoparticles described in the first aspect as the delivery carrier has superior properties because it has the characteristic properties of selective transfection for DC cells, and can obtain higher transfection efficiency and transfection expression when the transfection rate is the same or lower, and produce higher antibody titers.

[0089] The present invention provides a ribonucleic acid (RNA) vaccine built on the knowledge that RNA (e.g., messenger RNA (mRNA)) can safely direct the body's cellular machinery to produce virtually any protein of interest, from natural proteins to antibodies and other entirely novel proteins that can have therapeutic activity inside or outside cells. RNA (e.g., mRNA) vaccines can be used in a variety of settings depending on the prevalence of infection or the degree or level of unmet medical need.

[0090] The lipid compound nanoparticles described in the first aspect of the present invention or the nanoparticle complexes of the third aspect are used to prevent, treat and / or improve diseases selected from the following: cancer or tumor diseases, infectious diseases, such as (viral, bacterial or protozoal) infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenic diseases, i.e. (genetic) diseases, or general genetic diseases, diseases with a genetic genetic background and typically caused by a determined gene defect and inherited according to Mendel's laws, cardiovascular diseases, neuronal diseases, respiratory diseases, digestive system diseases, skin diseases, musculoskeletal disorders, connective tissue disorders, tumors, immunodeficiency, endocrine, nutritional and metabolic diseases, eye diseases and ear diseases.

[0091] The nucleic acid vaccine of the present invention can be administered by any route that produces a therapeutically effective result. The route includes (but is not limited to) intradermal, subcutaneous, intraperitoneal, oral, intramuscular, intranasal, intraocular, upper respiratory tract, intravenous, vaginal, and rectal administration. In some embodiments, the nucleic acid vaccine of the present invention is administered by injection.

[0092] Beneficial Effects

[0093] Compared with the prior art, the present invention has the following technical effects:

[0094] The compound, lipid compound nanoparticle or nucleic acid nanoparticle complex provided by the present invention has good biocompatibility, high transfection efficiency, low toxicity and excellent technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 shows the H NMR spectrum of compound L0111;

[0096] Figure 2 shows the H NMR spectrum of compound L0112;

[0097] Figure 3 shows the H NMR spectrum of compound L0113;

[0098] Figure 4 shows the H NMR spectrum of compound L0114;

[0099] Figure 5 shows the H NMR spectrum of compound L0115;

[0100] Figure 6 shows the H NMR spectrum of compound L0116;

[0101] Figure 7 shows the H NMR spectrum of compound L0117;

[0102] Figure 8 shows the H NMR spectrum of compound L0118;

[0103] Fig. 9 shows the H NMR spectrum of compound L0119;

[0104] Fig.10 shows the H NMR spectrum of compound L0120;

[0105] Fig.11 shows the H NMR spectrum of compound L0121;

[0106] Fig.12 shows the H NMR spectrum of compound L0122;

[0107] Fig.13 shows the H NMR spectrum of compound L0123;

[0108] Fig.14 shows the H NMR spectrum of compound L0124;

[0109] Fig.15 shows the H NMR spectrum of compound L0125;

[0110] Fig.16 shows the H NMR spectrum of compound L0126;

[0111] Fig.17 shows the H NMR spectrum of compound L0127;

[0112] Fig.18 shows the H NMR spectrum of compound L0128;

[0113] Fig.19 shows the H NMR spectrum of compound L0129;

[0114] Fig. 20 shows the H NMR spectrum of compound L0130;

[0115] Fig.21 shows the H NMR spectrum of compound L0131;

[0116] Fig. 22 shows the mass spectrum of compound L0132;

[0117] Fig.23 shows the H NMR spectrum of compound L0133;

[0118] Fig.24 shows the H NMR spectrum of compound L0134;

[0119] Fig.25 shows the H NMR spectrum of compound L0135;

[0120] Fig.26 shows the H NMR spectrum of compound L0136;

[0121] Fig. 27 The statistical diagram of the silencing efficiency of the nucleic acid nanoparticle complex loaded with EGFP-siRNA delivered to Hela-EGFP cells in Example 9 is shown;

[0122] Fig.28 The figure shows the expression of luciferase in mice detected by IVIS after intravenous injection of nucleic acid nanoparticle complexes carrying FLuc-mRNA in Example 10;

[0123] Fig.29 The figure shows the expression of luciferase in mice detected by IVIS after intraperitoneal injection of nucleic acid nanoparticle complexes carrying FLuc-mRNA in Example 10;

[0124] Fig.30 The figure shows the expression of luciferase in mice after intramuscular injection of nucleic acid nanoparticle complexes loaded with FLuc-mRNA detected by IVIS in Example 10;

[0125] Fig.31 The figure shows the expression of luciferase in mice detected by IVIS after subcutaneous injection of nucleic acid nanoparticle complexes loaded with FLuc-mRNA in Example 10;

[0126] Fig.32 The statistical graph of serum IgG antibody levels of mice after immunization with the nucleic acid nanoparticle complex loaded with SARS-CoV-2 S-mRNA in Example 11 is shown; the horizontal axis represents the different prescriptions on the 14th day and the 28th day after the first immunization, and the vertical axis represents the difference in OD values ​​of optical density at two wavelengths. The OD value is an indicator for judging the level of IgG antibodies in serum, reflecting the level of anti-S protein IgG in serum.

[0127] Definition of terms

[0128] Unless otherwise indicated, the following terms and phrases as used herein are intended to have the following meanings:

[0129] "Compounds of the present invention" means compounds of formula I or pharmaceutically acceptable salts, tautomers, polymorphs, isomers and solvates thereof. Similarly, the phrase "compounds of formula I" means compounds of the formula and pharmaceutically acceptable salts, tautomers, polymorphs, isomers and solvates thereof.

[0130] In the present invention, expressions such as "compound I" and "compound represented by formula I" refer to the same compound.

[0131] "V / V" means volume ratio.

[0132] IC 50 It represents the half inhibitory concentration.

[0133] The term "plurality" means at least 2, for example 2, 3, 4 or 5 etc.

[0134] In the present invention, the meanings of the atoms in the compound structure are as follows: F represents fluorine, Cl represents chlorine, and D represents deuterium.

[0135] The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0136] The terms "optional", "optional" or "optionally" mean that the subsequently described event or circumstance may but need not occur.

[0137] In the present invention, "room temperature" refers to ambient temperature, which is from about 10°C to about 40°C. In some embodiments, "room temperature" refers to a temperature from about 20°C to about 30°C; in other embodiments, "room temperature" refers to a temperature from about 25°C to about 30°C; in still other embodiments, "room temperature" refers to 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, etc.

[0138] "Alkyl" is a hydrocarbon containing positive carbon atoms, secondary carbon atoms, tertiary carbon atoms, or cyclic carbon atoms. For example, an alkyl group can have 1 to 20 carbon atoms (i.e., C1-C 20 alkyl), 1 to 10 carbon atoms (i.e., C1-C 10alkyl), 1 to 8 carbon atoms (i.e., C1-C8 alkyl), or 1 to 6 carbon atoms (i.e., C1-C6 alkyl). Examples of suitable alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), 1-propyl (i-Pr, i-propyl, -CH2CH2CH3), 2-propyl (i-Pr, i-propyl, -CH(CH3)2), 1-butyl (n-Bu, n-butyl, -CH2CH2CH2CH3), 2-methyl-1-propyl (i-Bu, i-butyl, -CH2CH(CH3)2), 2-butyl (s-Bu, s-butyl, -CH (CH3)CH2CH3), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH3)3), 1-pentyl (n-pentyl, -CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl ( -CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), 1-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH (CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3 or octyl (-(CH2)7CH3).

[0139] The term "jk pieces", "jk yuan" or "C j -C k " wherein j and k are independently any non-zero natural numbers, and k>j; for example, "1-4" means 1, 2, 3 or 4, "4-6 yuan" means 4 yuan, 5 yuan or 6 yuan; "C3-C6" means C3, C4, C5 or C6. And so on.

[0140] In the nucleic acid nanoparticle complex, the positive charge is usually provided by the ionizable nitrogen (N) in the ionizable lipid (such as the compound shown in Formula A provided by the present invention), and the negative charge is provided by the phosphate (P) in the nucleic acid molecule, and the two can be combined together by electrostatic adsorption. The nitrogen-phosphorus ratio (N / P) is the ratio of the molar number of ionizable N in the ionizable lipid to the molar number of P in the nucleic acid molecule. DETAILED DESCRIPTION

[0141] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0142] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0143] The term “×g” indicates how many times the acceleration due to gravity is a centrifugal acceleration. For example, “5000×g” indicates 5000 times the acceleration due to gravity is a centrifugal acceleration.

[0144] DMG-PEG2000 stands for 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol 2000; PEG-DMPE stands for dimyristoylphosphatidylethanolamine-polyethylene glycol; PEG-DPPC stands for dipalmitoylphosphatidylcholine-polyethylene glycol; mPEG-STA stands for methoxypolyethylene glycol-monostearic acid; mPEG-PS stands for methoxypolyethylene glycol-phosphatidylserine; mPEG-DPPE stands for dipalmitoylphosphatidylethanolamine-methoxy-polyethylene glycol; mPEG-DSPE stands for =Methoxy-polyethylene glycol-phosphatidylethanolamine; mPEG-DMPE means methoxypolyethylene glycol-1,2-tetradecanoylphosphatidylethanolamine; DOTAP means (2,3-dioleoyl-propyl)-trimethylamine sulfate; DOPE means 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DSPC means 1,2-distearoyl-sn-glycero-3-phosphocholine; Chol means cholesterol; DMPC means 1,2-dimyristoyl-sn-glycero-phosphocholine; PC means phosphatidylcholine; 20 indicates Tween 20; DPPC indicates 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; 80 represents Span 80. EDCI represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. DMAP represents 4-dimethylaminopyridine.

[0145] L0111 represents L0111 compound, L0112 represents L0112 compound, L0113 represents L0113 compound, and so on.

[0146] FLuc-mRNA indicates messenger RNA encoding firefly luciferase; EGFP-pDNA indicates plasmid encoding green fluorescent protein; EGFP-siRNA indicates small interfering RNA silencing the expression of enhanced green fluorescent protein gene; S-mRNA or Spike-mRNA indicates messenger RNA encoding S protein.

[0147] FLuc-mRNA manufacturer: Shanghai Hongene Biotech Corporation.

[0148] The specific information of FLuc-mRNA stock solution is as follows:

[0149] Product name: FLuc-mRNA (N1-Me-pseudo U);

[0150] Product description: 1939 nucleotides in length;

[0151] Modifications: Fully substituted with N1-Me-pseudo UTP;

[0152] Concentration: 1.0 mg / mL;

[0153] Storage environment: 1mM sodium citrate, pH 6.4;

[0154] Storage requirements: -40℃ or below.

[0155] Example 1: Preparation of compound L0111

[0156] The cationic lipid compound of the present invention is produced by any previously known synthesis method known to a person skilled in the art. The raw materials Compound 1, Compound 2, Compound 3, and Compound 4 in the preparation method can be purchased commercially or synthesized using conventional methods.

[0157] The simple synthesis method and specific process of the compound L0111 of the present invention are described as follows:

[0158]

[0159] Synthesis of compound 3: 5.766 g of cycloisopropyl malonate (compound 2) and 10.200 mL of lauroyl chloride (compound 1) were mixed with 30 mL of ultra-dry DCM and stirred for 5 min in an ice bath; 6.430 mL of pyridine and 16 mL of ultra-dry DCM were added and the mixture was reacted for 1 h in an ice bath; the reaction was continued after the ice bath was removed, and the consumption of cycloisopropyl malonate (PE / EA=1:1, v / v) was complete as monitored by TLC, and the reaction lasted for 16 h in total; the reaction system was diluted with a large amount of DCM and filtered with diatomaceous earth; the filtered organic phase was washed once with a saturated ammonium chloride solution and then twice with saturated brine, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to obtain a solution containing compound 3 (deep red liquid), which was directly used in the next step without separation and purification.

[0160] Synthesis of compound 5: 36.536 g of 1,6-hexanediol (compound 4) was mixed with compound 3 obtained in the previous step, heated for reaction, and the compound 3 (PE / EA=1:1, v / v) was completely consumed under TLC monitoring, and the reaction lasted for 18 h in total; after cooling, a large amount of petroleum ether was added to precipitate 1,6-hexanediol, filtered, and the filtrate was washed once with a saturated ammonium chloride solution and three times with saturated brine, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to obtain a dark red liquid, which was separated and purified by column chromatography (eluent was PE / EA=3:1 (v / v), Rf≈0.5), to obtain 5.417 g of compound 5. An appropriate amount of the obtained compound 5 was taken to detect the hydrogen spectrum and mass spectrum, and the results were as follows:

[0161] 1 H NMR(400MHz,Chloroform-d)δ4.13(t,J=6.8Hz,2H),3.64(t,J=6.4Hz,2H),3.42(s,2H),2.52(t,J=7.2Hz ,2H),1.70-1.62(m,2H),1.62-1.51(m,6H),1.47-1.33(m,4H),1.32-1.19(m,16H),0.87(t,J=6.8Hz,3H).

[0162] HRMS(ESI,m / z):[M+H] + calcd.For C 20 H 38 O4,343.28428; found 343.28497.

[0163] Synthesis of compound 6: 5.140 g of compound 5 was mixed with 130 mL of ultra-dry DCM; 6.255 mL of LTEA was added under N2 atmosphere, and then 0.187 g of DMAP dissolved in 10 mL of ultra-dry DCM was added, and stirred in an ice bath for 5 min; 4.523 g of TBSCl (tert-butyldimethylsilyl chloride) was dissolved in 20 mL of ultra-dry DCM, added under N2 atmosphere, and the ice bath was removed after sufficient stirring and dissolution, and the reaction was carried out at room temperature for 75 min; TLC monitored that compound 5 (PE / EA=5:1 (v / v)) was completely consumed, and then extracted with water, the organic phase was collected, and the aqueous phase was extracted once with DCM, and the organic phase and DCM phase were combined to obtain a combined solution, which was washed twice with saturated brine, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation, and separated and purified by column chromatography (eluent: PE / EA=20:1 (v / v), Rf≈0.5) to obtain 6.319 g of compound 6. An appropriate amount of the obtained compound 6 was taken for mass spectrometry testing, and the results were as follows:

[0164] HRMS(ESI,m / z):[M+H] + calcd.For C 26 H 52 O4Si,457.37076; found457.37067.

[0165] Synthesis of compound 7: 6.319g of compound 6 was mixed with 150mL of methanol and stirred for 5min in an ice bath; 0.630g of sodium borohydride was added and stirred for 2h in an ice bath; TLC monitored that compound 6 (PE / EA=20:1, v / v) was completely consumed, saturated sodium bicarbonate solution was added to quench the reaction under ice bath conditions, methanol was removed by rotary evaporation, a small amount of pure water was added, and the mixture was extracted twice with ethyl acetate, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation, and separated and purified by column chromatography (eluent: PE / EA=10:1 (v / v), Rf≈0.5), to obtain 2.932g of compound 7. An appropriate amount of the obtained compound 7 was taken to detect the hydrogen spectrum and mass spectrum, and the results are as follows:

[0166] 1 H NMR(400MHz,Chloroform-d)δ4.08(t,J=6.8Hz,2H),4.03-3.92(m,1H),3.58(t,J=6.4Hz,2H),2.99(d,J=3.6Hz,1H),2.48(dd,J =16.4,3.2Hz,1H),2.37(dd,J=16.4,8.8Hz,1H),1.67-1.57(m,2H),1.56-1.42(m,4H),1.41-1.19(m,22H),0.91-0.81(m,12H).

[0167] HRMS(ESI,m / z):[M+H] + calcd.For C 26 H 54 O4Si,459.38641; found459.38915.

[0168] Synthesis of compound 8: 1.373g of compound 7, 0.036g of DMAP, 2mL of ultra-dry DCM dissolved 6.255mL of TEA, added by injection, stirred under ice bath for 10min; 2mL of ultra-dry DCM dissolved hexanoyl chloride, added dropwise under N2 atmosphere, reacted in ice bath for 10min, then removed the ice bath, reacted at room temperature; TLC monitored that compound 7 (PE / EA=10:1, v / v) was completely consumed, reacted for 18h; saturated ammonium chloride solution was added for extraction, the organic phase was collected, washed twice with saturated brine, the organic phase was collected, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, separated and purified by column machine (PE / EA=20:1, v / v, Rf≈0.5), and compound 8 was obtained, with a yield of 1.100g and a yield of 65.8%. An appropriate amount of the obtained compound 8 was taken to detect the hydrogen spectrum and mass spectrum, and the results are as follows:

[0169] 1 H NMR(400MHz,Chloroform-d)δ5.28-5.15(m,1H),4.09-3.98(m,2H),3.57(t,J=6.4Hz,2H),2.64 -2.30(m,3H),2.24(t,J=7.6Hz,1H),1.66-1.44(m,8H),1.36-1.17(m,28H),0.91-0.83(m,16H).

[0170] HRMS(ESI,m / z):[M+H] + calcd.For C 32 H 64 O5Si,557.45958; found 557.45974.

[0171] Synthesis of compound 9: 1.100g of compound 8 was stirred and dissolved in 20mL of ultra-dry THF; 1.563g of tetrabutylammonium fluoride trihydrate was added to the above system; TLC monitored that compound 8 (PE / EA=25:1, v / v) was completely consumed, and the reaction lasted for 7h; saturated ammonium chloride solution was added to quench the reaction, and the water layer was extracted with DCM three times, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation; separated and purified by column chromatography (PE / EA=5:1, v / v, Rf≈0.45) to obtain compound 9, with a yield of 0.717g and a yield of 81.8%. An appropriate amount of the obtained compound 9 was taken to detect the mass spectrum, and the results were as follows:

[0172] HRMS(ESI,m / z):[M+H] + calcd.For C 26 H 50 O5,443.37310; found443.37283.

[0173] Synthesis of compound 10: 0.717g of compound 9 was taken and dissolved by stirring with 5mL of ultra-dry DCM; then 0.680g of sodium bicarbonate and 0.867g of (1,1,1-triacetoxy)-1,1-dihydro-1,2-benzidoyl-3(1H)-one were added and stirred at room temperature for 0.5h; TLC monitored that compound 9 (PE / EA=5:1, v / v) was completely consumed, saturated sodium thiosulfate solution was added to quench the reaction, and the aqueous layer was extracted three times with DCM, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation; separated and purified by column chromatography (PE / EA=10:1, v / v, Rf≈0.5), and compound 10 was obtained, with a yield of 0.263g and a yield of 36.9%. An appropriate amount of the obtained compound 10 was taken and the mass spectrum was detected, and the results were as follows:

[0174] HRMS(ESI,m / z):[M+H] + calcd.For C 26 H 48 O5,441.35745; found441.35687.

[0175] Synthesis of L0111: 0.336g of compound 10 was stirred and dissolved with 2mL ultra-dry THF; 0.027g of 4-amino-1-butanol and 0.163g of sodium triacetoxyborohydride were added, and the mixture was stirred at room temperature for 12h; a saturated sodium bicarbonate solution was added to quench the reaction, and the aqueous layer was extracted three times with DCM, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation; L0111 was separated and purified by column chromatography (DCM / Ultra=3:1, v / v, Rf≈0.5), and the yield was 0.131g, and the yield was 44.0%. An appropriate amount of the obtained L0111 was taken to detect the hydrogen spectrum and mass spectrum, and the results were as follows:

[0176] 1 H NMR(400MHz,Chloroform-d)δ5.21(p,J=6.4Hz,2H),4.05(t,J=6.8Hz,4H),3.57(s,2H),2.67-2 .30(m,9H),2.26(t,J=7.6Hz,4H),1.63-1.55(m,20H),1.36-1.22(m,52H),0.94-0.84(m,12H).

[0177] HRMS (ESI, m / z) [M+H] +calcd for C56H108NO9938.80186; found: 938.80203.

[0178] Example 2: Preparation of compound L0112

[0179]

[0180] Synthesis of compound 11: Take 0.690g of compound 7 and 0.010g of DMAP, dissolve 3.0mL of TEA in 2mL of ultra-dry DCM, inject and rinse, stir under ice bath for 10min; dissolve lauroyl chloride in 2mL of ultra-dry DCM, add dropwise under N2 atmosphere, react in ice bath for 10min, remove ice bath, react at room temperature; TLC monitors that compound 7 (PE / EA=10:1, v / v) is completely consumed, react for 18h; add saturated ammonium chloride solution to extract, collect organic phase, wash organic phase twice with saturated salt water, collect organic phase, dry over anhydrous sodium sulfate, concentrate by rotary evaporation, separate and purify by column machine, PE / EA=25:1, v / v, Rf≈0.5, obtain compound 11, yield 0.544g, yield 56.5%. Take appropriate amount of obtained compound 11, detect mass spectrum, the result is as follows:

[0181] HRMS(ESI,m / z):[M+H] + calcd.For C 38 H 76 O5Si,641.55348; found 641.55320.

[0182] Synthesis of compound 12: 0.544g of compound 11 was stirred and dissolved with 10mL of ultra-dry THF; mixed with 0.318g of tetrabutylammonium fluoride trihydrate and prepared with ultra-dry THF to a 1M solution of tetrabutylammonium fluoride trihydrate, and reacted at room temperature; TLC monitored that compound 8 (PE / EA=25:1, v / v) was completely consumed, and the reaction lasted for 18h; saturated ammonium chloride solution was added to quench the reaction, and the water layer was extracted three times with DCM, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation; separated and purified by column chromatography (PE / EA=5:1, v / v, Rf≈0.5), and compound 12 was obtained, with a yield of 0.384g and a yield of 85.7%. An appropriate amount of the obtained compound 12 was taken for mass spectrometry, and the results were as follows: HRMS (ESI, m / z): [M+H] + calcd.For C 32 H 62 O5,527.46700; found527.46789.

[0183] Synthesis of compound 13: 0.384 g of compound 12 was dissolved in 5 mL of ultra-dry DCM by stirring; 0.307 g of sodium bicarbonate and 0.383 g of (1,1,1-triacetoxy)-1,1-dihydro-1,2-benzidoyl-3(1H)-one were added and stirred at room temperature for 0.5 h; TLC monitored the complete consumption of compound 9 (PE / EA=5:1, v / v), saturated sodium thiosulfate solution was added to quench the reaction, the aqueous layer was extracted three times with DCM, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation; the mixture was separated and purified by column chromatography (PE / EA=8:1, v / v, Rf≈0.5) to obtain compound 13 with a yield of 0.108 g and a yield of 27.3%.

[0184] Synthesis of compound L0112: 0.108g of compound 13 was dissolved by stirring with 2mL ultra-dry THF; mixed with 0.008g of 4-amino-1-butanol and 0.044g of sodium triacetoxyborohydride, and stirred at room temperature for 12h; TLC monitoring (DCM / Ultra=3:1, v / v)(Ultra=DCM:methanol:ammonia water=75:22:3), there was a relatively concentrated point at Rf≈0.5, saturated sodium bicarbonate solution was added to quench the reaction, the water layer was extracted three times with DCM, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation; separated and purified by column chromatography (DCM / Ultra=3:1, v / v, Rf≈0.5), and compound L0112 was obtained, with a yield of 0.022g and a yield of 23.2%. An appropriate amount of the obtained compound L0112 was taken to detect the hydrogen spectrum and mass spectrum, and the results were as follows:

[0185] 1 H NMR(400MHz,Chloroform-d)δ5.20(p,J=6.2Hz,2H),4.04(t,J=6.8Hz,4H),3.54(t,J=4.4Hz,2H),2.60-2.47(m,4H),2.47-2.3 5(m,6H),2.25(t,J=7.6Hz,4H),1.61(dt,J=14.4,7.2Hz,16H),1.51-1.41(m,4H),1.38-1.20(m,76H),0.87(t,J=6.8Hz,12H);

[0186] HRMS (ESI, m / z) [M+H] + calcd for C68H132NO9:1106.98966; found:1106.99201.

[0187] Example 3: Preparation of Compound L0113

[0188]

[0189] Synthesis of compound 14: 1.377g of compound 7 and 0.039g of DMAP were mixed with the solution obtained by dissolving 1.5mL of LTEA in 2mL of ultra-dry DCM, and stirred for 10min under ice bath; 800mg of myristic acid chloride was dissolved in 2mL of ultra-dry DCM, and added dropwise under N2 atmosphere, reacted in ice bath for 10min, then the ice bath was removed and reacted at room temperature; TLC monitored that compound 7 (PE / EA=10:1, v / v) was completely consumed, and the reaction lasted for 18h; saturated ammonium chloride solution was added for extraction, the organic phase was collected, and the organic phase was washed twice with saturated brine, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation, separated and purified by column chromatography, PE / EA=50:1, v / v, Rf≈0.5, and compound 14 was obtained, with a yield of 1.572g and a yield of 78.3%. HRMS(ESI,m / z):[M+H] + calcd.For C 40 H 80 O5Si,669.58478; found669.58466.

[0190] Synthesis of compound 15: 1.572 g of compound 14 was placed in a three-necked flask, the straight mouth was connected to a two-way valve, the oblique mouth was connected to a rubber stopper, and 10 mL of ultra-dry THF was stirred and dissolved; 1.853 g of tetrabutylammonium fluoride trihydrate was taken and prepared into a 1M solution with ultra-dry THF, and reacted at room temperature; TLC monitored that compound 8 (PE / EA=25:1, v / v) was completely consumed, and the reaction lasted for 18 hours; saturated ammonium chloride solution was added to quench the reaction, and the water layer was extracted three times with DCM, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation; separated and purified by column chromatography, PE / EA=5:1, v / v, Rf≈0.5, to obtain compound 15, with a yield of 0.902 g and a yield of 69.2%. HRMS (ESI, m / z): [M+H] + calcd.For C 34 H 66 O5,555.49830; found 555.49822.

[0191] Synthesis of compound 16: 0.902g of compound 15 was dissolved by stirring with 5mL of ultra-dry DCM; then mixed with 0.686g of sodium bicarbonate and 0.861g of (1,1,1-triacetoxy)-1,1-dihydro-1,2-benzidoyl-3(1H)-one and stirred at room temperature for 0.5h; TLC monitored the complete consumption of compound 9 (PE / EA=5:1, v / v), saturated sodium thiosulfate solution was added to quench the reaction, the aqueous layer was extracted three times with DCM, the organic phase was collected, dried over anhydrous sodium sulfate and concentrated by rotary evaporation; separated and purified by column chromatography (PE / EA=8:1, v / v, Rf≈0.5), and compound 16 was obtained with a yield of 0.622g and a yield of 69.0%. HRMS (ESI, m / z): [M+H] + calcd.ForC 34 H 66 O5,553.48265; found 553.48342.

[0192] Synthesis of compound L0113: 0.133g of compound 16 was dissolved by stirring with 2mL ultra-dry THF; then mixed with 0.009g of 4-amino-1-butanol and 0.054g of sodium triacetoxyborohydride and stirred at room temperature for 12h; TLC monitoring (DCM / Ultra=3:1, v / v)(Ultra=DCM:methanol:ammonia water=75:22:3), there was a relatively concentrated point at Rf≈0.5, saturated sodium bicarbonate solution was added to quench the reaction, the water layer was extracted three times with DCM, the organic phase was collected, dried over anhydrous sodium sulfate and concentrated by rotary evaporation; separated and purified by column chromatography (DCM / Ultra=3:1, v / v, Rf≈0.5), and compound L0113 was obtained, with a yield of 0.062g and a yield of 53.4%. An appropriate amount of the obtained compound L0113 was taken to detect the hydrogen spectrum and mass spectrum, and the results were as follows:

[0193] 1H NMR (400MHz, Chloroform-d) δ5.21 (p, J = 6.4Hz, 2H), 4.05 (t, J = 6.8Hz, 4H), 3.58 (s, 2H), 2.75-2. 32(m,8H), 2.26(t,J=7.6Hz,4H), 1.66-1.54(m,20H), 1.43-1.18(m,80H), 0.88(t,J=6.8Hz,12H).

[0194] HRMS(ESI,m / z):[M+H] + calcd.For C 72 H 140 NO9,1163.05226; found 1163.05738.

[0195] Example 4: Preparation of Compound L0114

[0196]

[0197] Synthesis of compound 17: 1.045g of compound 5 was dissolved in 10mL of methanol and 40mL of methanol was added, and the mixture was stirred in an ice bath for 5min; 0.137g of sodium borohydride was added, and the mixture was stirred in an ice bath for 2h; TLC monitored that compound 5 (PE / EA=5:1, v / v) was completely consumed, and a saturated sodium bicarbonate solution was added to quench the reaction under ice bath conditions. After removing methanol by rotary evaporation, an appropriate amount of pure water was added, and the mixture was extracted twice with ethyl acetate. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The mixture was separated and purified by column chromatography (PE / EA=2:1, v / v, Rf≈0.35) to obtain compound 17 with a yield of 0.950g and a yield of 91.9%. HRMS (ESI, m / z): [M+H] + calcd.ForC 20 H 40 O4,345.29993; found 345.29975.

[0198] Synthesis of compound 18: 0.950g of compound 17 was dissolved in 30mL of ultra-dry DCM, 15mL of saturated sodium bicarbonate solution was added, and then 0.122g of potassium bromide and 0.045g of TEMPO were added, and stirred in an ice bath for 10min; 3mL of sodium hypochlorite aqueous solution (effective chlorine ≥5%) was added, and TLC monitoring was performed every 2min (PE / EA=5:1, v / v), and the reaction lasted for 10min; saturated sodium thiosulfate solution was added to quench the reaction, the aqueous phase was extracted with DCM and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and separated and purified by column chromatography (PE / EA=4:1, v / v, Rf≈0.5), to obtain compound 18, with a yield of 0.086g and a yield of 9.1%. HRMS (ESI, m / z): [M+H] + calcd.For C 20 H 38 O4,343.28428; found 343.28415.

[0199] Synthesis of compound L0114: 0.086g of compound 18 was taken and dissolved by stirring with 2mL ultra-dry THF; then 0.009g of 4-amino-1-butanol and 0.056g of sodium triacetoxyborohydride were added and stirred at room temperature for 12h; TLC monitoring (DCM / Ultra=3:1, v / v) (Ultra=DCM:methanol:ammonia water=75:22:3), there was a relatively concentrated point at Rf≈0.5, saturated sodium bicarbonate solution was added to quench the reaction, the water layer was extracted three times with DCM, the organic phase was collected, dried over anhydrous sodium sulfate and concentrated by rotary evaporation; separated and purified by column chromatography (DCM / Ultra=3:1, v / v, Rf≈0.5), and compound L0114 was obtained, with a yield of 0.043g and a yield of 55.1%. An appropriate amount of the obtained compound L0114 was taken to detect the hydrogen spectrum and mass spectrum, and the results were as follows:

[0200] 1H NMR (400MHz, Chloroform-d) δ4.07 (tt, J = 11.0, 5.6 Hz, 4H), 3.98 (tt, J = 8.0, 4.0 Hz, 2H), 3.56 (t, J = 5.2 Hz, 2H), 2.75-2.4 9(m,6H), 2.50-2.42(m,2H), 2.37(dd,J=16.0,9.2Hz,2H), 1.77-1.43(m,14H), 1.43-1.16(m,46H), 0.85(t,J=6.8Hz,6H).

[0201] HRMS(ESI,m / z):[M+H] + calcd.For C 44 H 87 NO7,742.65553; found 742.65491.

[0202] Example 5: Preparation of Compound L0115

[0203]

[0204] Take 14.070g of cycloisopropyl malonate (compound 2) and 24.826mL of lauroyl chloride (compound 1) and dissolve them in ultra-dry 300mL DCM, stir for 5min under ice bath; then add 15.706mL of pyridine, react for 1h under ice bath condition, remove the ice bath and react for 5h at room temperature; TLC monitors that cycloisopropyl malonate (PE / EA=1:1, v / v) is completely consumed, and the reaction is 6h in total; after rotary evaporation to remove DCM, add appropriate amount of ethyl acetate, and filter with diatomaceous earth; wash the filtered organic phase once with 100mL of saturated ammonium chloride solution and twice with saturated brine (100mL×2), collect the organic phase, dry it over anhydrous sodium sulfate and concentrate it by rotary evaporation to obtain compound 3 (red-black liquid), which is directly used in the next step without separation and purification.

[0205] 27.552 g of 1,4-butanediol (compound 19) was mixed with the compound 3 obtained in the previous step, and heated to react at a reaction temperature of 80-100°C. The compound 3 (PE / EA=1:1, v / v) was completely consumed as monitored by TLC, and the reaction lasted for 11.5 h in total. After cooling, 1500 mL of DCM was added, and the mixture was washed once with 300 mL of saturated ammonium chloride solution and twice with saturated brine (500 mL×2). After drying over anhydrous sodium sulfate, the mixture was concentrated by rotary evaporation to obtain a dark red liquid, which was separated and purified by column chromatography (PE / EA=3:1, v / v, Rf≈0.5) to obtain compound 20 with a yield of 7.247 g and a yield of 45.29%. HRMS (ESI, m / z): [M+Na] + calcd.For C 18 H 34 O4,337.23493; found 337.23532.

[0206] 7.247 g of compound 20 was dissolved in 200 mL of ultra-dry DCM by stirring; 9.611 mL of TEA was added under N2 atmosphere, 0.282 g of DMAP was dissolved in 300 mL of ultra-dry DCM and then added, and stirred in an ice bath for 5 min; 7.002 g of TBSCl was dissolved in 300 mL of ultra-dry DCM and then added under N2 atmosphere. After sufficient stirring and dissolution, the ice bath was removed. After reacting at room temperature for 75 min, TLC monitoring of compound 20 (PE / EA=5:1, v / v) was completely consumed. An appropriate amount of pure water was charged into a separatory funnel to extract the reaction system, and the organic phase was collected. After the aqueous phase was extracted with DCM, the organic phases were combined, washed twice with saturated brine (100 mL×2), dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The product was separated and purified by column chromatography (PE / EA=20:1, v / v, Rf≈0.5) to obtain compound 21 with a yield of 9.234 g and a yield of 93.44%. HRMS(ESI,m / z):[M+H] + calcd.For C 24H 48 O4Si,429.33946; found429.33914.

[0207] 2.240g of compound 21 was dissolved in methanol and stirred for 5min in an ice bath; 0.237g of sodium borohydride was added and stirred for 2h in an ice bath; TLC monitored that compound 21 (PE / EA=20:1, v / v) was completely consumed, saturated sodium bicarbonate solution was added to quench the reaction under ice bath conditions, methanol was removed by rotary evaporation, 150mL of pure water was added, and the mixture was extracted twice with ethyl acetate (50mL×2), the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation, and separated and purified by column chromatography (PE / EA=10:1, v / v, Rf≈0.5) to obtain compound 22 with a yield of 0.957g and a yield of 42.56%. HRMS (ESI, m / z): [M+H] + calcd.For C 24 H 50 O4Si,431.35511; found431.35559.

[0208] Take 0.957g of compound 22 and 0.027g of DMAP, dissolve 1.234mL of TEA in 50mL of ultra-dry DCM, and stir for 10min under ice bath; dissolve 0.770mL of lauroyl chloride in 20mL of ultra-dry DCM, add under N2 atmosphere, react in ice bath for 10min, remove ice bath, and react at room temperature; TLC monitors that compound 22 (PE / EA=10:1, v / v) is completely consumed, and react for 18h; add 30mL of saturated ammonium chloride solution to extract, collect the organic phase, wash the organic phase twice with saturated brine (20mL×2), collect the organic phase, dry over anhydrous sodium sulfate, and concentrate by rotary evaporation, separate and purify by column chromatography (PE / EA=25:1, v / v, Rf≈0.5), and obtain compound 23, with a yield of 1.095g and a yield of 80.5%. HRMS(ESI,m / z):[M+H] + calcd.For C 36 H 72 O5Si,613.52218; found613.52265.

[0209] 1.095g of compound 23 was dissolved by stirring with 30mL of ultra-dry THF; it was mixed with 1.415g of tetrabutylammonium fluoride trihydrate and prepared with ultra-dry THF to a 1M solution of tetrabutylammonium fluoride trihydrate, and reacted at room temperature; TLC monitored that compound 23 (PE / EA=25:1, v / v) was completely consumed, and the reaction lasted for 6h; saturated ammonium chloride solution was added to quench the reaction, the organic phase was collected, washed twice with saturated brine (20mL×2), dried over anhydrous sodium sulfate, and concentrated by rotary evaporation; it was separated and purified by column chromatography (PE / EA=5:1, v / v, Rf≈0.5) to obtain compound 24, with a yield of 0.417g and a yield of 46.7%. HRMS (ESI, m / z): [M+H] + calcd.For C 30 H 58 O5,499.43570; found499.43604.

[0210] 0.417 g of compound 24 was taken and dissolved in 10 mL of ultra-dry DCM; 0.356 g of sodium bicarbonate and 0.472 g of (1,1,1-triacetoxy)-1,1-dihydro-1,2-benzidoyl-3(1H)-one were added and stirred at room temperature for 0.5 h; TLC monitored that compound 24 (PE / EA=5:1, v / v) was completely consumed, saturated sodium thiosulfate solution was added to quench the reaction, and the aqueous layer was extracted three times with DCM. The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation; it was separated and purified by column chromatography (PE / EA=10:1, v / v, Rf≈0.5) to obtain compound 25 with a yield of 0.233 g and a yield of 56.1%. 1 H NMR (400MHz, Chloroform-d) δ9.78 (s, 1H), 5.26-5.15 (m, 1H), 4.09 (t, J = 6.4Hz, 2H), 2.55 (q, J = 6.8Hz, 4H), 2.26 (t, J =7.6Hz,2H), 2.03-1.90(m,2H), 1.69-1.51(m,4H), 1.36-1.15(m,34H), 0.87(t,J=6.8Hz,6H).HRMS(ESI,m / z):[M+H] + calcd.For C 30 H 56 O5,497.42005; found497.41992.

[0211] Take 0.119 g (compound 25) and stir and dissolve it with 10 mL ultra-dry THF; then add 0.010 g 4-amino-1-butanol and 0.052 g sodium triacetoxyborohydride, and stir at room temperature for 12 h; TLC monitoring (DCM / Ultra=3:1, v / v) (Ultra=DCM:methanol:ammonia water=75:22:3), there are two closely concentrated points at Rf≈0.5, add saturated sodium bicarbonate solution to quench the reaction, collect the organic phase, extract the aqueous layer three times with DCM, collect the organic phase, dry it over anhydrous sodium sulfate, and concentrate it by rotary evaporation; separate and purify it with a column machine (DCM / Ultra=3:1, v / v, Rf≈0.5) to obtain compound L0115 with a yield of 0.091 g and a yield of 86.7%. 1 H NMR (400MHz, Chloroform-d) δ5.26-5.13(m,2H), 4.06(t,J=6.4Hz,4H), 3.60-3.53(m,2H), 2.70-2.37(m,10H) , 2.26(t,J=7.6Hz,4H), 1.78-1.43(m,20H), 1.36-1.17(m,68H), 0.87(t,J=6.8Hz,12H).HRMS(ESI,m / z):[M+H] + calcd.For C 64 H 123 NO9,1050.92706; found1050.92618.

[0212] Example 6: Preparation methods of compound L0116, compound L0117, compound L0118, compound L0119, compound L0120, compound L0121, compound L0122, compound L0123, compound L0124, compound L0125, compound L0126, compound L0127, compound L0128, compound L0129 and compound L0130

[0213] The compounds L0116, L0117, L0118, L0119, L0120, L0121, L0122, L0123, L0124, L0125, L0126, L0127, L0128, L0129, L0130, L0131, L0132, L0133, L0134, L0135 and L0136 of the present invention are obtained by reductive amination of commercially available amines and corresponding aldehyde compounds. The synthesis methods thereof are referred to L0111-L0115.

[0214] According to the structural characteristics of the target product, 0.1mmol of commercially available amine (as shown in Compound A1-Compound A10, respectively) and 0.13*nmmol of aldehyde (as shown in Compound 10, Compound 13, Compound 16, Compound 18, or Compound 25, respectively, where n is the number of active hydrogen atoms of the amine) were dissolved in 2mL of tetrahydrofuran, 0.13*nmmol of sodium triacetoxyborohydride was added to the above solution, and stirred at room temperature for 12h. 5mL of saturated sodium carbonate solution was added to quench the reaction, and 10mL of DCM was extracted twice. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The residue was purified by silica gel column chromatography to obtain the target products L0116-L0136.

[0215]

[0216] Appropriate amounts of the obtained compounds L0116, L0117, L0118, L0119, L0120, L0121, L0122, L0123, L0124, L0125, L0126, L0127, L0128, L0129, L0130, L0131, L0132, L0133, L0134, L0135 and L0136 were taken respectively, and their hydrogen spectra and mass spectra were detected. The results are as follows:

[0217] Compound L0116: 1 H NMR(400MHz,Chloroform-d)δ5.23-5.06(m,2H),3.98(t,J=6.8Hz,4H),3.82-3.72(m,1H),3.67(dd,J=11.6,4.0Hz,1H),3.63-3.52(m,1H),3.45(dd ,J=11.6,4.4Hz,1H),2.83-2.31(m,10H),2.20(t,J=7.6Hz,4H),1.58-1.4 6(m,12H),1.44-1.08(m,56H),0.87-0.77(m,12H).HRMS(ESI,m / z):[M+H] + calcd.For C 55 H 105 NO 10 , 940.78112; found 940.78070.

[0218] Compound L0117: 1H NMR(400MHz,Chloroform-d)δ5.29-5.15(m,2H),4.05(t,J=6.8Hz,4H),3.73-3.57(m,2H),2.80-2.68(m,2H),2.66-2 .46(m,8H),2.26(t,J=7.6Hz,4H),1.67-1.51(m,14H),1.51-1.10(m,54H),0.93-0.84(m,12H).HRMS(ESI,m / z):[M+H] + calcd.For C 54 H 103 NO9,910.77056; found 910.77051.

[0219] Compound L0118: 1 H NMR(400MHz,Chloroform-d)δ5.26-5.15(m,2H),4.04(t,J=6.8Hz,4H),2.63-2.36(m,16H),2.36-2.32(m,2H),2.29 -2.23(m,6H),1.73-1.50(m,14H),1.49-1.39(m,4H),1.39-1.15(m,52H),0.93-0.82(m,12H).HRMS(ESI,m / z):[M+H] + calcd.For C 60 H 115 N3O8, 1006.87569; found 1006.87518.

[0220] Compound L0119: 1 H NMR(400MHz,Chloroform-d)δ5.28-5.14(m,2H),4.04(t,J=6.8Hz,4H),2.67-2.32(m,17H),2.32-2.22(m, 6H),1.77-1.49(m,12H),1.49-1.39(m,4H),1.39-1.14(m,52H),0.93-0.82(m,12H).HRMS(ESI,m / z):[M+H] + calcd.For C 59 H 113 N3O8, 992.86004; found 992.86032.

[0221] Compound L0120: 1H NMR(400MHz,Chloroform-d)δ5.29-5.16(m,2H),4.07(t,J=6.4Hz,4H),3.85-3.67(m,2H),3.50(dd,J=11.2,4.0Hz,1H),2.82 -2.36(m,10H),2.26(t,J=7.6Hz,4H),1.70-1.46(m,16H),1.35-1.23(m,68H),0.88(t,J=6.8Hz,12H).HRMS(ESI,m / z):[M+H] + calcd.For C 63 H 121 NO 10 , 1052.90632; found 1052.90719.

[0222] Compound L0121: 1 H NMR(400MHz,Chloroform-d)δ5.27-5.15(m,2H),4.07(t,J=6.4Hz,4H),3.57(t,J=5.2Hz,2H),2.72-2.40(m,9H ),2.26(t,J=7.6Hz,4H),1.70-1.43(m,16H),1.37-1.18(m,68H),0.87(t,J=6.8Hz,12H).HRMS(ESI,m / z):[M+H] + calcd.ForC 62 H 119 NO9, 1022.89576; found 1022.89627.

[0223] Compound L0122: 1 H NMR(400MHz,Chloroform-d)δ5.26-5.12(m,2H),4.13-4.00(m,4H),3.63(t,J=5.2Hz,4H),2.73-2.43(m,16H) ,2.25(t,J=7.6Hz,4H),1.78-1.46(m,18H),1.41-1.13(m,68H),0.86(t,J=6.8Hz,12H).HRMS(ESI,m / z):[M+H] + calcd.ForC 67 H 130 N2O 10 , 1123.98449; found 1123.98449.

[0224] Compound L0123:1 1H NMR (400 MHz, Chloroform-d) δ 5.28 - 5.15 (m, 2H), 4.06 (t, J = 6.4 Hz, 4H), 3.63 (t, J = 6.4 Hz, 2H), 2.70 - 2.31 (m, 10H), 2.25 (t, J = 7.6 Hz, 4H), 1.69 - 1.43 (m, 20H), 1.42 - 1.17 (m, 72H), 0.87 (t, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 66 H 127 NO9, 1078.95836; found 1078.96087.

[0225] Compound L0124: 1 1H NMR (400 MHz, Chloroform-d) δ 5.28 - 5.16 (m, 2H), 4.05 (t, J = 6.8 Hz, 4H), 3.63 (d, J = 6.8 Hz, 4H), 3.03 (d, J = 8.0 Hz, 1H), 2.57 (pd, J = 15.2, 6.8 Hz, 8H), 2.26 (t, J = 7.6 Hz, 4H), 1.61 (pd, J = 7.2, 4.3 Hz, 12H), 1.43 - 1.15 (m, 56H), 0.93 - 0.83 (m, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 55 H 105 NO 10 , 940.78112; found 940.78279.

[0226] Compound L0125: 1 1H NMR (400 MHz, Chloroform-d) δ 5.27 - 5.14 (m, 2H), 4.03 (t, J = 6.8 Hz, 4H), 3.62 (t, J = 6.4 Hz, 2H), 2.91 - 2.66 (m, 6H), 2.59 - 2.48 (m, 4H), 2.25 (t, J = 7.6 Hz, 4H), 1.77 - 1.48 (m, 20H), 1.45 - 1.19 (m, 56H), 0.87 (q, J = 6.8 Hz, 12H). HRMS (ESI, m / z): [M+H] + calcd. For C 58 H 111 NO9, 966.83316; found 966.83516.

[0227] Compound L0126: 1 H NMR (400MHz, Chloroform-d) δ5.19 (p, J = 6.4Hz, 5H), 4.12-3.98 (m, 10H), 2.78-2.44 (m, 40H), 2.32-2. 17(m,10H),1.77-1.49(m,30H),1.48-1.16(m,220H),0.86(t,J=6.8Hz,30H).HRMS(ESI,m / z):[M+2H] + calcd.For C 180 H 345 N5O 20 ,m / z:1450.31560,found 1449.8233.

[0228] Compound L0127: 1 H NMR(400MHz,Chloroform-d)δ5.21(p,J=6.4Hz,5H),4.04(t,J=6.8Hz,10H),2.73-2.32(m,40H),2.26(t, J=7.6Hz,10H),1.68-1.49(m,30H),1.39-1.19(m,140H),0.88(q,J=6.4Hz,30H).HRMS(ESI,m / z):[M+3H] + calcd.For C 210 H 402 N4O 24 ,780.33749,found:780.34277.

[0229] Compound L0128: 1 H NMR(400MHz,Chloroform-d)δ5.20(p,J=6.4Hz,5H),4.12-4.01(m,10H),2.70-2.42(m,36H),2.26(t ,J=7.6Hz,10H),2.21-2.17(m,4H),1.67-1.50(m,30H),1.51-1.15(m,200H),0.87(t,J=6.8Hz,30H).

[0230] Compound L0129: 11H NMR (400 MHz, Chloroform-d) δ 5.27 - 5.14 (m, 3H), 4.03 (t, J = 6.8 Hz, 6H), 2.75 - 2.48 (m, 10H), 2.25 (t, J = 7.6 Hz, 6H), 1.78 - 1.44 (m, 24H), 1.41 - 1.18 (m, 78H), 0.87 (q, J = 6.8 Hz, 18H). HRMS (ESI, m / z): [M+H] + calcd. For C 78 H 147 NO 12 , 1291.09960; found 1291.10025.

[0231] Compound L0130: 1 1H NMR (400 MHz, Chloroform-d) δ 5.20 (p, J = 6.4 Hz, 3H), 4.05 (t, J = 6.8 Hz, 6H), 2.63 - 2.48 (m, 6H), 2.39 (t, J = 7.2 Hz, 6H), 2.25 (t, J = 7.6 Hz, 6H), 1.68 - 1.51 (m, 18H), 1.50 - 1.39 (m, 6H), 1.35 - 1.16 (m, 102H), 0.87 (t, J = 6.8 Hz, 18H). HRMS (ESI, m / z): [M+H] + calcd. For C 90 H 171 NO 12 , 1459.28740; found 1459.28864.

[0232] Compound L0131: 1 1H NMR (400 MHz, Chloroform-d) δ 5.27 - 5.14 (m, 3H), 4.03 (t, J = 6.8 Hz, 6H), 2.63 - 2.43 (m, 18H), 2.37 - 2.30 (m, 2H), 2.25 (t, J = 7.6 Hz, 4H), 1.75 - 1.53 (m, 18H), 1.53 - 1.41 (m, 6H), 1.41 - 1.14 (m, 78H), 0.91 - 0.81 (m, 18H). HRMS (ESI, m / z): [M+H] + calcd. For C 84 H 159 N3O 12 , 1403.19965; found 1403.19718.

[0233] Compound L0132: HRMS (ESI, m / z): [M+H] + calcd.For C 59 H 113 NO 10 ,996.84372;found996.84448.

[0234] Compound L0133: 1 H NMR(400MHz,Chloroform-d)δ5.26-5.16(m,2H),4.05(t,J=6.8Hz,4H),3.84-3.66(m,2H),3.50(dd,J=11.2,4.0Hz,1H),2.83 -2.39(m,10H),2.26(t,J=7.6Hz,4H),1.70-1.40(m,16H),1.39-1.14(m,60H),0.87(t,J=6.8Hz,12H).HRMS(ESI,m / z):[M+H] + calcd.For C 59 H 113 NO 10 , 996.84372; found 996.84466.

[0235] Compound L0134: 1 H NMR(400MHz,Chloroform-d)δ5.20(p,J=6.4Hz,2H),4.03(t,J=6.8Hz,4H),3.59(t,J=5.2Hz,2H),2.83-2.42(m,1 0H),2.25(t,J=7.6Hz,4H),1.79-1.49(m,20H),1.39-1.20(m,60H),0.86(t,J=6.8Hz,12H).HRMS(ESI,m / z):[M+H] + calcd.For C 60 H 115 NO9, 994.86446; found 994.86665.

[0236] Compound L0135: 1H NMR(400MHz,Chloroform-d)δ5.24-5.14(m,2H),4.03(t,J=6.8Hz,4H),3.63(t,J=6.4Hz,2H),2.93-2.62(m,6H),2.60-2 .47(m,4H),2.25(t,J=7.6Hz,4H),1.74-1.47(m,20H),1.44-1.18(m,64H),0.86(t,J=6.8Hz,12H).HRMS(ESI,m / z):[M+H] + calcd.For C 62 H 119 NO9, 1022.89576; found 1022.89860.

[0237] Compound L0136: 1 H NMR(400MHz,Chloroform-d)δ5.26-5.15(m,2H),4.04(t,J=6.8Hz,4H),3.60(t,J=5.2Hz,2H),2.73-2.64(m,2H),2.63-2 .45(m,8H),2.26(t,J=7.6Hz,4H),1.72-1.43(m,16H),1.40-1.19(m,60H),0.87(t,J=6.8Hz,12H).HRMS(ESI,m / z):[M+H] + calcd.For C 58 H 111 NO9, 966.83316; found 966.83601.

[0238] Example 7: Preparation of Nucleic Acid Nanoparticle Complex

[0239] Take a nucleic acid drug stock solution (wherein the nucleic acid can be FLuc mRNA, EGFP pDNA, EGFP siRNA, SpikemRNA) and dilute it with a buffer solution to a nucleic acid concentration of 0.04 mg / mL (the buffer can be: PBS buffer with a pH value of 3 to 6 and a concentration of 1 to 200 mM, citric acid-sodium citrate buffer, citric acid-disodium hydrogen phosphate buffer, HEPES buffer, sodium acetate buffer, Tris buffer, preferably pH = 4, PBS buffer with a concentration of 10 mM, 20 mM, 50 mM, citric acid-sodium citrate buffer; In this embodiment, a citric acid-sodium citrate buffer with a pH of 4 and a concentration of 50 mM is used) as the aqueous phase.

[0240] The nucleic acid nanoparticle complex was prepared according to the prescription in Table 1. The required PEG derivatives, phospholipids, cholesterol analogs and lipid compounds were taken out of the refrigerator and balanced to room temperature. The PEG derivatives, phospholipids, cholesterol analogs and lipid compounds were weighed and dissolved in ethanol (PEG derivatives were dissolved in ethanol, and the dissolution concentration range was: 1 mg / mL to 10 mg / mL; phospholipids were dissolved in ethanol, and the dissolution concentration range was: 1 mg / mL to 20 mg / mL; cholesterol analogs were dissolved in ethanol, and the dissolution concentration range was: 1 mg / mL to 20 mg / mL; lipid compounds were dissolved in ethanol, and the dissolution concentration range was: 1 mg / mL to 30 mg / mL). Ultrasonic dispersion was used to dissolve the dissolved PEG derivatives, phospholipids, cholesterol analogs and lipid compounds. The corresponding volumes of solutions of the nucleic acid nanoparticles and lipid compounds are taken according to the proportions described in Table 1, and then an appropriate amount of ethanol is added to mix (the amount of ethanol added is adaptively adjusted according to the component ratio of each prescription nucleic acid nanoparticle complex and the ratio of the aqueous phase and the organic phase in the subsequent operation), and an ethanol solution containing PEG derivatives, phospholipids, cholesterol analogs and lipid compounds is prepared as the organic phase. The volume ratio of the aqueous phase to the organic phase is 3:1, and the aqueous phase and the organic phase are quickly mixed using a nanoparticle preparation instrument at a flow rate ratio of 3:1. The ethanol and water in the mixed solution are removed by dialysis or ultrafiltration, and nuclease-free water is added to make the nucleic acid concentration 0.04 mg / mL. Finally, the nucleic acid nanoparticle complex prescription described in Table 1 is obtained, which is stored at 2-8°C for use.

[0241] Prescription Rp.58 prepared using commercially available ionizable cationic lipid molecule SM-102 (CAS: 2089251-47-6) and prescription Rp.69 prepared using commercially available ionizable cationic lipid molecule ALC-0315 (CAS: 2036272-55-4) were used as controls.

[0242] Table 1: Nucleic acid nanoparticle complex formulation

[0243]

[0244]

[0245]

[0246] Note: The nitrogen-phosphorus ratio of the lipid compound to the nucleic acid in Table 1 represents the ratio of the molar amount of ionizable nitrogen atoms in the lipid compound (i.e., the compound provided by the present invention, such as at least one of compounds L0111-L0136, or ALC-0315 or SM-102) in the nucleic acid nanoparticle complex to the molar amount of phosphorus atoms in the nucleic acid.

[0247] Example 8: Characterization of the nucleic acid nanoparticle complex of the present invention

[0248] Particle size and potential: The nucleic acid nanoparticle complex was prepared according to the preparation method described in Example 7, and the dynamic light scattering particle size (size), surface potential (Zeta Potential) and polydispersity (PDI) of the nucleic acid nanoparticle complex were tested at 25°C using a Malvern Zetasizer Nano ZSE.

[0249] The results are shown in Table 2, which indicate that the particle size of the nucleic acid nanoparticle complex of the present invention ranges from 68 nm to 210 nm, has good dispersibility, and the surface charge of the nanocomplex ranges from -12 mV to +26 mV.

[0250] Encapsulation efficiency: Using FLuc-mRNA as the model mRNA, the nucleic acid nanoparticle complex was prepared according to the preparation method described in Example 7, and the mRNA encapsulation efficiency of each formulation was determined using the Quant-iT RiboGreen RNA detection kit (ThermoFische). The specific method was determined according to the standard procedure of the Quant-iT RiboGreen RNA kit. The results are shown in Table 2. The formulations all had good encapsulation effects on mRNA, and the encapsulation efficiency was above 85%.

[0251] Table 2: Characterization of nucleic acid nanoparticle complexes

[0252]

[0253]

[0254]

[0255] Example 9: In vitro cell transfection experiment and cytotoxicity study of nucleic acid nanoparticle complexes

[0256] 1) Experiments of in vitro transfection of DC2.4 (mouse dendritic cells), 4T1 (mouse breast cancer cells), 293T (human renal epithelial cells), and HeLa (human cervical cancer cells) with nucleic acid nanoparticle complexes encapsulating FLuc-mRNA:

[0257] The cell suspension in logarithmic growth phase was cultured at 4×10 4The cells were divided into 96-well plates at a density of 10 cells per well and placed in a 37°C, 5% CO2 incubator for static culture. After 24 hours, the cell wells were removed, the complete culture medium was aspirated, and 180 μL of opti-MEM was added. FLuc-mRNA was prepared according to the preparation method of different prescriptions described in Example 7, and the nucleic acid nanoparticle complex mixture was diluted with PBS. After standing, a nucleic acid nanoparticle complex containing 200 ng FLuc-mRNA was added to each well, and each sample was repeated in 4 wells. After 4 hours of administration, the culture medium in the 96-well plate was replaced with complete culture medium. Continue to culture for 24 hours, aspirate the complete culture medium, rinse it with PBS, add D-Luciferin working solution to each 96-well plate, continue to culture in a 37°C incubator for 5 minutes, and use Omega-FLuostar microplate reader to image and test the fluorescence expression intensity of FLuc-mRNA. According to the manufacturer's instructions, Lipofectamine2000 (Invitrogen) was used as a positive control group. The relative transfection efficiency is calculated as follows:

[0258] Relative transfection efficiency (%) = fluorescence intensity of cells transfected with nucleic acid nanoparticle complexes / fluorescence intensity of cells transfected with Lipo2000 × 100%

[0259] Results: The transfection efficiency of FLuc-mRNA of some prescriptions on DC2.4 (mouse dendritic cells), 4T1 (mouse breast cancer cells), 293T (human renal epithelial cells), and HeLa (human cervical cancer cells) cells is shown in Table 3. Conclusion: Among them, Rp.03, Rp.09, Rp.14, Rp.15, Rp.21, Rp.33, Rp.34, Rp.35, Rp.39, and Rp.54 have better transfection effects.

[0260] Table 3: Relative transfection efficiency of FLuc-mRNA in DC2.4, 4T1, 293T and HeLa cells with different formulations in Example 8

[0261]

[0262] 2) Toxicity experiment of in vitro transfection of DC2.4 (mouse dendritic cells), 4T1 (mouse breast cancer cells), 293T (human renal epithelial cells), and HeLa (human cervical cancer cells) with nucleic acid nanoparticle complexes encapsulating FLuc-mRNA:

[0263] The cell suspension in logarithmic growth phase was cultured at 4×10 4The cells were divided into 96-well plates at a density of 10 cells per well and placed in a 37°C, 5% CO2 incubator for static culture. After 24 hours, the cell wells were removed, the complete culture medium was aspirated, and opti-MEM was added. FLuc-mRNA was prepared according to the preparation method of different prescriptions described in Example 7 to prepare nucleic acid nanoparticle complexes, and then the nucleic acid nanoparticle complex mixture was diluted with PBS. After standing, nucleic acid nanoparticle complexes containing 200ng FLuc-mRNA were added to each well, and each sample was repeated 4 wells. After 4 hours of administration, the culture medium in the 96-well plate was replaced with complete culture medium. Continue to culture for 48 hours, aspirate the complete culture medium, and rinse it with PBS. The cell wells without prescription were used as negative controls, and the CCK-8 culture medium wells without cells were used as blank controls. 90μL of serum-free culture medium and 10μL of CCK-8 mixed solution were added to each well, and incubated in the incubator for 2 to 4 hours. The absorbance at 450nm was measured using an Omega-FLuostar microplate reader. Cell viability calculation formula:

[0264] Cell viability*%=[A(drug added)-A(blank)] / [A(no drug added)-A(blank)]×100%;

[0265] A (drug addition): absorbance of each well with DC2.4 cells, prescription solution, and CCK-8 solution added;

[0266] A (blank): absorbance of each well with only CCK-8 solution added;

[0267] A (no drug added): absorbance of each well with DC2.4 cells and CCK-8 solution added;

[0268] *Cell viability: cell proliferation activity or cytotoxic activity.

[0269] The results are shown in Table 4. Conclusion: The results show that the cell survival rate is above 80%, indicating that the nucleic acid nanoparticle complexes of different formulations provided by the present invention have no obvious cytotoxicity and have good biocompatibility, and can be used for subsequent animal in vivo experiments.

[0270] Table 4: Survival rate of cells after different prescriptions were used to treat different cells in Example 8

[0271]

[0272] 3) Experiments on in vitro transfection of DC2.4 (mouse dendritic cells) and 4T1 (mouse breast cancer cells) with nucleic acid nanoparticle complexes containing EGFP-pDNA:

[0273] The cell suspension in logarithmic growth phase was cultured at 4×10 4The cells were divided into 96-well plates at a density of cells per well and placed in a 37°C, 5% CO2 incubator for static culture. After 24 hours, the cell well plate was removed, the complete culture medium was aspirated, and opti-MEM was added. EGFP-pDNA was taken and nucleic acid nanoparticle complexes were prepared according to the preparation methods of different prescriptions described in Example 7, and the nucleic acid nanoparticle complex mixture was diluted with PBS. After standing, nucleic acid nanoparticle complexes containing 200ng FLuc-mRNA were added to each well, and each sample was repeated in 4 wells. 4 hours after administration, the culture medium in the 96-well plate was replaced with complete culture medium. The culture was continued for 24 hours, the cells were digested and collected, the fluorescence intensity of the FITC channel of live cells in each well was detected by flow cytometry, and the geometric mean of the fluorescence intensity of EGFP-positive cells in the duplicate wells was calculated. According to the manufacturer's instructions, Lipofectamine2000 (Invitrogen) was used as a positive control group. The relative transfection efficiency was calculated as follows:

[0274] Relative transfection efficiency (%) = geometric mean of fluorescence intensity of cells transfected with nucleic acid nanoparticle complexes / geometric mean of fluorescence intensity of cells transfected with Lipo2000 × 100%

[0275] The results are shown in Table 5. Conclusion: The results show that the nucleic acid nanoparticle complex encapsulating EGFP-pDNA shows a good expression level at the cellular level.

[0276] Table 5: Relative transfection efficiency of EGFP-pDNA in DC2.4 and 4T1 cells by different prescriptions in Example 7

[0277]

[0278] 4) Gene expression silencing experiment in vitro by transfecting HeLa-EGFP cells (polyclonal cell line stably expressing EGFP fluorescent protein) with nucleic acid nanoparticle complexes encapsulating EGFP-siRNA (using EGFP-siRNA as model siRNA):

[0279] The cell suspension in logarithmic growth phase was diluted to 1×10 5The density of cells per well was divided into 24-well plates and placed in a 37°C, 5% CO2 incubator for static culture. After 24 hours, EGFP-siRNA was taken to prepare nucleic acid nanoparticle complexes according to the preparation methods of different prescriptions described in Example 7, and the nucleic acid nanoparticle complex mixture was diluted with PBS. After standing, nucleic acid nanoparticle complexes containing 160ng EGFP-siRNA were added to each well, and each sample was repeated 3 wells. 4 hours after administration, the culture medium in the 96-well plate was replaced with complete culture medium. Continue to culture for 18-24 hours, digest and collect cells, detect the fluorescence intensity of the FITC channel of live cells in each well by flow cytometer, and calculate the geometric mean of the fluorescence intensity of EGFP-positive cells in duplicate wells. The siRNA silencing efficiency (i.e., the percentage reduction in the average fluorescence intensity of cells) is calculated as follows:

[0280] siRNA silencing efficiency (%) = (geometric mean fluorescence intensity of cells not transfected with nucleic acid nanoparticle complexes - geometric mean fluorescence intensity of cells transfected with nucleic acid nanoparticle complexes) / geometric mean fluorescence intensity of cells not transfected with nucleic acid nanoparticle complexes × 100%

[0281] The results are as follows Fig. 27 Conclusion: The results show that the nucleic acid-lipid compound encapsulating EGFP-siRNA showed good gene silencing effect in cell transfection, among which Rp.03, Rp.14, Rp.15, Rp.19, Rp.21, Rp.28, Rp.33, Rp.34, Rp.35, Rp.39 and Rp.54 had a silencing efficiency of more than 50% in delivering siRNA in cells, which was better than other prescriptions.

[0282] Example 10: Small animal fluorescence imaging to detect transfection of nucleic acid nanoparticle complexes in mice

[0283] Three BALB / c mice were included in each group. FLuc-mRNA was used as the model mRNA. A nucleic acid nanoparticle complex containing FLuc-mRNA was prepared according to the preparation method of the prescription described in Example 7. The experimental group used an insulin needle to inject 75 μL of the nucleic acid nanoparticle complex prescription containing 3 μg FLuc-mRNA into each mouse. NC represents the blank control group, and 75 μL of PBS buffer was injected intramuscularly with an insulin needle. When the administration method was intravenous injection, the injection site was the mouse tail vein. When the administration method was intraperitoneal injection, the injection site was the mouse abdominal cavity. When the administration method was intramuscular injection, the injection site was the mouse thigh muscle. When the administration method was subcutaneous injection, the injection site was subcutaneous on the back of the mouse. Detection was performed 3 hours after intravenous injection, 6 hours after intraperitoneal injection, 24 hours after intramuscular injection, and 24 hours after subcutaneous injection. Take an appropriate amount of substrate D-Luciferin, dilute it with PBS to prepare a solution with a concentration of 15 mg / mL, keep it away from light, and inject 200 μL of substrate into each mouse. Place the mouse in a small animal anesthesia box, open the ventilation valve, and release isoflurane to anesthetize the mouse. 5 minutes after substrate injection, use a small animal in vivo imaging system (PerkinElmer, IVIS Lumina SeriesⅢ) to perform whole-body in vivo imaging bioluminescence image detection on the mouse. Take bioluminescent images of mice.

[0284] The results are as follows Figure 28 to Figure 31 As shown, the nucleic acid nanoparticle complexes in the experimental group showed luciferase expression in the whole body in vivo imaging. The greater the fluorescence intensity, the more luciferase expression. Conclusion: The nucleic acid nanoparticle complexes in each experimental group encapsulating FLuc-mRNA have good luciferase expression in mice.

[0285] like Fig.28 As shown, in the intravenous administration experimental group, all prescriptions were effectively expressed in mice after administration. The expression of luciferase in prescriptions Rp.02, Rp.03, Rp.04, Rp.06, Rp.07, Rp.09, Rp.22, Rp.23, Rp.24, Rp.25, Rp.26, Rp.40, Rp.48, Rp.49, Rp.59, Rp.62, Rp.63, Rp.64, and Rp.65 was better than that in other prescriptions, and was significantly higher than that in prescription Rp.58 prepared by commercially available cationic lipid SM-102.

[0286] like Fig.29 As shown, in the experimental group of intraperitoneal injection, the formula was effectively expressed in the mice after the formula was administered, indicating that the formula of the nucleic acid nanoparticle complex in the experimental group had a significant effect.

[0287] like Fig.30As shown, among the prescriptions in the intramuscular injection experimental group, all prescriptions were effectively expressed in mice after administration. The expression of luciferase in prescriptions Rp.01, Rp.12, Rp.13, Rp.14, Rp.15, Rp.17, Rp.18, Rp.19, Rp.30, Rp.48, and Rp.49 was better than that in other prescriptions, and was significantly higher than that in prescription Rp.58 prepared by commercially available cationic lipid SM-102.

[0288] like Fig.31 As shown, among the prescriptions in the subcutaneous injection experimental group, all prescriptions were effectively expressed in mice after administration. The expression of luciferase in prescriptions Rp.14, Rp.15, Rp.19, Rp.28, and Rp.29 was better than that in other prescriptions, and was significantly higher than that in prescription Rp.58 prepared by commercially available cationic lipid SM-102.

[0289] Example 11: Evaluation of humoral immune effects of nucleic acid nanoparticle complexes in mice

[0290] The SARS-CoV-2 S-mRNA was used as a model mRNA. The SARS-CoV-2 S-mRNA was provided by Hongene Biotech Corporation. The nucleotide sequence of the SARS-CoV-2 S-mRNA (cap1 structure, N1-me-pseudo U modified) is shown in the S-mRNA in the sequence list.

[0291] The specific information of S-mRNA stock solution is as follows:

[0292] Product Name: COVID-19 Spike Protein, Full Length-mRNA;

[0293] Product description: 4088 nucleotides in length;

[0294] Modifications: Fully substituted with N1-Me-pseudo UTP;

[0295] Concentration: 1.0 mg / mL;

[0296] Storage environment: 1mM sodium citrate, pH 6.4;

[0297] Storage requirements: -40℃ or below.

[0298] Experimental process:

[0299] Step 1: The first immunization of mice: On day 0, 5-6 week old female BALB / c mice were given 5 mice in each group and injected intramuscularly with 125 μL PBS (blank control), 5 μg naked S-mRNA (negative control), and 125 μL of nucleic acid nanoparticle complex formula Rp.12, Rp.14, Rp.15, Rp.17, Rp.19, Rp.30, Rp.38, and Rp.58 loaded with 5 μg S-mRNA. They were also injected intravenously with 125 μL of nucleic acid nanoparticle complex formula Rp.03, Rp.09, and Rp.23 loaded with 5 μg S-mRNA, and subcutaneously with 125 μL of nucleic acid nanoparticle complex formula Rp.19 loaded with 5 μg S-mRNA.

[0300] Step 2: First serum collection: On day 14, blood was collected from the outer canthus of the mice. After the blood coagulated at 4°C for 1 hour, it was centrifuged at 5000×g for 5 minutes at 4°C, and the supernatant was collected. It was then centrifuged at 10000×g for 5 minutes at 4°C, and the supernatant was added to eight consecutive PCR tubes and aliquoted and stored at -20°C for later use.

[0301] Step 3: Second immunization of mice: On the 14th day, blood was collected from the lateral canthus of the mice and the first immunization process was repeated.

[0302] Step 4: Second serum collection: On day 28, i.e. 14 days after the second immunization, blood was collected from the outer canthus of the mice. Repeat step 2 to store mouse serum.

[0303] Step 5: ELISA to detect serum IgG content: Dilute the S protein in PBS, and coat the ELISA plate with 100 μL of diluent (containing 0.5 or 1 μg S protein) per well, and coat overnight at 4°C. Discard the liquid in the plate, add 200 μL PBST to each well to wash the plate 3 times, and then add 200 μL of PBS blocking solution containing 5% to 10% BSA to each well and block it on a shaker at room temperature for 2 hours. Discard the blocking solution, add 200 μL PBST to each well to wash the plate once, and then add 100 μL of serum diluted 200 times with PBS, and incubate it on a shaker at room temperature for 2 hours. Discard the serum, add 200 μL PBST to each well to wash the plate 3 times, and then add 100 μL of antibody diluent (antibody and PBS diluted at a ratio of 1:1000) to each well, and incubate it on a shaker at room temperature for 1 hour. The antibody was discarded, 200 μL PBST was added to each well and the plate was washed three times. Then 50 μL TMB colorimetric solution was added to each well for reaction in the dark. After the positive control well turned dark blue or reacted for 10 minutes, 50 μL 2M sulfuric acid or 50 μL 1M phosphoric acid was added to each well to terminate the reaction. The optical density at 450 nm and 630 nm was detected by a microplate reader, and the OD value difference was calculated to reflect the level of anti-S protein IgG in the serum.

[0304] The results are as follows Fig.32Conclusion: The results showed that the OD values ​​of prescriptions Rp.03, Rp.09, Rp.12, Rp.14, Rp.15, Rp.17, Rp.19, Rp.23, Rp.30, Rp.38, and Rp.58 after the second immunization were significantly higher than those of the blank control group and the naked mRNA negative control group, causing significant immune responses. Prescriptions Rp.03, Rp.09, Rp.12, Rp.14, Rp.15, Rp.17, Rp.19, Rp.30, and Rp.38 were comparable to the commercial ionizable cationic lipid molecule SM-102 prescription Rp.58, suggesting that these prescriptions of nucleic acid nanoparticle complexes have strong serum conversion efficiency and humoral immune activation function.

[0305] Example 12: Toxicity evaluation of nucleic acid nanoparticle complexes in mice

[0306] Sample: The nucleic acid nanoparticle complex containing FLuc-mRNA was prepared according to the preparation method of the prescription described in Example 7. An equal volume of PBS buffer was used as a control group.

[0307] Test animals: clean grade Balb / c mice, female, 6-8 weeks old, weighing 22-25 g.

[0308] Acute toxicity experiment in mice: 3 female mice were used to prepare mRNA formulations Rp.65, Rp.66, Rp.67, Rp.68, and Rp.69 containing different ionizable lipids according to the method of Example 7. The dosage was 50 μg / mouse (based on mRNA content), and an equal volume of PBS buffer was used as the control group. The administration method was tail vein injection. After administration, the mice were observed continuously for 1 week, during which the physiological conditions of the mice, the time of onset of poisoning symptoms, the degree of symptom development, the development process, the time of death, the characteristics before death, and the number of dead mice and other related information were recorded.

[0309] After 24 hours of administration, mice in the ALC-0315 prescription group Rp.69 died one after another. The Rp.65, Rp.66, Rp.67, Rp.68 groups containing the compound provided by the present invention and the PBS administration group had no obvious toxic reactions in one week, which reflects the safety of the compound provided by the present invention.

[0310] The method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein within the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.

Claims

1. A compound, the structure of which is shown in formula A, ， in, a is an integer from 1 to 10; b is an integer from 1 to 9; c is an integer from 1 to 9; R1 is C 11 ~C 20 alkyl; R2 is -C(=O)Ra; Ra is C5~C 20 alkyl; R3 is a hydroxyl group, or ; R4 is or ; Rb is C 11 -C 20 alkyl; w is an integer from 1 to 9; z is an integer from 1 to 9; X1 is 0; Each X2 is independently selected from O; Y1 is selected from O; Y2 is selected from O.

2. The compound according to claim 1, wherein the compound represented by formula A is selected from the compound represented by formula I, the compound represented by formula II or the compound represented by formula IV, 、 or ； in, In the compound represented by formula II, w is an integer of 1-9; z is an integer of 1-9; In the compound represented by formula I, R3 is a hydroxyl group.

3. The compound according to claim 2, wherein the compound represented by formula A is selected from the compound represented by formula I, wherein: R1 is unsubstituted C 11 ~C 15 A straight-chain alkyl group, R2 is -C(=O)Ra, Ra is an unsubstituted C5~C 15 a is an integer of 1 to 6, b is an integer of 3 to 5, and c is an integer of 3 to 5.

4. The compound according to claim 2, wherein the compound represented by formula A is selected from the compound represented by formula I, wherein: R1 is unsubstituted C 11 A straight-chain alkyl group, R2 is -C(=O)Ra, Ra is an unsubstituted C5~C 13 a is an integer of 1 to 6, b is an integer of 3 to 5, and c is an integer of 3 to 5.

5. The compound according to any one of claims 2 to 4, wherein the compound represented by formula A is selected from the compound represented by formula I, and the compound represented by formula I is selected from compound L0111, compound L0112, compound L0113, compound L0115, compound L0117, compound L0121, compound L0123, compound L0125, compound L0134, compound L0135 or compound L0136, ， ， ， ， ， ， ， ， ， ， 。 6. The compound according to claim 2, wherein the compound represented by formula A is selected from the compound represented by formula II, wherein: a is an integer of 1 to 5, b is an integer of 3 to 9, c is an integer of 1 to 5, w is an integer of 1 to 5, z is an integer of 5 to 9, and Ra is an unsubstituted C5 to C 15 Straight chain alkyl, Rb is unsubstituted C 11 ~C 15 Straight chain alkyl.

7. The compound according to claim 2, wherein the compound represented by formula A is selected from the compound represented by formula II, wherein: a is an integer of 1 to 3, b is an integer of 4 to 6, c is an integer of 1 to 3, w is an integer of 1 to 3, z is an integer of 5 to 8, and Ra is an unsubstituted C5 to C 13 Straight chain alkyl, Rb is unsubstituted C 11 ~C 13 Straight chain alkyl.

8. The compound according to claim 2, wherein the compound represented by formula A is selected from the compound represented by formula II, wherein: a is 1, b is 5, c is 1, w is 1, z is 6, Ra is unsubstituted C5~C 13 Straight chain alkyl, Rb is unsubstituted C 11 Straight chain alkyl or unsubstituted C 12 Straight chain alkyl.

9. The compound according to claim 2 or 6, wherein the compound represented by formula A is selected from the compound represented by formula II, and the compound represented by formula II is selected from compound L0126, compound L0127 or compound L0128, ， ， 。 10. The compound according to claim 2, wherein the compound represented by formula A is selected from the compound represented by formula IV, wherein: a is an integer of 1 to 5, b is an integer of 3 to 9, c is an integer of 3 to 9, z is an integer of 5 to 9, and Ra is an unsubstituted C5 to C 15 Straight chain alkyl, Rb is unsubstituted C 11 ~C 15 Straight chain alkyl.

11. The compound according to claim 2, wherein the compound represented by formula A is selected from the compound represented by formula IV, wherein: a is an integer of 1 to 3, b is an integer of 4 to 6, c is an integer of 4 to 6, z is an integer of 5 to 8, and Ra is an unsubstituted C5 to C 13 Straight chain alkyl, Rb is unsubstituted C 11 ~C 13 Straight chain alkyl.

12. The compound according to claim 2, wherein the compound represented by formula A is selected from the compound represented by formula IV, wherein: a is 1, b is 5, c is 5, z is 6, and Ra is an unsubstituted C5~C 13 Straight chain alkyl, Rb is unsubstituted C 11 ~C 15 Straight chain alkyl.

13. The compound according to claim 2 or 10, wherein the compound represented by formula A is selected from the compound represented by formula IV, wherein: The compound represented by formula IV is selected from compound L0131, 。 14. A compound selected from: ， 。 15. A lipid compound nanoparticle comprising the following components: the compound according to any one of claims 1 to 14 and auxiliary materials.

16. The lipid compound nanoparticles according to claim 15, wherein the auxiliary material is selected from at least one of PEG derivatives, lipids, lipidoids, alcohols, sugars or inorganic salts.

17. The lipid compound nanoparticle according to claim 16, wherein the PEG derivative is selected from at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, PEG-modified stearic acid, and PEG-modified phosphatidylserine.

18. The lipid compound nanoparticle of claim 16, wherein the PEG derivative comprises 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)], dilauroylphosphatidylethanolamine-polyethylene glycol, dimyristoylphosphatidylethanolamine-polyethylene glycol, dipalmitoylphosphatidylcholine polyethylene glycol, dipalmitoylphosphatidylethanolamine-polyethylene glycol, PEG-distearylglycerol, PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide, PEG-dipalmitoylphosphatidylethanolamine or PEG-1,2-dimyristyloxypropyl-3-amine.

19. The lipid compound nanoparticle according to claim 16, wherein the PEG derivative comprises at least one of DMG-PEG2000, mPEG-DSPE, mPEG-STA, mPEG-PS, mPEG-DMPE, and mPEG-DPPE.

20. The lipid compound nanoparticle according to claim 16, wherein the lipid comprises a phospholipid or a sterol.

21. The lipid compound nanoparticle according to claim 16, wherein the phospholipid comprises at least one of phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-diondecanoyl-sn-glycero-phosphocholine, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; The sterol includes at least one of cholesterol, lanosterol, 5α-cholestane-3β-ol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid or α-tocopherol.

22. The lipid compound nanoparticle according to any one of claims 15-21, comprising the compound according to any one of claims 1-14, a PEG derivative and a lipid, wherein the lipid is selected from at least one of a phospholipid and a sterol.

23. The lipid compound nanoparticles according to any one of claims 15 to 21, wherein the content of the compound according to any one of claims 1 to 14 is 14.8 mol% to 70.0 mol%, calculated based on the total molar amount of each component in the lipid compound nanoparticles.

24. The lipid compound nanoparticle according to claim 22, wherein the content of the PEG derivative is 0.4 mol%-10 mol%, calculated based on the total molar amount of each component in the lipid compound nanoparticle.

25. The lipid compound nanoparticles according to claim 22, wherein the content of the phospholipid is 5.0 mol%-30.0 mol%, calculated based on the total molar amount of each component in the lipid compound nanoparticles.

26. The lipid compound nanoparticles according to claim 22, wherein the content of the sterol is 10.0 mol%-75.0 mol% calculated based on the total molar amount of each component in the lipid compound nanoparticles.

27. The lipid compound nanoparticles according to claim 22, wherein the content of the sterol is 15.0 mol%-75.0 mol%, calculated based on the total molar amount of each component in the lipid compound nanoparticles.

28. The lipid compound nanoparticles according to claim 22, wherein the molar ratio of PEG derivative: phospholipid: sterol: the compound according to any one of claims 1 to 14 is (0.4-10.0): (5.0-30.0): (10.0-75.0): (14.8-70.0).

29. The lipid compound nanoparticles according to claim 22, wherein the molar ratio of PEG derivative: phospholipid: sterol: the compound according to any one of claims 1 to 14 is (0.4-10.0): (5.0-30.0): (15.0-75.0): (14.8-70.0).

30. The lipid compound nanoparticles according to claim 22, wherein the molar ratio of PEG derivative: phospholipid: sterol: compound according to any one of claims 1 to 14 in the lipid compound nanoparticles is 2.50: 16.00: 16.50: 65.00, 1.00: 5.00: 64.00: 30.00, 0.40: 8.00: 56.60: 35.00, 1.00: 8.00: 61.00: 30.00, 1.20: 12.00: 38.30: 48.50, 1.70: 9.00: 40.80: 48.50, 2.50: 16.00: 21.50: 60.00, 1.20: 9.00 : 47.80 : 42.00,1.00 : 16.00 : 34.50 :48.50,2.50 : 8.00 : 41.00 : 48.50,1.50 : 8.00 : 25.50 : 65.00,2.50 : 11.50 : 51.00 : 35.00,1.50 : 8.00 : 30.50 : 60.00,1.00 : 5.00 : 62.00 : 32.00,0.60 :5.00 : 54.40 : 40.00,1.50 : 16.00 : 47.50 : 35.00,1.63 : 12.99 : 45.38 : 40.00,1.20 : 8.00 : 55.8 : 35.00,1.30 : 8.00 : 55.70 : 35.00,0.40 : 5.00 : 59.60 : 35.00,1.00 : 20.00 : 37.00 : 42.00,1.00 : 25.00 : 34.00 : 40.00,3.20 : 12.00 : 29.80 : 55.00,1.00 : 8.00 : 53.00 : 38.00,2.50 : 9.50 : 33.00 : 55.00,10.00 : 16.00 : 54.00 : 20.00,3.00 : 30.00 : 42.00 : 25.00,3.20 : 16.80 : 10.00 : 70.00,3.00 : 17.00 : 25.00 : 55.00,3.20 : 16.80 : 15.00 : 65.00,4.20 : 11.00 : 70.00 : 14.80,6.20 : 6.80 : 75.00 : 15.00,1.50 : 11.50 : 38.50: 48.50,7.50 : 9.61 : 35.56 : 47.33, 3.00 : 9.50 : 32.50 : 55.00, 0.95 : 7.58 : 26.47 : 65.00 or 1.40 : 11.15 : 38.95 : 48.

50. .

31. The lipid compound nanoparticles according to claim 22, wherein the molar ratio of PEG derivative: phospholipid: sterol: the compound according to any one of claims 1 to 14 in the lipid compound nanoparticles is (0.4-1.5): (8.0-11.5): (38.5-56.6): (35.0-48.5).

32. The lipid compound nanoparticles according to claim 22, wherein the molar ratio of PEG derivative: phospholipid: sterol: compound according to any one of claims 1 to 14 in the lipid compound nanoparticles is 0.40: 8.00: 56.60: 35.00, 1.50: 11.50: 38.50: 48.50 or 1.40: 11.15: 38.95: 48.

50.

33. A nucleic acid nanoparticle complex comprising: Nucleic acid and at least one of the lipid compound nanoparticles selected from any one of claims 15-32.

34. The nucleic acid-nanoparticle complex according to claim 33, wherein the ratio of the molar amount of ionizable nitrogen atoms in the compound according to any one of claims 1 to 14 to the molar amount of phosphorus atoms in the nucleic acid in the nucleic acid-nanoparticle complex is 5 to 50.

35. A pharmaceutical composition comprising the lipid compound nanoparticles according to any one of claims 15 to 32 or the nucleic acid nanoparticle complexes according to any one of claims 33 to 34, and pharmaceutically acceptable excipients.

36. Use of the compound of any one of claims 1 to 14, the lipid compound nanoparticles of claims 15 to 32, the nucleic acid nanoparticle complex of any one of claims 33 to 34, or the pharmaceutical composition of claim 35 in the preparation of a product for in vivo delivery of nucleic acids.

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