Transfection reagent based on blank lipid nanoparticles as well as preparation method and application of transfection reagent

Abstract

The invention provides a transfection reagent based on blank lipid nanoparticles as well as a preparation method and application of the transfection reagent, and belongs to the technical field of biological medicines. The transfection reagent based on the blank lipid nanoparticles comprises the following components: (1) the blank lipid nanoparticles; (2) a biologically active ingredient; the blank lipid nanoparticles comprise ionizable lipid, phospholipid, cholesterol and polyethylene glycol conjugated lipid. The preparation process of the blank lipid nanoparticles and the transfection reagent is simple, and the preparation can be completed without the help of equipment. The transfection reagent based on the blank lipid nanoparticles is prepared from the blank lipid nanoparticles and nucleic acid, the dosage can be flexibly adjusted according to the requirements of a user, the transfection effect is stable, various cells (such as 293T, Hela, HepG2, TWO3 and MCF7) can be transfected, the cytotoxicity is low, and the transfection effect is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a transfection reagent based on blank lipid nanoparticles, a preparation method thereof, and an application thereof. Background Art

[0002] Inserting foreign DNA into bacteria to express the desired protein has become a conventional technique, such as synthesizing human insulin in Escherichia coli. However, since bacteria are different from mammalian (eukaryotic) cells and lack the enzymes and organelles responsible for protein processing and modification (e.g., glycosylation, disulfide bond formation, etc.), synthesizing recombinant proteins in bacteria is subject to multiple limitations. Bacteria cannot fold larger proteins into the correct 3D structure to ensure the biological activity of the protein. Delivering effective genes into eukaryotic cells for expression can well solve this dilemma.

[0003] Transfection is a process of delivering foreign nucleic acids into eukaryotic cells to change the genetic composition of host cells. The above-mentioned nucleic acids include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and small non-coding RNAs, such as siRNA, shRNA, and miRNA. Currently, there is a huge and growing demand for efficient gene delivery agents.

[0004] Common commercially available transfection reagents include the Lipofectamine series from ThermoFisher, such as 2000, 3000, Lipofectamine LTX, Plus, RNAiMAX (CN106318971A, CN107057061A); Lipofection from Invitrogen; the DharmaFECT series from Doharmacon; DOTAP from Roche; HiPerfect from Qiagen; Nanofectamine from GE Healthcare; and Escort IV liposomes from SigmaAldrich.

[0005] The working principle of these commercially available transfection reagents is generally to use cationic polymers or cationic lipids to bind to anionic substances such as nucleic acids, and transport the nucleic acid substances into cells. Sometimes, other reagents (such as peptides) must be added as adjuvants to perform effective transfection. They not only have a complex composition and relatively high toxicity of cationic materials to cells, but also these reagents do not have good transfection effects on all cell types, and the selectivity of the reagents for cell types is relatively high. Therefore, there is still an urgent need for transfection reagents with lower toxicity than existing transfection reagents and generally having transfection effects on various types of cells.

[0006] Patent CN105143456A discloses lipid nanoparticles for transfection, comprising a transfection reagent composition and an anionic macromolecule. The transfection reagent composition comprises: (a) one or more cationic lipids or their pharmaceutically acceptable salts; (b) one or more neutral lipids; (c) one or more sterols; and (d) one or more surfactants. Although this patent can effectively deliver siRNA OGN and other anionic macromolecules to primary cells that are difficult to transfect in vitro and to target cells in vivo, the lipid nanoparticles for transfection need to adopt a special and complex mixing process to obtain a suitable product.

[0007] The present invention provides a transfection reagent, which is prepared by mixing lipid nanoparticles and nucleic acids in a solution state, with a simple composition, a concise process and high reproducibility. The transfection reagent provided by this patent can efficiently transfect a variety of cells and has low toxicity to cells, and has a better transfection effect compared with commercially available transfection reagents. Summary of the Invention

[0008] Aiming at the problems existing in the prior art, the present invention provides a transfection reagent based on blank lipid nanoparticles, its preparation method and application. The transfection reagent based on blank lipid nanoparticles of the present invention comprises: (1) blank lipid nanoparticles; (2) biologically active components; the composition of the blank lipid nanoparticles comprises: ionizable lipids, phospholipids, cholesterol and polyethylene glycol-conjugated lipids. The preparation processes of the blank lipid nanoparticles and the transfection reagent are simple and can be completed without the aid of equipment. The transfection reagent is prepared from blank lipid nanoparticles and nucleic acids, and the dosage can be flexibly adjusted according to the needs of users, and the transfection effect is stable. It can transfect a variety of cells (such as 293T, Hela, HepG2, TWO3, MCF7), has low cytotoxicity and good transfection effect; in cell transfection, it has a better transfection effect compared with commercially available transfection reagents.

[0009] To achieve the above object, in the first aspect, the present invention provides a transfection reagent based on blank lipid nanoparticles, comprising: (1) blank lipid nanoparticles; (2) biologically active components; the composition of the blank lipid nanoparticles comprises: 5-70 mol% of a first ionizable lipid, 0-30 mol% of a second ionizable lipid, 5-50 mol% of phospholipids, 10-70 mol% of cholesterol and 0-15 mol% of polyethylene glycol-conjugated lipids; in the transfection reagent, the dosage of the biologically active component is 0.1%-50% (w / w) of the total amount of the blank lipid nanoparticles and the biologically active component.

[0010] In a preferred embodiment, the composition of the blank lipid nanoparticles comprises: 10 - 60 mol% of a first ionizable lipid, 0 - 30 mol% of a second ionizable lipid, 5 - 30 mol% of a phospholipid, 15 - 70 mol% of cholesterol, and 0 - 10 mol% of a polyethylene glycol-conjugated lipid;

[0011] In a preferred embodiment, the biologically active ingredient comprises at least one of nucleic acid and polypeptide.

[0012] In a preferred embodiment, the nucleic acid is DNA and / or RNA, including at least one of siRNA, miRNA, saRNA, sgRNA, dsRNA, shRNA, smRNA, ssRNA, mRNA, circRNA, snRNA, crRNA, IncRNA, snoRNA, piRNA, pDNA, ssDNA, circular or linear DNA, DNA microloop, msDNA.

[0013] In a preferred embodiment, the polypeptide is selected from polypeptides comprising 2 - 50 amino acids.

[0014] In a preferred embodiment, the first ionizable lipid is selected from compounds having the general formula (1) or pharmaceutically acceptable salts, stereoisomers, and tautomers thereof;

[0015]

[0016] wherein, R1, R2, and R3 are independently of each other H, C 5-40 a straight-chain or branched-chain alkyl group, C 5-40 a straight-chain or branched-chain alkenyl group, C 5-40 a straight-chain or branched-chain alkynyl group, a 3 - 6-membered saturated or partially unsaturated cycloalkyl group having 1 - 3 side chains, or a 6 - 10-membered aromatic group having 1 - 3 side chains; the side chains are independently selected from C 10-30 a straight-chain or branched-chain alkyl group, C 10-30 a straight-chain or branched-chain alkenyl group, C 10-30 a straight-chain or branched-chain alkynyl group; provided that at most one of R1, R2, and R3 is H;

[0017] M is selected from -NR4R5, a saturated or partially unsaturated 3 - 6-membered heterocyclic group containing at least one nitrogen atom, a 6 - 10-membered heteroaryl group containing at least one nitrogen atom, and the heterocyclic group and heteroaryl group are unsubstituted or substituted by one or more -OH, carboxyl group, amino group, oxo group, or halogen;

[0018] R4 and R5 are independently of each other H, C 1-6 a straight-chain or branched-chain alkyl group, C 2-6 a straight-chain or branched-chain alkenyl group, or C 2-6A linear or branched alkynyl group, wherein said C 1-6 A linear or branched alkyl group, C 2-6 A linear or branched alkenyl group or C 2-6 The linear or branched alkynyl group is unsubstituted or substituted with one or more -OH, carboxyl, amino, amide, amidine, guanidine or halogen groups;

[0019] G1, G2, G3 are each independently -O-, -S-, -NR6-, -S-S-, -C(=O)-, -C(=S)-, -C(=O)O-, -CH(OH)-, -

[0020] OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -

[0021] -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR6-, -NR6-S(=O)2-, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-; wherein each R6, R 13 are each independently selected from H, hydroxy, C 1-30 A linear or branched alkyl or cycloalkyl group, C 2-30 A linear or branched alkenyl group;

[0022] L1 is selected from -X1- or -(CR7R8) m -X1-, wherein each X1 is independently selected from -O-, -S-, -NR 14 -, -S-S-, -C(=O)-, -C(=S)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -, -NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-, -OC(=O)NR 14 -, -NR 14 C(=O)NR 15 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR14 -,-NR 14 -S(=O)2-,-P(=O)(OR 14 )O-,-OP(=O)(OR 14 )-or-OP(=O)(OR 14 )O-;wherein,m is an integer from2to6,R 7、 R8are each independently H,hydroxy,halogen,C 1-6 linear or branched alkyl or cycloalkyl,C 2-6 linear or branched alkenyl,each R 14 ,R 15 are each independently selected from H,C 1-30 linear or branched alkyl or cycloalkyl,C 2-30 linear or branched alkenyl;

[0023] L2is-(CR9R 10 ) n -or-(CR9R 10 ) n -X2-(CR 11 R 12 ) k -,wherein X2is selected from-O-,-S-,-NR 16 -,-S-S-,-C(=O)-,-C(=S)-,-C(=O)O-,-OC(=O)-,-C(=O)NR 16 -,-NR 16 C(=O)-,-OC(=O)O-,-NR 16 C(=O)O-,-OC(=O)NR 16 -,-NR 16 C(=O)NR 17 -,-C(=O)S-,-C(=S)S-,-SC(=S)-,-SC(=O)-,-OC(=O)S-,-SC(=O)O-,-SC(=O)S-,-OS(=O)2O-,-S(=O)2O-,-OS(=O)2-,-S(=O)2-,-S(=O)2-NR 16 -NR 16 -S(=O)2-,-P(=O)(OR 16 )O-,-OP(=O)(OR 16 )-or-OP(=O)(OR 16 )O-;n is an integer from1to6;k is an integer from1to6;R 9、 R 10、 R 11、 R 12 are each independently H,hydroxy,halogen,C 1-6a straight-chain or branched alkyl or cycloalkyl group, C 2-6 a straight-chain or branched alkenyl group, each R 16 and R 17 are independently selected from H, C 1-30 a straight-chain or branched alkyl or cycloalkyl group, C 2-30 a straight-chain or branched alkenyl group;

[0024] wherein the alkyl, cycloalkyl, and alkenyl groups described for R4 to R 17 are unsubstituted or substituted with one or more groups selected from hydroxy, mercapto, amino, substituted amino, and halogen;

[0025] The salt does not include quaternary ammonium salts.

[0026] In a preferred embodiment, R1, R2, and R3 are independently one of the following groups:

[0027]

[0028] wherein Y is absent or is C 1-30 a straight-chain or branched alkyl or cycloalkyl group, C 2-20 a straight-chain or branched alkenyl group, C 2-20 a straight-chain or branched alkynyl group; R1' and R2' are independently H, C 1-30 a straight-chain or branched alkyl group, C 2-30 a straight-chain or branched alkenyl group, C 2-30 a straight-chain or branched alkynyl group, and the total carbon chain length of Y, R1', and R2' is 8 - 40.

[0029] In a preferred embodiment, R1, R2, and R3 are independently selected from the following groups:

[0030]

[0031] wherein R1' and R2' are independently H, C 1-30 a straight-chain or branched alkyl group, C 2-30 a straight-chain or branched alkenyl group, C 2-30 a straight-chain or branched alkynyl group, and the total carbon chain length of R1' and R2' is 8 - 30.

[0032] In a preferred embodiment, R1, R2, and R3 are independently selected from any one of the following groups:

[0033]

[0034] In a preferred embodiment, G1, G2, and G3 are, independently of one another, -O-, -S-, -NR6-, -S-S-, -C(=O)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -P(=O)(OR6)O-, -OP(=O)(OR6)-, or -OP(=O)(OR6)O-.

[0035] In a preferred embodiment, L1 is selected from -(CR7R8) m -X1-, where X1 is selected from -O-, -S-, -NR 14 -, -S-S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 14 -, -NR 14 C(=O)-, -OC(=O)O-, -NR 14 C(=O)O-, -OC(=O)NR 14 -, -NR 14 C(=O)NR 15 -, -P(=O)(OR 14 )O-, -OP(=O)(OR 14 )-, or -OP(=O)(OR 14 )O-.

[0036] In a preferred embodiment, L2 is -(CR9R 10 ) n -X2-(CR 11 R 12 ) k -, where X2 is selected from -O-, -S-, -NR 16 -, -S-S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -, -NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 -, -NR 16 C(=O)NR 17 -, -P(=O)(OR 16 )O-, -OP(=O)(OR 16 )-, or -OP(=O)(OR 16 )O-.

[0037] In a preferred embodiment, M is selected from the following structures:

[0038]

[0039] wherein m' and n' are independently integers from 0 to 6, and R1" and R2" are independently H, C 1-6 alkyl, C 2-6 alkenyl, guanidyl, amidino, amido, aliphatic amine, 3- to 10-membered nitrogen-containing heterocycle; the nitrogen-containing heterocycle is selected from pyrrole, imidazole, pyridine, pyrazole, triazole, oxazole, isoxazole, thiophene, isothiazole, pyridazine, pyrazine, piperazine, indole, benzimidazole, carbazole, quinoline, isoquinoline, purine and pyrimidine and their tautomeric forms, which are unsubstituted or optionally substituted by one or more organic groups selected from hydroxy, mercapto, amino, substituted amino, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 6-14 aryl.

[0040] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1A):

[0041]

[0042] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1B):

[0043]

[0044] In a preferred embodiment, the compound of formula (1) is selected from the compounds of formula (1C):

[0045]

[0046] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1D):

[0047]

[0048] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1E)

[0049]

[0050] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1F):

[0051]

[0052] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1G):

[0053]

[0054] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1H):

[0055]

[0056] In a preferred embodiment, M is selected from any one of the following groups:

[0057]

[0058] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1I):

[0059]

[0060] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1J):

[0061]

[0062] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1K):

[0063]

[0064] In a preferred embodiment, Y is absent, and the compound of formula (1) is selected from the compounds represented by formula (1L):

[0065]

[0066] wherein R1' and R2' are independently selected from H, C1-30 straight-chain or branched-chain alkyl, C2-30 straight-chain or branched-chain alkenyl, C2-30 straight-chain or branched-chain alkynyl, and the total carbon chain length of R1' and R2' is 8-40.

[0067] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1M):

[0068]

[0069] In a preferred embodiment, the compound of formula (1) is selected from:

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (II) with a compound of formula (III):

[0099]

[0100] wherein Xa and Xb are groups containing a leaving group or a nucleophilic group, and Xa and Xb form L1 through a nucleophilic reaction or a condensation reaction.

[0101] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (IV) with a compound of formula (V):

[0102]

[0103] wherein Xc and Xd are groups containing a leaving group or a nucleophilic group, and Xc and Xd form L2 through a nucleophilic reaction or a condensation reaction.

[0104] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (VI):

[0105]

[0106] wherein Xe is a group containing a leaving group or a nucleophilic group, Xf is a compound containing a leaving group or a nucleophilic group, and Xe and Xf form M through a nucleophilic reaction or a condensation reaction.

[0107] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of sequentially reacting a compound of formula (VII) with a compound of formula (VIII), a compound of formula (IX), and a compound of formula (X):

[0108]

[0109] wherein Xg, Xh, Xi, Xj, Xk, Xl are groups containing a leaving group or a nucleophilic group, and Xg and Xj form G1 through a nucleophilic reaction or a condensation reaction, Xh and Xk form G2 through a nucleophilic reaction or a condensation reaction, and Xi and Xl form G3 through a nucleophilic reaction or a condensation reaction.

[0110] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (XI) with a compound of formula (XII):

[0111]

[0112] Step of reacting the compound of formula (XIII) with the compound of formula (XII):

[0113]

[0114] wherein Xm is a group containing a nucleophilic group, and Xm and C═C-L of the compound of formula (XII) 2a form X1-L2 through an addition reaction.

[0115] In a preferred embodiment, R1, R2, and R3 are each independently a group as follows:

[0116]

[0117] wherein the meanings of Y, R1', and R2' are the same as before;

[0118] wherein it further includes the step of forming the tail chains R1, R2, and R3:

[0119]

[0120] wherein X is a leaving group.

[0121] In a preferred embodiment, the second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, or tautomer thereof:

[0122]

[0123] wherein N1 is NH or O;

[0124] R a is selected from C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 cycloalkyl, C6-C 24 alcohol, C6-C 24 short-chain polyoxyethylene; the C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 cycloalkyl, C6-C 24 alcohol, C6-C 24 short-chain polyoxyethylene is in a straight-chain or branched-chain structure; R b and R c are each independently selected from C1-C 12 alkyl, C2-C 12 alkenyl, C1-C 12 alkynyl, C3-C 12 cycloalkanyl, C6-C 12 aryl, C1-C 12 alkyl alcohol, C1-C 12heterocyclic group, alkylamine;

[0125] The alkylamine is wherein, R a ’ is C1-C 12 alkyl, and the R b ’ and R b ” are each independently selected from H, C1-C6 alkylamine, R c ” is selected from C1-C6 alkyl which is unsubstituted or substituted by amino group, and R c ”’ is H, or -R c ’-A1’-R c ”-NH2;

[0126] Provided that when A1’ is -CO-NH-, -NH-CO- or -CO-O-, R c ’ is C1-C6 alkyl; when A1’ is -CO-, R c ’ does not exist.

[0127] In a preferred embodiment, R b and R c are each independently selected from C1-C 12 alkyl alcohol, alkylamine.

[0128] In a preferred embodiment, provided that: when R a is C6-C 24 alkenyl, A1 is NH, and R b and R c are each independently selected from C1-C 12 alkyl alcohol;

[0129] When R a is branched C6-C 24 alkyl, A1 is NH, and R b and R c are each independently selected from C1-C 12 alkyl alcohol, alkylamine;

[0130] R a is straight-chain C6-C 24 alkyl, A1 is NH or O, and R b and R c are each independently selected from alkylamine;

[0131] When R a is straight-chain C6-C 24 alkyl, A1 is O, and R b and R c are each independently selected from C1-C 12 alkyl alcohol;

[0132] When R a is C6-C 24 short-chain polyoxyethylene, A1 is NH or O, and R b and R c are each independently selected from alkylamines;

[0133] Or when R a is C6-C 24 alkyl alcohol, A1 is NH or O, and R b and R c are each independently selected from alkylamines.

[0134] In a preferred embodiment, R a is selected from the following compound structures:

[0135]

[0136] In a preferred embodiment, R b and R c are selected from the following compound structures:

[0137]

[0138] R d is selected from C1-C6 alkanes or cycloalkanes.

[0139] In a preferred embodiment, the compound of formula (2) is selected from at least one of the following compounds:

[0140]

[0141]

[0142] In a preferred embodiment, the method for preparing the second ionizable lipid comprises the following reaction steps: R a ” -NH2 ① reacts with an α,β-unsaturated carbonyl compound ② to form an ionizable lipid compound ③:

[0143]

[0144] wherein, R a ” -NH2 is R a -NH2 or

[0145] R b ’ and R b ” are simultaneously H, or R b ’ and R b ” are simultaneously C1-C6 amines, or Rb ’ and R b ” is simultaneously -R c ’-A1’-R c ”-NH2.

[0146] In a preferred embodiment, the method for preparing the second ionizable lipid comprises:

[0147] 1) R a ” -NH2① reacts with an α,β-unsaturated carbonyl compound ④ to form a compound ⑤;

[0148] 2) The compound ⑤ reacts with a nucleophile ⑥ to form an ionizable lipid compound ③;

[0149]

[0150] wherein the nucleophile ⑥ is R b -NH2 or R b -OH; R a ” -NH2 is selected from R a -NH2 or

[0151] R b ’ and R b ” are simultaneously H, or R b ’ and R b ” are simultaneously C1-C6 amines, or R b ’ and R b ” are simultaneously -R c ’-A1’-R c ”-NH2; Z2 is a leaving group, and Z2 reacts with NH2 to obtain A1.

[0152] In a preferred embodiment, the method for preparing the second ionizable lipid comprises:

[0153] 1) R a ” -NH2① reacts with an α,β-unsaturated carbonyl compound ② to form a compound ⑦;

[0154] 2) The compound ⑦ reacts with an α,β-unsaturated carbonyl compound ⑧ to form a compound ⑨;

[0155] 3) The compound ⑨ reacts with a nucleophile ⑩ to form an ionizable lipid compound

[0156]

[0157] wherein, R a ” -NH2 is Ra -NH2, Z3 is a leaving group, and A3 reacts with Z3 to obtain A1.

[0158] In a preferred embodiment, in Formula 2a - Formula 2c, at least one nucleophile undergoes a Michael addition reaction with the β-carbon atom of at least one α,β-unsaturated carbonyl compound to form the ionizable lipid compound with two branches containing carbon-carbon bonds, carbon-oxygen bonds, carbon-nitrogen bonds, carbon-sulfur bonds or carbon-selenium bonds.

[0159] In a preferred embodiment, Formula 2a - Formula 2b further includes the step of performing an iterative reaction using the compound of Formula (2) with a terminal amino group obtained by the reaction of Formula 2a - Formula 2b as a raw material according to Step 1 in Formula 2a or Steps 1 - 2 in Formula 2b;

[0160] The compound of Formula (2) with a terminal amino group is

[0161] Wherein, R b ’ and R b ” are simultaneously H, or R b ’ and R b ” are simultaneously C1 - C6 amines, or R b ’ and R b ” are simultaneously -R c ’-A1’-R c ”-NH2.

[0162] In a preferred embodiment, the reaction formula of Formula 2a - Formula 2c further includes the step of reacting with the compound of Formula (2) with a terminal amino group obtained by the reaction formula of Formula 2a - Formula 2c as a raw material ;

[0163] The compound of Formula (2) with a terminal amino group is

[0164] Wherein, R b ’ and R b ” are simultaneously H, or R b ’ and R b ” are simultaneously C1 - C6 amines, or R b ’ and R b ” are simultaneously -R c ’-A1’-R c ”-NH2.

[0165] The leaving group described above refers to the leaving part in a nucleophilic reaction or a condensation reaction, including but not limited to: H,

[0166] OH, H2O, halogens (e.g., F, Cl, Br, and I), cyanate anions, inorganic acids (e.g., nitric acid, sulfuric acid, phosphoric acid), carboxylic acids (e.g., acetic acid, trifluoroacetic acid, benzoic acid, etc.), sulfonic acids (e.g., methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, p-nitrobenzenesulfonic acid, etc.), carbon dioxide (CO2), nitrogen (N2), imidazole, alkoxy groups (R-O-), amino groups (-NHR, where R is an alkyl or aryl group from which H has been removed), phenoxy groups, tertiary carbocations (e.g., tert-butyl cation), carbocations stabilized by unsaturated systems or heteroatoms, or various protecting groups described above.

[0167] The nucleophilic groups described above refer to molecules or ions that can provide an electron pair to form a new chemical bond in a chemical reaction. Common nucleophilic groups include: hydroxide (HO - ), ammonia (NH3), hydroxylamine (NH2OH), hydrazine (NH2-NH2), substituted hydrazines, nucleophilic halogens (such as Cl - , Br - or I - ), hydride ion (H - ), azide anion (N3 - ), cyanate anion (CN - ), alcohol or alkoxide anions (e.g., alcohols from which the hydroxyl hydrogen has been removed), amino groups (including primary, secondary, and tertiary amines) or amine anions, carbanions (e.g., carbanions in organometallic reagents such as Grignard reagents, organolithium reagents, Gilman reagents, etc.), thiol or thiolate anions, thioethers, enols or enolate anions, vinyl ethers, enamines, carboxylic acids or carboxylate anions, alkyl or aryl phosphines (e.g., triphenylphosphine), aromatic heterocycles with lone pairs of electrons (e.g., pyridine), etc.

[0168] In a preferred embodiment, the starting materials used in the reaction process further contain protecting groups, and the reaction steps include protection and / or deprotection steps.

[0169] In a preferred embodiment, for the transfection reagent based on blank lipid nanoparticles, the lipid nanoparticles further comprise 0 - 60 mol% of other ionizable lipids, and the other ionizable lipids are selected from at least one of the following compounds:

[0170]

[0171]

[0172]

[0173]

[0174] In a preferred embodiment, the phospholipids include at least one of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diuvacenoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesteryl succinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE).

[0175] In a preferred embodiment, the steroid or its derivative includes at least one of cholesterol, cholesteryl stearate, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, fucosterol, tomatine, ursolic acid, α-tocopherol.

[0176] In a preferred embodiment, the polyethylene glycol-conjugated lipid comprises at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0177] In a preferred embodiment, the polyethylene glycol-conjugated lipid comprises at least one of PEG-distearyloxypropyl (PEG-DSA), PEG-c-DOMG, PEG-DPPC, polyethylene glycol dimethacrylate (PEG-DMA), 1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol (PEG-DMG), dipalmitin-polyethylene glycol (DPG-PEG), 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol (DSG-PEG), 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), dipalmitoylphosphatidylethanolamine-polyethylene glycol (DPPE-PEG), distearoylphosphatidylethanolamine-polyethylene glycol (PEG-DSPE), dilauroylphosphatidylethanolamine-polyethylene glycol (PEG-DLPE), and dimyristoylphosphatidylethanolamine-polyethylene glycol (PEG-DMPE) lipids.

[0178] In a preferred embodiment, in the transfection reagent, the nucleic acid molecule is encapsulated inside the lipid nanoparticle and / or adsorbed on the surface of the lipid nanoparticle to form a complex.

[0179] In a second aspect, the present invention provides a method for preparing the aforementioned transfection reagent based on blank lipid nanoparticles, which is characterized by comprising the step of mixing the blank lipid nanoparticles with nucleic acid in a solvent.

[0180] In a preferred embodiment, the mixing methods include manual mixing, vortex mixing, stirring mixing, and microchannel mixing.

[0181] In a preferred embodiment, the concentration of the nucleic acid after mixing is 5 - 1000 ng / μl.

[0182] In a preferred embodiment, the solvent is at least one of water, an aqueous solution of an organic solvent, and a buffer salt solution.

[0183] In a preferred embodiment, the pH of the buffer salt solution is 1 - 9, the concentration of the buffer salt is 0.1 - 200 mM, and the aqueous solution of the organic solvent is an alcohol solution with a volume concentration < 50%.

[0184] In a preferred embodiment, the alcohol is ethanol.

[0185] In a preferred embodiment, the buffer salt solution is selected from at least one of citrate solution, acetate solution, tartrate solution, phosphate solution, carbonate solution, Tris-HCl solution, and sodium chloride solution.

[0186] In a preferred embodiment, at least one of culture medium, sugar, glycerol, DMSO, salt, antibiotic, and surfactant is further added to the transfection reagent based on blank lipid nanoparticles.

[0187] In a third aspect, the present invention provides the application of the aforementioned transfection reagent based on blank lipid nanoparticles in in vitro transfection.

[0188] In a preferred embodiment, the application in in vitro transfection is the application in the preparation of products for in vitro transfection.

[0189] In a fourth aspect, the present invention provides an in vitro transfection method, which mixes the aforementioned transfection reagent based on blank lipid nanoparticles with cells for transfection.

[0190] In a fifth aspect, the present invention provides the application of the aforementioned transfection reagent based on blank lipid nanoparticles in the preparation of drugs for in vitro transfection, which mixes the aforementioned transfection reagent based on blank lipid nanoparticles with cells for transfection.

[0191] In a preferred embodiment, the cells include eukaryotic cells and / or prokaryotic cells; the prokaryotic cells are bacterial cells; the eukaryotic cells are at least one of animal cells, plant cells, algal cells, and fungal cells.

[0192] In a preferred embodiment, the survival rate of the cells is ≥80%.

[0193] Compared with the prior art, the present invention has the following beneficial effects:

[0194] 1. The transfection reagent based on blank lipid nanoparticles of the present invention includes: (1) blank lipid nanoparticles; (2) biologically active components; the composition of the blank lipid nanoparticles includes: ionizable lipid, phospholipid, cholesterol, and polyethylene glycol-conjugated lipid. The preparation processes of the blank lipid nanoparticles and the transfection reagent are simple and can be completed without the aid of equipment.

[0195] 2. The transfection reagent based on blank lipid nanoparticles proposed by the present invention is prepared from blank lipid nanoparticles and nucleic acids, can be flexibly adjusted in dosage according to the needs of users, and has stable transfection effects.

[0196] 3. The transfection reagent based on blank lipid nanoparticles proposed by the present invention can transfect a variety of cells (such as 293T, Hela, HepG2, TWO3, MCF7, etc.), with low cytotoxicity and good transfection effect; in cell transfection, compared with commercially available transfection reagents, the transfection effect is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0197] Figure 1 shows the luciferase activity of blank lipid nanoparticle - Luciferase pDNA complex in different cells;

[0198] Figure 2 shows the luciferase activity of blank lipid nanoparticle - Luciferase mRNA complex in different cells;

[0199] Figure 3 shows the transfection of blank lipid nanoparticle - eGFP mRNA complex in 293T cells;

[0200] Figure 4 shows the transfection of blank lipid nanoparticle - eGFP mRNA complex in Hela cells;

[0201] Figure 5 shows the transfection of blank lipid nanoparticle - eGFP mRNA complex in HepG2 cells;

[0202] Figure 6 shows the transfection of blank lipid nanoparticle - siRNA - cy3 complex in different cells DETAILED DESCRIPTION OF THE INVENTION

[0203] The synthesis process of the present invention can accommodate multiple functional groups, so various substituted starting materials can be used. These processes generally provide the desired final compound at the end or near the end of the entire process, although in some cases it may be necessary to further convert the compound into its pharmaceutically acceptable salt. The compounds of the present invention can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or intermediates that are easily prepared, by adopting standard synthetic methods and procedures known to those skilled in the art or that are obvious to those skilled in the art based on the teachings herein. Standard synthetic methods and procedures for preparing organic molecules, as well as functional group transformations and operations, can be obtained from relevant scientific literature or from standard textbooks in the art. The following description of the synthetic methods is designed to illustrate rather than limit the general procedures for preparing the compounds of the present invention.

[0204] The compounds of the present invention having the various formulas described herein can be prepared from commercially available starting materials or starting materials that can be prepared using literature processes according to the processes described in the corresponding general synthetic routes. The variables (such as R1, R2, and R3, etc.) in each general synthetic route are defined as herein. Those of ordinary skill in the art should note that in the reaction procedures and synthetic schemes described herein, the order of certain steps can be varied, such as the introduction and removal of protecting groups.

[0205] In the reaction schemes described herein, multiple stereoisomers can be produced. When no specific stereoisomer is indicated, this should be understood to include all possible stereoisomers produced by the reaction. Those of ordinary skill in the art should recognize that the reaction can be optimized to preferentially obtain one isomer, or new schemes can be designed to produce a single isomer. If a mixture is produced, techniques such as preparative thin-layer chromatography, preparative HPLC, preparative chiral HPLC, or preparative SFC can be used to separate the isomers.

[0206] (1) Synthesis of the first ionizable lipid of Preparation Example 1

[0207] The general synthetic route of the first ionizable lipid is as in General Synthetic Routes 1-5.

[0208] General Synthetic Route 1

[0209]

[0210] Wherein, M' is M or M with a protecting group.

[0211] As described in General Synthetic Route 1 above, Boc-amino tris(hydroxymethyl)methane reacts with an acid (Compound 2) in a condensation reaction to form Compound 3. Step 1 can be carried out in an organic solvent (such as dichloromethane (DCM)) in the presence of, for example, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) and 4-dimethylaminopyridine (DMAP). Step 1 can be carried out at room temperature for 24 hours.

[0212] Then, the Boc protecting group of Compound 3 is removed to form Compound 4. Step 2 can be carried out in an organic solvent (such as DCM) under the catalysis of an acid (such as trifluoroacetic acid).

[0213] Then, Compound 4 undergoes a condensation reaction with Compound 5 to obtain Compound 6. Step 3 can be carried out in an organic solvent (such as DCM or DMF) under the catalysis of EDCl and DMAP or dicyclohexylcarbodiimide (DCC).

[0214] If the M' group in Compound 6 contains the above-mentioned protecting group, the protecting group is removed to obtain the target lipid compound. Step 4 is carried out under the reaction conditions for removing the selected protecting group.

[0215] General Synthetic Route 2

[0216]

[0217] Wherein M' is M or M with a protecting group; A is O, NH or S.

[0218] As described in General Synthetic Route 2 above, Compound 1 and Compound 2 undergo a condensation reaction to obtain Compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of EDCl and DMAP.

[0219] Then, Compound 3 is deprotected from the tert-butoxycarbonyl group to obtain Compound 4. Step 2 can be carried out in an organic solvent (such as DCM) in the presence of an acid (such as trifluoroacetic acid) and a cation scavenger (such as triisopropylsilane (TiPS)).

[0220] Then, Compound 4 and a Compound 5 undergo a condensation reaction to obtain Compound 6. Step 3 can be carried out in an organic solvent (such as DCM or DMF) in the presence of EDCl and DMAP or DCC.

[0221] If the M' group in Compound 6 contains the above-mentioned protecting group, the protecting group is removed to obtain the target lipid compound. Step 4 is carried out under the deprotection reaction conditions of the selected protecting group.

[0222] General Synthetic Route 3

[0223]

[0224] Wherein, X is a halogen, such as Cl, Br or I; R4' is R4 or R4 with a protecting group; R5' is R5 or R5 with a protecting group.

[0225] As described in General Synthetic Route 3 above, Boc-aminotris(hydroxymethyl)methane and Compound 2 undergo a condensation reaction to form Compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0226] Then, Compound 3 is deprotected from the Boc group to form Compound 4. Step 2 can be carried out in an organic solvent (such as DCM) under the catalysis of an acid (such as trifluoroacetic acid).

[0227] Subsequently, Compound 4 and a halogen-substituted aldehyde (Compound 5) undergo a condensation and reduction reaction to obtain Compound 6. Step 3 can be carried out in an organic solvent (such as dichloroethane (DCE)) in the presence of a reducing agent (such as sodium triacetoxyborohydride (NaBH(OAc)3)).

[0228] Subsequently, Compound 6 undergoes a nucleophilic reaction with an amine (Compound 7) to form Compound 8. Step 4 can be carried out in an organic solvent (such as in DMF) in the presence of a base (such as a non-nucleophilic organic base (e.g., triethylamine, iPr2EtN) or an inorganic base (e.g., K2CO3)) and a catalyst (KI or NaI).

[0229] If there are the above-mentioned protecting groups in the R4’ and / or R5’ groups of Compound 8, the protecting groups are removed to obtain the target lipid compound. Step 5 is carried out under the deprotection reaction conditions of the selected protecting group.

[0230] General Synthetic Route 4

[0231]

[0232] Wherein, X is a halogen, such as Cl, Br or I; R4’ is R4 or R4 containing a protecting group; R5’ is R5 or R5 containing a protecting group.

[0233] As described in General Synthetic Route 4 above, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with Compound 2 to form Compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0234] Subsequently, the Boc protecting group of Compound 3 is removed to form Compound 4. Step 2 can be carried out in an organic solvent (such as DCM) under the catalysis of an acid (such as trifluoroacetic acid).

[0235] Subsequently, Compound 4 undergoes a condensation reaction with a halogen-substituted Compound 5 to obtain Compound 6. Step 3 can be carried out in an organic solvent such as DCM or DMF under the catalysis of EDCl and DMAP or DCC.

[0236] Subsequently, Compound 6 undergoes a nucleophilic reaction with an amine (Compound 7) to form Compound 8. Step 4 can be carried out in an organic solvent (such as DMF) in the presence of a base (such as a non-nucleophilic organic base (e.g., triethylamine, iPr2EtN) or an inorganic base (e.g., K2CO3)) and a catalyst (KI or NaI).

[0237] If the R4’ and / or R5’ groups of compound 8 have the above-mentioned protecting groups, the protecting groups are removed to obtain the target lipid compound. Step 5 is carried out under the deprotection reaction conditions of the selected protecting group.

[0238] General synthetic route 5

[0239]

[0240] wherein M pro is M or M containing a protecting group.

[0241] As described in the above general synthetic route 5, Boc-aminotris(hydroxymethyl)methane undergoes a condensation reaction with compound 2 to form compound 3. Step 1 can be carried out in an organic solvent (such as DCM) in the presence of, for example, EDCl and DMAP. Step 1 can be carried out at room temperature for 24 hours.

[0242] Then, the Boc protecting group of compound 3 is removed to form compound 4. Step 2 can be carried out in an organic solvent (such as DCM) under the catalysis of an acid (such as trifluoroacetic acid).

[0243] Then, compound 4 and compound 5 undergo a condensation and reduction reaction to obtain compound 6. Step 3 can be carried out in an organic solvent (such as dichloroethane (DCE)) in the presence of a reducing agent (such as sodium triacetoxyborohydride (NaBH(OAc)3)).

[0244] If the M pro group of compound 6 contains the above-mentioned protecting group, the protecting group is removed to obtain the target lipid compound. Step 4 is carried out under the deprotection reaction conditions of the selected protecting group.

[0245] In addition, it should also be understood that any specific embodiment of the present invention within the scope of the prior art may be explicitly excluded from any one or more claims. Since these embodiments are considered to be known to those of ordinary skill in the art, they can be excluded, even if such exclusion is not explicitly stated herein.

[0246] All cited sources, such as the references, publications, databases, database entries, and technologies cited herein, are incorporated herein by reference, even if not explicitly stated in the citation. In the case of a conflict between the cited source and the statements of the present application, the statements of the present application shall prevail.

[0247] (1) Synthesize a compound according to formula (1), (1A), (1B), (1C), (1D), (1E), (1F), (1G), (1H), (1I), (1J), (1K), (1L), or (1M)

[0248] A. General considerations

[0249] It should be noted that the raw materials used in the present invention are all ordinary commercially available products, and their sources are not specifically limited.

[0250] The following described process route can be used to synthesize the compound 1001-3422 of the present invention.

[0251] The following abbreviations are used in this article:

[0252] THF: Tetrahydrofuran

[0253] MeCN: Acetonitrile

[0254] MeOH: Methanol

[0255] PE: Petroleum ether

[0256] EA: Ethyl acetate

[0257] DMF: N,N-Dimethylformamide

[0258] EDCl: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0259] LAH: Lithium aluminum hydride

[0260] DCM: Dichloromethane

[0261] DMAP: 4-Dimethylaminopyridine

[0262] LDA: Lithium diisopropylamide

[0263] rt: Room temperature

[0264] DCE: 1,2-Dichloroethane

[0265] n-BuLi: n-Butyllithium

[0266] i-Pr2EtN: N,N-Diisopropylethylamine

[0267] B. Intermediate synthesis

[0268] Intermediate A:

[0269]

[0270] Intermediate A is obtained by the following synthesis process route:

[0271]

[0272] Dissolve tris(hydroxymethyl)aminomethane (50.0 g) and di-tert-butyl dicarbonate (Boc2O) (99.1 g) in a mixed solvent of methanol (300 mL) / H2O (30 mL), and react at room temperature for 72 h. Purify by silica gel column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain the white solid compound intermediate A (N-Boc-aminotris(hydroxymethyl)methane) (75.0 g). LCMS (ESI) calcd for C9H 19 NO 5， [M+H] + m / z 222.13, found 222.25.

[0273] Intermediate B:

[0274]

[0275] Intermediate B is obtained by the following synthetic process route:

[0276]

[0277] Add diethyl 2-ethyl-1,3-propanedicarboxylate (29.0 g), THF (90 mL), DMF (30 mL) to a single-necked flask. Add NaH (3.7 g) under an ice bath. Stir at room temperature for 30 min under nitrogen protection. Add bromopentadecane (15.0 g). React at 80 °C for 2 h under nitrogen protection. Concentrate under reduced pressure to remove THF. Drop the reaction solution into ice water. Add ethyl acetate and stir. Filter through diatomaceous earth. Collect the organic phase and then separate the layers. Wash the organic phase with saturated brine. Separate the organic phase and purify by silica gel column chromatography (PE:EA = 100:1 - 50:1) to obtain diethyl 2-ethyl-2-pentadecylmaleate (16.5 g). Add EtOH (50 mL), H2O (50 mL), KOH (11.3 g) to diethyl 2-ethyl-2-pentadecylmaleate (8.0 g). React at 90 °C for 12 h. Concentrate under reduced pressure to remove EtOH. Add dilute hydrochloric acid to adjust the reaction system to pH = 4 - 5. Add water and ethyl acetate and stir to separate the layers. Wash the organic phase with saturated brine. Separate the organic phase and purify by column chromatography (PE:EA = 5:1 - 1:1) to obtain 2-ethyl-2-pentadecylmaleic acid (5.2 g). React 2-ethyl-2-pentadecylmaleic acid (10.0 g) under open conditions at 170 °C for 6 h. Cool to room temperature. Add water and ethyl acetate and stir to separate the layers. Wash the organic phase with saturated brine. Separate the organic phase, concentrate, and purify by column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain Intermediate B (2-ethylheptadecanoic acid) (8.2 g). 1H NMR (400 MHz, CDCl3) δ 2.31 (tt, J = 8.6, 5.3 Hz, 1H), 1.71 - 1.46 (m, 4H), 1.28 (s, 26H), 0.96 (t, J = 7.4 Hz, 3H), 0.90 (t, J = 6.7 Hz, 3H).

[0278] Intermediate C:

[0279]

[0280] Intermediate C is obtained by the following synthetic process route:

[0281]

[0282] Add capric acid (50 g) and the solvent THF (500 mL) to a three-necked flask. After cooling the system to 0 °C, slowly add the reactant NaH (23.22 g). Stir for 1 h at 0 °C under nitrogen protection, then slowly dropwise add LDA (62.19 g) and continue to stir for 1 h at 0 °C under nitrogen protection. Finally, after dropwise adding the reactant iodo-nonane (88.52 g), raise the temperature to room temperature and stir overnight. Dilute with 1 L of DCM and wash with saturated NH4Cl solution and water respectively. The organic layer is dried over anhydrous Na2SO4, filtered, concentrated, and purified by silica gel column chromatography (PE:EA = 50:1) to finally obtain intermediate C (2-octylundecanoic acid) (11 g). 1H NMR (400 MHz, Chloroform-d) δ 2.37 (tt, J = 8.7, 5.1 Hz, 1H), 1.71 - 1.58 (m, 2H), 1.48 (dt, J = 13.3, 6.7 Hz, 2H), 1.29 (d, J = 9.4 Hz, 25H), 0.90 (t, J = 6.8 Hz, 6H).

[0283] Intermediate D:

[0284]

[0285] Intermediate D was obtained by the following synthetic process route:

[0286]

[0287] Add tridecanoic acid (79.5 g) and the solvent THF (800 mL) to a three-necked flask. After cooling the system to 0 °C, slowly add NaH (22.25). Stir the reaction solution for 1 h at 0 °C under nitrogen protection, then slowly dropwise add LDA (317.87 g) to the reaction system; continue to stir the reaction solution for 1 h at 0 °C under nitrogen protection. After dropwise adding the reactant iodo-n-hexane (94.39), raise the temperature to room temperature and stir overnight. Dilute the reaction solution with 1 L of DCM and wash with saturated NH4Cl solution and water respectively. The organic layer is dried over anhydrous Na2SO4, filtered, concentrated, and subjected to silica gel column chromatography (PE:EA = 50:1) to obtain intermediate D (2-hexyltridecanoic acid) (26 g). 1H NMR (400 MHz, Chloroform-d) δ 2.36 (tt, J = 8.7, 5.4 Hz, 1H), 1.63 (ddd, J = 14.3, 8.7, 5.5 Hz, 2H), 1.49 (dq, J = 13.5, 6.6 Hz, 2H), 1.29 (d, J = 10.1 Hz, 26H), 0.90 (t, J = 6.6 Hz, 6H).

[0288] Intermediate E:

[0289]

[0290] The intermediate E is obtained through the following synthetic process route:

[0291]

[0292] Dissolve tris(hydroxymethyl)aminomethane (10.0 g) and tert-butyl acrylate (21.1) in EtOH (150 mL). Under nitrogen protection, react at 45 °C for 30 h, then concentrate under reduced pressure to remove EtOH. Add 100 mL × 3 of the solvent (PE:EA = 20:1) for slurrying, filter to obtain the white solid compound intermediate E (11.5 g). 1H NMR (400 MHz, CDCl3) δ 3.60 (s, 6H), 2.84 (t, J = 5.9 Hz, 2H), 2.47 (t, J = 5.8 Hz, 2H), 1.48 (s, 9H).

[0293] Intermediate F:

[0294]

[0295] The intermediate F is obtained through the following synthetic process route:

[0296]

[0297] Add (2-aminoethyl)carbamic acid tert-butyl ester (2.7 g), MeCN (90 mL), benzyl 2-bromoethyl ether (7.99 g), and K2CO3 (11.65 g) to a single-necked flask. React at 80 °C overnight, then add water and ethyl acetate for stirring and liquid separation. Wash the organic phase with saturated brine, separate the organic phase, concentrate, and purify by silica gel column chromatography (PE:EA = 10:1 - 5:1) to obtain compound 2 (5.5 g). Add dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL) to compound 2, stir at room temperature for 3 h, then concentrate under reduced pressure to obtain compound 3, which is intermediate F (6 g). LCMS (ESI) calcd for C 20 H 28 N2O2, [M + H] + m / z

[0298] 329.22, found 329.24.

[0299] Intermediate G:

[0300]

[0301] The intermediate G is obtained through the following synthetic process route:

[0302]

[0303] To a single-necked flask, add Compound 1 (3.0 g), MeCN (90 mL), 3-benzyloxypropyl bromide (9.45 g), K2CO3 (12.94 g). After reacting overnight at 80 °C, add water and ethyl acetate, stir, and separate the layers. Wash the organic phase with saturated brine, separate the organic phase, concentrate it, and purify it by column chromatography (PE:EA = 10:1 - 5:1) to obtain Compound 2 (6.9 g). To Compound 2 (6.9 g), add Dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL). After stirring at room temperature for 3 h, concentrate under reduced pressure to obtain Intermediate G (7.5 g). LCMS (ESI) calcd for C 22 H 32 N2O2, [M+H] + m / z 357.25, found 357.51。

[0304] Intermediate H:

[0305]

[0306] Intermediate H is obtained by the following synthetic route:

[0307]

[0308] To a single-necked flask, add Compound 1 (1.5 g), MeCN (90 mL), 4-bromobutyl benzyl ether (5.02 g), K2CO3 (6.47 g). After reacting overnight at 80 °C, add water and ethyl acetate, stir, and separate the layers. Wash the organic phase with saturated brine, separate the organic phase, concentrate it, and purify it by column chromatography (PE:EA = 20:1 - 10:1) to obtain Compound 2 (4.1 g). To Compound 2 (4.1 g.), add Dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL). After stirring at room temperature for 3 h, concentrate under reduced pressure to obtain Intermediate H (4.9 g). LCMS (ESI) calcd for C 24 H 36 N2O2, [M+H] + m / z 385.28, found 385.56。

[0309] Intermediate I:

[0310]

[0311] Intermediate I is obtained by the following synthetic route:

[0312]

[0313] To a single-necked flask, add compound 1 (3.9 g), MeCN (90 mL), benzyl 2-bromoethyl ether (5.78 g), K2CO3 (15.46 g). After reacting overnight at 80 °C, add water and ethyl acetate, stir, and separate the layers. Wash the organic layer with saturated brine, separate the organic layer, concentrate it, and purify it by column chromatography (PE:EA = 20:1 - 10:1) to obtain compound 2 (6.8 g). To compound 2 (6.8 g), add Dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL). After stirring at room temperature for 3 h, concentrate under reduced pressure to obtain intermediate I (6.5 g). LCMS (ESI) calcd for C 12 H 20 N2O, [M+H] + m / z 209.16, found 209.31.

[0314] Intermediate J:

[0315]

[0316] Intermediate J is obtained by the following synthetic route:

[0317]

[0318] To a single-necked flask, add butyric acid (5.0 g), THF (100 mL). Add NaH (2.73 g) at 0 °C, slowly dropwise add LDA (56.8 mL), react at room temperature for 30 min, add 1-bromotridecane, continue to react overnight at room temperature, add ice water and ethyl acetate, stir, and separate the layers. Wash the organic layer with saturated brine, separate the organic layer, concentrate it, and purify it by column chromatography (PE:EA = 10:1 - 5:1) to obtain intermediate J (5.0 g). LCMS (ESI) calcd for C 17 H 34 O2, [M+H] + m / z

[0319] 271.26, found 271.46.

[0320] Intermediate K:

[0321]

[0322] Intermediate K is obtained by the following synthetic route:

[0323]

[0324] Add reactant 1 (24 g), Imidazole (19.00 g) and solvent DCM (200 mL) into a three-necked flask. After cooling to 0 °C, slowly add reactant TBDMSCI (38.57 g). Stir at 25 °C for 4 h, then dilute with 300 mL of DCM. Wash the organic phase twice with 1 L of water and then dry with anhydrous Na2SO4. After filtration and concentration, add silica gel and elute through a column with 1:20 DCM:MeOH to collect the sample and concentrate to obtain intermediate K (28.00 g). 1H NMR (400 MHz, CDCl3) δ 4.89 (s, 1H), 3.58 (t, J = 6.1 Hz, 2H), 2.66 (t, J = 7.3 Hz, 2H), 2.45 (s, 3H), 1.67 - 1.40 (m, 4H), 0.84 (s, 9H).

[0325] Intermediate L:

[0326]

[0327] Intermediate L is obtained through the following synthetic process route:

[0328]

[0329] Add compound DMSO (3.58 g) and anhydrous solvent DCM (30 mL) into a three-necked flask. Cool the system to -78 °C and slowly add oxalyl chloride (2.91 g). Stir the reaction solution at -78 °C under N2 protection for 10 minutes, then slowly drip in compound 1 (3 g). Continue to stir at -78 °C under N2 protection for 1 h. Finally, drip in TEA (9.28 g). Continue to stir at -78 °C under N2 protection for 0.5 h, then dilute with 100 mL of DCM and wash with saturated NH4Cl solution and water respectively. Dry the organic layer with anhydrous Na2SO4, filter and concentrate to obtain intermediate L (3.0 g). LCMS (ESI) calcd for C 11 H 14 O3, [M + H] + m / z 195.09, found 195.23.

[0330] Intermediate M:

[0331]

[0332] Intermediate M is obtained through the following synthetic process route:

[0333]

[0334] Add intermediate K (2.0 g), compound 1 (1.79 g) and solvent ACN (20 mL) into a sealed tube. Add reactants K2CO3 (3.81 g) and KI (1.52 g). Stir overnight at 70 °C under N2 protection, then dilute with 100 mL of EA, and wash with saturated NH4Cl solution and water respectively. Dry the organic layer with anhydrous Na2SO4, filter and concentrate, then add silica gel and elute through a column with 15:1 DCM:MeOH. Collect the sample and concentrate to obtain compound 2 (2.0 g). Add reactant 2 (1.80 g) and solvent DCM (20 mL) into a three-necked flask. Replace the system with nitrogen and cool to -78 °C, then add DIBAL-H (1.00 g) dropwise. Stir at -78 °C for 4 h, then quench the reaction by dropping methanol and sodium carbonate solution into the reaction solution. Then extract with DCM (100 mL). Dry the organic layer with anhydrous sodium sulfate, filter and concentrate to obtain intermediate M (1.36 g). LCMS(ESI) calcd for C 15 H 33 NO2Si,[M+H] + m / z 288.52, found 288.23。

[0336] C, Compound 1003, synthesized according to General Synthetic Route 1

[0337] Structural formula:

[0338] Chemical formula: C 63 H 122 N2O7

[0339] Molecular weight: 1019.68

[0340] Step 1: Synthesize compound 3 in General Synthetic Route 1

[0341]

[0342] Add compound 1 (intermediate A) (1.0 g), DCM (20 mL), DMAP (2.2 g), and EDCI (3.4 g) to compound 2 (intermediate B) (4.3 g). Stir at room temperature for 12 h under nitrogen protection, then add water and dichloromethane and stir for liquid separation. Wash the organic phase with saturated brine, separate the organic phase, dry the organic phase with anhydrous sodium sulfate again, concentrate, and purify by silica gel column chromatography (PE:EA = 40:1 - 30:1) to obtain compound 3 (2.6 g).

[0343] Step 2: Synthesize compound 4 in General Synthetic Route 1

[0344]

[0345] DCM (15 mL) and TFA (5 mL) were added to Compound 3 (2.6 g). After stirring at room temperature for 3 h, the mixture was concentrated and purified by silica gel column chromatography (PE:EA = 10:1 - 5:1) to obtain Compound 4 (3.2 g).

[0346] Step 3: Synthesis of Compound 6 in General Synthetic Route 1

[0347]

[0348] DCM (20 mL), Boc-glycine (Compound 5, 578 mg), and DCC (6.8 g) were added to Compound 4 (3.2 g). After stirring at room temperature under nitrogen protection for 12 h, water and ethyl acetate were added and stirred for liquid separation. The organic phase was washed with saturated brine, the organic phase was separated, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (PE:EA = 5:1 - 2:1) to obtain Compound 6 (2.2 g).

[0349] Step 4: Compound 1003

[0350]

[0351] DCM (15 mL) and TFA (5 mL) were added to Compound 6 (2.2 g). After stirring at room temperature for 12 h, the mixture was concentrated and purified by column chromatography (DCM:MeOH = 30:1 - 20:1) to obtain Compound 1003 (1.3 g). 1 HNMR (400 MHz, CDCl3) δ 4.49 (d, J = 2.6 Hz, 6H), 3.28 (s, 2H), 2.32 (tt, J = 8.6, 5.5 Hz, 3H), 1.62 (q, J = 7.1 Hz, 12H), 1.27 (d, J = 3.4 Hz, 78H), 0.94 - 0.86 (m, 18H).

[0352] D. Compound 1002, synthesized according to General Synthetic Route 1

[0353] Structural formula:

[0354] Chemical formula: C 60 H 116 N2O7

[0355] Molecular weight: 977.60

[0356] Synthesized according to General Synthetic Route 1, with a process similar to that of Compound 1003, except that Intermediate C was used as Compound 2 in General Synthetic Route 1. 11H NMR (400 MHz, CDCl3) δ 4.48 (s, 6H), 3.27 (s, 2H), 2.37 (tt, J = 8.5, 5.5 Hz, 3H), 1.64 (d, J = 6.9 Hz, 6H), 1.50 - 1.42 (m, 6H), 1.27 (s, 72H), 0.90 (t, J = 6.7 Hz, 18H).

[0357] E. Compound 1004 was synthesized according to General Synthetic Route 1

[0358] Structural formula:

[0359] Chemical formula: C 54 H 104 N2O7

[0360] Molecular weight: 893.43

[0361] Synthesized according to General Synthetic Route 1, similar to the synthesis process of Compound 1003, except that palmitic acid was used as Compound 2 in General Synthetic Route 1 1 1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 4.45 (s, 6H), 3.74 (s, 2H), 2.35 (t, J = 7.6 Hz, 6H), 1.60 (p, J = 6.9 Hz, 6H), 1.27 (s, 72H), 0.90 (t, J = 6.7 Hz, 9H).

[0362] F. Compound 1001 was synthesized according to General Synthetic Route 1

[0363] Structural formula:

[0364] Chemical formula: C 59 H 108 N2O7

[0365] Molecular weight: 957.52

[0366] Synthesized according to General Synthetic Route 1, similar to the synthesis process of Compound 1003, except that (9Z)-9-hexadecenoic acid was used as Compound 2 in General Synthetic Route 1, and 5-(N,N-dimethylamino)valeric acid was used as Compound 5 in General Synthetic Route 1 11H NMR (400 MHz, CDCl3) δ 5.42 - 5.30 (m, 6H), 4.51 - 4.38 (m, 6H), 3.19 (dt, J = 11.3, 5.6 Hz, 2H), 2.97 (t, J = 5.9 Hz, 6H), 2.46 (t, J = 6.6 Hz, 1H), 2.35 (td, J = 7.6, 3.1 Hz, 5H), 2.20 (t, J = 7.6 Hz, 2H), 2.03 (d, J = 6.2 Hz, 12H), 1.89 - 1.81 (m, 3H), 1.74 (q, J = 7.8 Hz, 2H), 1.67 - 1.51 (m, 6H), 1.31 (d, J = 7.8 Hz, 48H), 0.90 (t, J = 6.7 Hz, 9H).

[0367] G. Compound 1014 was synthesized according to General Synthetic Route 2

[0368] Structural formula:

[0369] Chemical formula: C 70 H 137 N3O9

[0370] Molecular weight: 1164.88

[0371] Step 1: Synthesis of Intermediate 3 of General Synthetic Route 2

[0372]

[0373] Intermediate 3 was synthesized according to the method shown in Step 1 of General Synthetic Route 1, with the difference that Intermediate E was used as Compound 1 in General Synthetic Route 1

[0374] Step 2: Synthesis of Compound 4 of General Synthetic Route 2

[0375]

[0376] To Compound 3 (4.8 g), DCM (15 mL), TFA (12 mL), and TiPS (3 mL) were added. After reacting at room temperature for 12 h, water and dichloromethane were added and stirred for liquid separation. The organic phase was washed with saturated brine, the organic phase was separated and concentrated, and purified by silica gel column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain Compound 4 (3.3 g).

[0377] Step 3: Synthesis of Compound 6 of General Synthetic Route 2

[0378]

[0379] Compound 4 (4.0 g), DCM (80 mL), EDCI (1.1 g), 1-hydroxybenzotriazole (HOBt) (0.78 g), and DIEA (2.5 g) were added to compound 5 (1.9 g). After stirring at room temperature for 12 h, water and dichloromethane were added and stirred, followed by liquid separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 30:1 - 20:1) to obtain compound 6 (4.0 g).

[0380] Step 4: Compound 1014

[0381]

[0382] MeOH (30 mL), DCM (10 mL), and Pd / C (4.73 g (10%)) were added to compound 6 (4.0 g). After stirring overnight at room temperature under hydrogen, it was filtered through diatomaceous earth, concentrated, and purified by silica gel column chromatography (DCM:MeOH 30:1 - 15:1) to obtain compound 1014 (2 g). 1 H (400 MHz, CDCl3) δ 7.38 (t, J = 5.8 Hz, 1H), 4.14 - 4.05 (m, 6H), 3.58 (t, J = 4.9 Hz, 4H), 3.31 (q, J = 5.7 Hz, 2H), 2.89 (t, J = 6.3 Hz, 2H), 2.64 (q, J = 5.2 Hz, 6H), 2.29 (dq, J = 10.8, 4.4 Hz, 6H), 1.66 - 1.37 (m, 12H), 1.24 (d, J = 4.3 Hz, 78H), 0.87 (t, J = 7.1 Hz, 18H).

[0383] H. Compound 1015 was synthesized according to General Synthetic Route 2

[0384] Structural formula:

[0385] Chemical formula: C 72 H 141 N3O9

[0386] Molecular weight: 1192.93

[0387] Synthesized according to General Synthetic Route 2, similar to the synthesis process of compound 1014, except that intermediate G was used as compound 5 in General Synthetic Route 2 1H (400 MHz, CDCl3) δ 7.58 (t, J = 5.7 Hz, 1H), 4.15 - 4.01 (m, 6H), 3.70 (t, J = 5.4 Hz, 4H), 3.39 (q, J = 5.7 Hz, 2H), 2.86 (t, J = 6.2 Hz, 2H), 2.57 (dt, J = 20.5, 5.8 Hz, 6H), 2.36 - 2.22 (m, 5H), 1.76 - 1.67 (m, 4H), 1.62 - 1.35 (m, 13H), 1.23 (d, J = 4.6 Hz, 78H), 0.92 - 0.80 (m, 18H).

[0388] I. Compound 1020, synthesized according to General Synthetic Route 2

[0389] Structural formula:

[0390] Chemical formula: C 74 H 145 N3O9

[0391] Molecular weight: 1220.99

[0392] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that intermediate H is used as Compound 5 in General Synthetic Route 2.

[0393] J. Compound 1025, synthesized according to General Synthetic Route 2

[0394] Structural formula:

[0395] Chemical formula: C 69 H 135 N3O8

[0396] Molecular weight: 1134.85

[0397] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that intermediate I is used as Compound 5 in General Synthetic Route 2. 1 H (400 MHz, CDCl3) δ 6.98 (t, J = 5.7 Hz, 1H), 4.10 (m, J = 3.8 Hz, 6H), 3.63 (t, J = 5.2 Hz, 2H), 3.35 (q, J = 5.8 Hz, 2H), 2.89 (t, J = 6.1 Hz, 2H), 2.57 (q, J = 5.3 Hz, 4H), 2.32 (s, 3H), 2.28 (q, J = 4.1 Hz, 5H), 1.65 - 1.41 (m, 13H), 1.24 (d, J = 4.4 Hz, 78H), 0.87 (t, J = 7.1 Hz, 18H).

[0398] Compound 1005, synthesized according to General Synthetic Route 2

[0399] Structural formula:

[0400] Chemical formula: C 68 H 133 N3O7

[0401] Molecular weight: 1104.83

[0402] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that N,N-dimethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1 H NMR(400MHz,CDCl3)δ4.19 - 4.07(m,6H),3.40(d,J=5.7Hz,2H),2.95 - 2.87(m,2H),2.82(s,4H),2.64 - 2.55(m,2H),2.38(s,6H),2.33 - 2.29(m,3H),1.63 - 1.55(m,6H),1.49 - 1.44(m,2H),1.26(d,J=5.4Hz,78H),0.89(td,J=7.0,2.8Hz,18H).

[0403] L. Compound 1006, synthesized according to General Synthetic Route 2

[0404] Structural formula:

[0405] Chemical formula: C 53 H 103 N3O7

[0406] Molecular weight: 894.42

[0407] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that myristic acid is used as Compound 2 in General Synthetic Route 2 and N,N-dimethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1 HNMR(400MHz,CDCl3)δ7.99(t,J=5.8Hz,1H),4.13(s,6H),3.60(q,J=5.3Hz,2H),3.38 - 3.34(m,2H),3.00(s,6H),2.94(t,J=5.8Hz,2H),2.45(t,J=5.8Hz,2H),2.35(t,J=7.6Hz,6H),1.66 - 1.56(m,6H),1.33 - 1.23(m,60H),0.90(t,J=6.8Hz,9H).

[0408] Compound 1007, synthesized according to General Synthetic Route 2

[0409] Structural formula:

[0410] Chemical formula: C 68 H 133 N3O7

[0411] Molecular weight: 1104.83

[0412] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that Intermediate D is used as Compound 2 in General Synthetic Route 2 and N,N-dimethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1 HNMR(400MHz,Chloroform-d)δ7.13(t,J=5.0Hz,1H),4.13(s,6H),3.33(q,J=5.6Hz,2H),2.92(t,J=6.0Hz,2H),2.47 - 2.28(m,7H),2.24(s,6H),1.60(dq,J=14.7,7.2Hz,6H),1.47(dd,J=14.2,6.7

[0413] Hz,6H),1.27(d,J=3.4Hz,78H),0.89(td,J=6.7,2.5Hz,18H).

[0414] Compound 1008, synthesized according to General Synthetic Route 2

[0415] Structural formula:

[0416] Chemical formula: C 68 H 133 N3O7

[0417] Molecular weight: 1104.01

[0418] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that Intermediate C is used as Compound 2 in General Synthetic Route 2 and N,N-dimethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1HNMR(400MHz, Chloroform-d) δ 4.13(s, 6H), 3.33(q, J=5.6Hz, 2H), 2.92(t, J=5.9Hz, 2H), 2.51 - 2.26(m, 7H), 2.24(s, 6H), 1.59(ddt, J=14.8, 10.9, 6.4Hz, 6H), 1.47(tq, J=11.0, 5.4Hz, 6H), 1.26(s, 77H), 0.89(t, J=6.7Hz, 18H).

[0419] O. Compound 1009, synthesized according to General Synthetic Route 2

[0420] Structural formula:

[0421] Chemical formula: C 62 H 121 N3O7

[0422] Molecular weight: 1020.66

[0423] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that Intermediate J is used as Compound 2 in General Synthetic Route 2 and N,N-dimethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1 H(400MHz, CDCl3) δ 7.04(t, J=5.0Hz, 1H), 4.12(m, J=3.3Hz, 6H), 3.30(q, J=5.6Hz, 2H), 2.91(t, J=6.0Hz, 2H), 2.39(t, J=6.0Hz, 2H), 2.33 - 2.25(m, 5H), 2.22(s, 6H), 1.76 - 1.54(m, 12H), 1.24(m, J=3.7Hz, 66H), 0.90 - 0.84(m, 18H).

[0424] P. Compound 1011, synthesized according to General Synthetic Route 2

[0425] Structural formula:

[0426] Chemical formula: C 70 H 137 N3O7

[0427] Molecular weight: 1132.88

[0428] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that N,N-diethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 11H NMR (400 MHz, CDCl3) δ 4.19 - 4.08 (m, 6H), 3.31 (q, J = 5.8 Hz, 2H), 2.92 (t, 2H), 2.57 (t, J = 6.9 Hz, 6H), 2.37 - 2.26 (m, 5H), 1.62 (ddd, J = 18.3, 9.0, 4.3 Hz, 12H), 1.27 (d, J = 4.7 Hz, 78H), 1.04 (t, J = 7.1 Hz, 6H), 0.94 - 0.86 (m, 18H).

[0429] Q. Compound 1012 was synthesized according to General Synthetic Route 2

[0430] Structural formula:

[0431] Chemical formula: C 72 H 141 N3O7

[0432] Molecular weight: 1160.93

[0433] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that N,N - diethylethylenediamine was used as Compound 5 in General Synthetic Route 2. 1 1H NMR (400 MHz, CDCl3) δ 6.59 (s, 1H), 4.18 - 4.07 (m, 6H), 3.29 (q, J = 5.7 Hz, 2H), 2.91 (t, J = 6.2 Hz, 2H), 2.54 (t, J = 6.1 Hz, 2H), 2.40 (t, J = 7.5 Hz, 4H), 2.35 - 2.27 (m, 5H), 1.63 - 1.42 (m, 16H), 1.27 (m, J = 4.5 Hz, 78H), 0.93 - 0.85 (m, 24H).

[0434] R. Compound 1013 was synthesized according to General Synthetic Route 2

[0435] Structural formula:

[0436] Chemical formula: C 74 H 145 N3O7

[0437] Molecular weight: 1188.99

[0438] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that N,N - diethylethylenediamine was used as Compound 5 in General Synthetic Route 2. 11H NMR (400 MHz, CDCl3) δ 4.10 (d, J = 4.1 Hz, 6H), 3.26 (q, J = 5.8 Hz, 2H), 2.89 (t, J = 6.2 Hz, 2H), 2.52 (d, J = 6.6 Hz, 2H), 2.41 (t, J = 7.4 Hz, 4H), 2.33 - 2.25

[0439] (m, 5H), 1.60 (d, J = 11.8 Hz, 16H), 1.24 (d, J = 4.5 Hz, 82H), 0.96 - 0.79 (m, 24H).

[0440] S. Compound 1029 was synthesized according to General Synthetic Route 2

[0441] Structural formula:

[0442] Chemical formula: C 68 H 132 N2O8

[0443] Molecular weight: 1105.81

[0444] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that N,N - diethyl - 2 - hydroxyethylamine was used as Compound 5 in General Synthetic Route 2. 1 1H NMR (400 MHz, CDCl3) δ 4.16 (t, J = 5.8 Hz, 2H), 4.13 - 4.05 (m, 6H), 2.86 (t, J = 6.3 Hz, 2H), 2.55 (t, J = 5.8 Hz, 2H), 2.44 (t, J = 6.3 Hz, 2H), 2.35 - 2.20 (m, 9H), 1.60 - 1.39 (m, 11H), 1.24 (d, J = 4.5 Hz, 80H), 1.02 - 0.79 (m, 18H).

[0445] T. Compound 1111 was synthesized according to General Synthetic Route 2

[0446] Structural formula:

[0447] Chemical formula: C 70 H 136 N4O7

[0448] Molecular weight: 1145.88

[0449] Synthesized according to General Synthetic Route 2, similar to the synthetic process of Compound 1014, except that Intermediate C is used as Compound 2 in General Synthetic Route 2 and tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate is used as Compound 5 in General Synthetic Route 2. 1H NMR (400 MHz, Chloroform-d) δ 4.28 (s, 6H), 3.41 (s, 2H), 3.31 (s, 4H), 3.22 (s, 3H), 2.97 - 2.80 (m, 5H), 2.70 (s, 5H), 2.46 - 2.32 (m, 4H), 1.60 (t, J = 7.1 Hz, 6H), 1.54 - 1.40 (m, 6H), 1.27 (s, 78H), 0.89 (t, J = 6.7 Hz, 18H).

[0450] U. Compound 1118, synthesized according to General Synthetic Route 2

[0451] Structural formula:

[0452] Chemical formula: C 71 H 139 N3O8

[0453] Molecular weight: 1162.91

[0454] Synthesized according to General Synthetic Route 2, similar to the synthetic process of Compound 1014, except that Intermediate C is used as Compound 2 in General Synthetic Route 2 and Intermediate K is used as Compound 5 in General Synthetic Route 2. 1 H NMR (400 MHz, Chloroform-d) δ 4.11 (s, 6H), 3.61 (s, 2H), 3.42 (d, J = 33.5 Hz, 2H), 2.91 (t, J = 6.2 Hz, 3H), 2.36 (dd, J = 9.2, 3.6 Hz, 6H), 1.47 (dd, J = 13.9, 6.2 Hz, 14H), 1.27 (s, 71H), 0.90 (t, J = 6.7 Hz, 18H).

[0455] V. Compound 1010, synthesized according to General Synthetic Route 3

[0456] Structural formula:

[0457] Chemical formula: C 67 H 133 N3O6

[0458] Molecular weight: 1076.82

[0459] Steps 1 and 2: Synthesize Compound 3 and Compound 4 of General Synthetic Route 3

[0460]

[0461] According to Steps 1 and 2 of General Synthesis Route 1, the synthesis process is the same as that of Compound 1003.

[0462] Step 3: Synthesize Compound 6 of General Synthesis Route 3

[0463]

[0464] Compound 4 (600 mg), chloroacetaldehyde (Compound 5, 242 mg (40%)), and NaBH(OAc)3 (394 mg) were added to DCE (60 mL). After reacting overnight at room temperature, water and dichloromethane were added and stirred for liquid separation. The organic phase was washed with saturated brine, and the organic phase was separated, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 30:1 - 15:1) to obtain Compound 6 (270 mg).

[0465] Step 4: Synthesize Compound 1010

[0466]

[0467] To Compound 6 (270 mg), MeCN (20 mL), N,N - dimethylethylenediamine (Compound 7, 229 mg), KI (43 mg), and K2CO3 (179 mg) were added. After reacting overnight at 70 °C under nitrogen protection, purification by silica gel column chromatography gave Compound 1010 (16 mg). 1 H (400 MHz, CDCl3) δ 4.10 (m, J = 2.9 Hz, 6H), 2.76 - 2.64 (m, 6H), 2.40 (t, J = 6.2 Hz, 2H), 2.29 (ddd, J = 8.5, 5.6, 2.9 Hz, 3H), 2.22 (s, 6H), 1.97 (s, 6H), 1.50 - 1.41 (m, 6H), 1.24 (d, J = 4.2 Hz, 78H), 0.87 (t, J = 7.1 Hz, 18H).

[0468] W. Compound 1059, synthesized according to General Synthesis Route 3

[0469] Structural formula:

[0470] Chemical formula: C 67 H 132 N2O7

[0471] Molecular weight: 1077.80

[0472] Synthesized according to the general synthetic route 3, which is similar to the synthesis process of compound 1010, except that intermediate C and intermediate L are used as compound 2 and compound 5 in the general synthetic route 3 respectively. In addition, the functional group transformation of compound 6 in the general synthetic route 3 needs to be carried out through the following process steps:

[0473]

[0474] To intermediate 6a (1.5 g), add solvents MeOH (5 mL) and THF (5 mL), and add reactants Pd(OH)2 / C (10%) (0.46 g) and Pd / C (10%) (0.35 g). Under H2 condition, react overnight at 25 °C, then filter with diatomaceous earth, concentrate the filtrate, and perform silica gel column chromatography (PE:EA 30:1 - 10:1) to obtain intermediate 6b (520 mg). To intermediate 6b (470 mg), add DCM (5 mL), and dropwise add SOCl2 (1064.26 mg, 8.946 mmol) at 0 °C. Transfer to room temperature and react for 3 hours, then extract the reaction solution with water and DCM, concentrate the organic phase, and perform silica gel column chromatography (PE:EA 20:1) to obtain intermediate 6c (181 mg). To intermediate 6c (170 mg), add dimethylamine (143.39 mg), DMF (2 mL), potassium carbonate (43.96 mg), and potassium iodide (26.39 mg). Stir the reaction solution at 70 °C, then filter, concentrate, and perform silica gel column chromatography (DCM:MeOH 20:1) to obtain compound 1059 (91 mg, yield 53.22%). 1 H NMR (400 MHz, Chloroform - d) δ4.11 (s, 6H), 3.51 (dt, J = 18.3, 5.6 Hz, 4H), 2.81 (dd, J = 10.3, 5.1 Hz, 2H), 2.50 (t, J = 6.0 Hz, 2H), 2.42 - 2.32 (m, 3H), 2.28 (s, 6H), 2.08 - 1.98 (m, 1H), 1.53 - 1.38 (m, 7H), 1.27 (s, 77H), 0.90 (t, J = 6.7 Hz, 18H).

[0475] X. Compound 1112, synthesized according to the general synthetic route 3

[0476] Structural formula:

[0477] Chemical formula: C 69 H 136 N2O6

[0478] Molecular weight: 1089.85

[0479] Synthesized according to the general synthetic route 3, similar to the synthetic process of compound 1010, with the difference that intermediate C and 6-bromohexanal are used instead of compound 2 and compound 5 in the general synthetic route 3 respectively. 1 H NMR(400MHz,Chloroform-d)δ4.11(s,6H),3.11(s,1H),2.57(t,J=6.8Hz,2H),2.44-2.19(m,12H),2.04(q,J=6.5Hz,2H),1.53-1.38

[0480] (m,13H),1.27(s,92H),0.90(t,J=6.7Hz,20H).

[0481] Y. Compound 1113, synthesized according to the general synthetic route 4

[0482] Structural formula:

[0483] Chemical formula: C 71 H 138 N2O8

[0484] Molecular weight: 1147.89

[0485] Steps 1 and 2: Synthesize compound 3 and compound 4 of the general synthetic route 4

[0486]

[0487] According to steps 1 and 2 of the general synthetic route 1, the same as the synthetic process of compound 1003.

[0488] Step 3: Synthesize compound 6 of the general synthetic route 4

[0489]

[0490] Add DIEA (0.30 g) and solvent DCM (20 mL) to compound 4. Under nitrogen protection, cool to 0 °C and then add 5-bromovaleryl chloride (compound 5, 0.27 g). Stir at 25 °C for 2 h. Dilute with 100 mL of DCM and then wash the organic phase twice with 100 mL of water. Dry the organic phase with anhydrous Na2SO4, filter and concentrate, and then perform silica gel column chromatography (PE:EA 20:1) to obtain compound 6 (800 mg).

[0491] Step 4: Synthesize compound 8 of the general synthetic route 4

[0492]

[0493] Add compound 6 (750.00 mg), intermediate K (compound 7, 217.31 mg) and solvent DMF (20 mL) into a three-necked flask. Add reactants K2CO3 (184.16 mg) and KI (110.60 mg). Stir at 25 °C for 16 h, then raise the temperature to 100 °C and stir for 3 h. After diluting the reaction solution with 200 mL of EA, wash the organic phase twice with 300 mL of saturated brine; dry the organic phase with anhydrous Na2SO4, filter and concentrate, add silica gel, and column chromatograph with 20:1 DCM:MeOH to collect the sample and concentrate to obtain compound 8 (700.00 mg).

[0494] Step 5: Synthesis of compound 1113

[0495]

[0496] Add compound 8 (700.00 mg) and solvent THF (5 mL) into a three-necked flask. Add dioxane hydrochloride solution (1 M) (5.55 mL). Stir the reaction solution at 25 °C for 2 h, then dilute with 50 mL of EA, wash the organic phase with saturated NaHCO3 solution and water, dry the organic phase with anhydrous Na2SO4, filter and concentrate, and perform silica gel column chromatography (MeOH:DCM 1:20) to obtain compound 1113 (201.1 mg, yield 31.59%). 1 H NMR (400 MHz, CDCl3) δ 4.50 - 4.39 (m, 6H), 3.59 (t, J = 4.6 Hz, 2H), 2.49 (s, 3H), 2.35 - 2.26 (m, 6H), 2.14 (t, J = 7.5 Hz, 2H), 1.75 - 1.69 (m, 4H), 1.66 - 1.43 (m, 17H), 1.27 (d, J = 3.7 Hz, 78H), 0.94 - 0.87 (m, 18H).

[0497] Z. Compound 1115, synthesized according to general synthetic route 4

[0498] Structural formula:

[0499] Chemical formula: C 67 H 131 N3O7

[0500] Molecular weight: 1090.80

[0501] Synthesized according to general synthetic route 4, similar to the synthesis process of compound 1010, except that 2-bromoacetyl chloride and N,N-dimethylethylenediamine are used instead of compound 5 and compound 7 in general synthetic route 4 respectively. 1HNMR(400MHz,CDCl3)δ7.42(s,1H),4.48(tt,J=11.9,5.7Hz,6H),3.27(s,2H),3.13(s,2H),3.01(s,2H),2.83(s,6H),2.38 - 2.25

[0502] (m,3H),1.55(dtt,J=36.3,14.1,7.3Hz,12H),1.27(d,J=3.3Hz,78H),0.90(td,J=7.2,3.4Hz,18H).

[0503] AA. Compound 1114, synthesized according to General Synthetic Route 5

[0504] Structural formula:

[0505] Chemical formula: C 70 H 138 N2O7

[0506] Molecular weight: 1119.88

[0507] Steps 1 and 2: Synthesize Compounds 3 and 4 of General Synthetic Route 5

[0508]

[0509] Same as the synthesis process of Compound 1003 in Steps 1 and 2 of General Synthetic Route 1.

[0510] Step 3: Synthesize Compound 6 of General Synthetic Route 5

[0511]

[0512] Add Compound 4 (500 mg), intermediate M (Compound 5, 298.68 mg), reactant STAB (328.68 mg) and DCE (10 mL) to a three - necked flask. After stirring the reaction solution in N2 at 25 °C for 16 h, add 100 mL of water to the reaction solution, extract twice with EA (100 mL), combine the organic phases, concentrate, add silica gel and elute through a column with 1:20 DCM:MeOH, and collect the sample and concentrate to obtain Compound 6 (500 mg).

[0513] Step 4: Synthesize Compound 1114

[0514]

[0515] Compound 6 (450 mg) and solvent THF (5 mL) were added to a three-necked flask, and dioxane hydrochloride solution (1 M) (3.65 mL) was added. After stirring at 25 °C for 2 h, it was diluted with 50 mL of EA, and the organic phase was washed with 50 mL of saturated NaHCO3 solution and water. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated, and then subjected to silica gel column chromatography (MeOH:DCM 1:20) to obtain compound 1114 (107 mg, yield 24.84%). 1 H NMR (400 MHz, CDCl3) δ 4.16 - 4.07 (m, 6H), 3.60 (t, J = 4.8

[0516] Hz, 2H), 2.62 (t, J = 7.0 Hz, 2H), 2.49 (s, 3H), 2.31 (tt, J = 8.4, 5.5 Hz, 6H), 1.70 (d, J = 4.6 Hz, 3H), 1.64 - 1.39 (m, 18H), 1.27 (d, J = 4.0 Hz, 78H), 0.90 (t, J = 7.1 Hz, 18H).

[0517] Z. Compound 1120 was synthesized according to the general synthetic route 5

[0518] Structural formula:

[0519] Chemical formula: C 72 H 142 N2O7

[0520] Molecular weight: 1147.90

[0521] Synthesized according to the general synthetic route 5, similar to the synthesis process of compound 1114, except that intermediate C and 6-bromohexanal were used instead of compound 2 and compound 5 in the general synthetic route 5 respectively. 1H NMR (400 MHz, Chloroform-d) δ 4.11 (s, 6H), 3.63 (s, 2H), 2.57 (t, J = 6.8 Hz, 4H), 2.37 (ddd, J = 8.5, 7.1, 4.3 Hz, 5H), 1.67 - 1.41 (m, 24H), 1.27 (s, 79H), 0.90 (t, J = 6.7 Hz, 18H).

[0522] (2) Synthesis of the second ionizable lipid in Preparation Example 2

[0523] 1. Preparation of compound 6001

[0524] Structural formula:

[0525] Molecular weight: 499.43

[0526] Add 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide to 6.4 mL of ethanol, stir and heat to 70 °C. After 3.5 h, add an additional 0.8 g of hydroxyethyl acrylamide. Continue the reaction for 16 h and then pass through a reverse column to obtain Compound 6002.

[0527] Add 0.7 g of Compound 6002, 0.4 g of palladium on carbon, and 28 mL of tetrahydrofuran to a reaction flask and stir. Replace with hydrogen and maintain pressure with a hydrogen balloon. After 16 h, filter and pass through a normal phase column (methanol - DCM system) to obtain 200 mg of Compound 6001. 1 HNMR(400MHz,CDCl3)δ7.54(t,J=5.5Hz,2H),3.79 - 3.68(m,4H),3.42(dd,J=10.0,5.4Hz,4H),2.89(t,J=6.0Hz,4H),2.55(dt,J=11.8,6.8Hz,6H),1.54(s,2H),1.27(d,J=11.2Hz,32H),0.91(t,J=6.8Hz,3H).

[0528] 2. Preparation of Compound 6002

[0529] Structural formula:

[0530] Molecular weight: 497.42

[0531] Add 0.8 g of oleylamine and 0.8 g of hydroxyethyl acrylamide to 6.4 mL of ethanol, stir and heat to 70 °C. After 3.5 h, add an additional 0.8 g of hydroxyethyl acrylamide. Continue the reaction for 16 h, then concentrate the organic solvent, pass through a reverse column, and lyophilize to obtain a total of 400 mg of Compound 6002. 1 H NMR(400MHz,CDCl3)δ7.31(s,2H),5.38(dd,J=13.0,7.4Hz,2H),3.79 - 3.66(m,4H),3.41(dd,J=10.1,5.4Hz,4H),2.82 - 2.72(m,4H),2.44(dd,J=15.1,9.2Hz,6H),2.10 - 1.99(m,4H),1.46(s,2H),1.29(s,24H),0.90(t,J=6.8Hz,3H).

[0532] 3. Preparation of Compound 6003

[0533] Structural formula:

[0534] Molecular weight: 445.34

[0535] 4.00 g of tetradecylamine, 6.53 g of 2-hydroxyethyl acrylate and 80 mL of tert-butanol were added to a reaction flask, stirred and heated to 70 °C. After 29 h, the organic solvent was concentrated, passed through a reverse column, and freeze-dried to obtain 1.5 g of Compound 6003. 1 1H NMR (400 MHz, CDCl3) δ 4.29 (t, J = 7.5 Hz, 4H), 3.89 - 3.75 (m, 4H), 2.81 (t, J = 6.2 Hz, 4H), 2.50 (ddd, J = 23.2, 14.1, 7.1 Hz, 6H), 1.46 (s, 2H), 1.28 (s, 24H), 0.91 (t, J = 6.3 Hz, 3H).

[0536] 4. Preparation of Compound 6004

[0537] Structural formula:

[0538] Molecular weight: 553.48

[0539] 0.8 g of oleylamine, 1.5 g of n-butyl acrylate and 5.6 mL of n-butanol were added to a reaction flask, stirred and heated to 100 °C. After 4 h, 1 mL of n-butyl acrylate was added, and the reaction continued for 1 h. Then it was passed through a normal-phase column (petroleum ether - ethyl acetate system), 0.4 g of sodium hydroxide, 1 mL of water and 10 mL of methanol were added, and stirred for hydrolysis for 30 min. 1 mL of concentrated hydrochloric acid and 10 mL of methanol were added to the mixed solution to adjust the pH to neutral, evaporated to dryness, dissolved in DCM, dried over anhydrous magnesium sulfate, filtered, evaporated the solvent, 10 mL of DCM, 1.06 g of 4-amino-1-butanol, 0.81 g of HOBT and 2.3 g of EDCI were added, and reacted at room temperature for 18 h. After concentrating the organic solvent, it was passed through a reverse column and freeze-dried to obtain 240 mg of Compound 6004. 1 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 16.6 Hz, 2H), 5.44 - 5.31 (m, 2H), 3.72 (d, J = 16.5 Hz, 4H), 3.31 (d, J = 5.6 Hz, 4H), 3.17 (s, 4H), 2.84 (s, 2H), 2.69 (s, 4H), 2.09 - 2.01 (m, 4H), 1.67 (d, J = 2.7 Hz, 10H), 1.37 - 1.26 (m, 24H), 0.91 (t, J = 6.8 Hz, 3H).

[0540] 5. Preparation of Compound 6005

[0541] Structural formula:

[0542] Molecular weight: 597.54

[0543] Add 25.00 g of 11 - hentriacontanone, 62.05 g of ammonium acetate and 500 mL of methanol to a reaction flask and stir. Add 6.55 g of sodium cyanoborohydride. After reacting for 16 h, add 250 mL of water and 250 mL of DCM. Separate the layers. Extract the aqueous phase with 50 mL of DCM and combine the organic phases. Pass through a normal phase column (methanol - DCM system) to obtain 21.00 g of compound 6005 - A in total.

[0544] Add 21.00 g of compound 6005 - A, 58 mL of n - butyl acrylate and 100 mL of n - butanol latex to a reaction flask and stir. Heat the temperature to 100 °C. Add an additional 10 mL of n - butyl acrylate. After 16 h, pass through a column (petroleum ether - ethyl acetate system) to obtain compound 6005 - B

[0545] 31.00 g.

[0546] Prepare a solution by adding 31.00 g of compound 6005 - B, 6.55 g of sodium hydroxide, 310 mL of methanol and 31 mL of water to a reaction flask and stir. After 20 min, add 150 mL of THF. Stir and heat the temperature to 50 °C. After 30 min, add a solution prepared by mixing 16.13 g of concentrated hydrochloric acid and 160 mL of methanol and stir. After 30 min, evaporate the solvent by rotary evaporation. Add 300 mL of DCM to dissolve, dry with anhydrous magnesium sulfate, filter, and evaporate the solvent by rotary evaporation to obtain 27.00 g of an oily compound 6005 - C.

[0547] Add 27.00 g of compound 6005 - C, 25.00 g of 4 - amino - 1 - butanol, 41.85 g of EDCI, 14.74 g of HOBT and 310 mL of DCM to a reaction flask and stir. After 18 h, concentrate the organic solvent and pass through a column (methanol - DCM system) to remove the excess 4 - amino - 1 - butanol compound to obtain 6.80 g of compound 6005. 1 H NMR (400 MHz, CDCl3) δ 3.70 (t, J = 5.6 Hz, 4H), 3.28 (t, J = 5.6 Hz, 4H), 2.74 (t, J = 6.0 Hz, 4H), 2.40 - 2.45 (m, 1H), 2.34 (t, J = 6.4 Hz, 4H), 1.63 - 1.65 (m, 8H), 1.25 - 1.35 (m, 36H), 0.91 (t, J = 6.4 Hz, 6H).

[0548] 6. Preparation of Compound 6006

[0549] Structural formula:

[0550] Molecular weight: 553.48

[0551] 1.00 g of oleylamine, 0.47 g of hydroxyethyl acrylamide and 8 mL of ethanol were added to a reaction flask, and the mixture was stirred and heated to 70 °C. After 13 h, the solvent was evaporated and the residue was purified by column chromatography (methanol-DCM system) to obtain 800 mg of intermediate 1. 1.5 mL of n-butyl acrylate and 5 mL of n-butanol were added, and the mixture was stirred and heated to 100 °C. After 3 h, the solvent was evaporated and the residue was purified by column chromatography (methanol-DCM system). A solution prepared by adding 0.4 g of sodium hydroxide to a mixture of 10 mL of methanol and 1 mL of water was added, and the mixture was stirred for 1 h. Then, a solution prepared by adding 1 mL of concentrated hydrochloric acid to 8 mL of methanol was added, and the mixture was stirred for 30 min. The solvent was evaporated, and the residue was dissolved in 100 mL of DCM, dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated. 0.45 g of 6-amino-1-hexanol, 0.27 g of HOBT, 0.78 g of EDCI and 10 mL of DCM were added and stirred. After 14 h, the organic solvent was concentrated and the residue was purified by column chromatography (methanol-DCM system) to obtain 300 mg of compound 6006. 1 H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 6.95 (s, 1H), 5.43 - 5.28 (m, 2H), 3.74 - 3.67 (m, 2H), 3.64 (t, J = 6.3 Hz, 2H), 3.39 (dd, J = 10.0, 5.3 Hz, 2H), 3.24 (dd, J = 12.9, 6.7 Hz, 2H), 2.74 (t, J = 5.8 Hz, 4H), 2.48 - 2.42 (m, 2H), 2.41 - 2.33 (m, 4H), 2.01 (dd, J = 13.6, 6.8 Hz, 4H), 1.66 - 1.17 (m, 34H), 0.89 (t, J = 6.7 Hz, 3H).

[0552] 7. Compound 6007

[0553] Structural formula:

[0554] Molecular weight: 953.78

[0555]

[0556] At room temperature, add octadecylamine (50 g) to a three-necked flask. After adding MeOH (200 mL) and stirring to mix, cool the mixture to 5 °C in an ice-water bath. Then slowly add methyl acrylate (31.5 g) dropwise, stir to mix, and restore to room temperature. The reaction solution is reacted at room temperature for 4 h; concentrated under reduced pressure and purified by column chromatography (PE:EA = 20:1 - 10:1) to obtain product 2 (68 g). At room temperature, add product 2 (15.0 g), MeOH (100 mL), and ethylenediamine (1.0 g) to a single-necked flask, heat to 60 °C, and maintain this temperature to react overnight; concentrate under reduced pressure, add 30 mL of toluene, heat to 60 °C to dissolve, slowly cool to room temperature, and evaporate to dryness under reduced pressure to obtain compound 3 (15 g). At room temperature, add compound 3 (2 g) to a single-necked flask. After adding MeOH (20 mL) and stirring to dissolve, then slowly add methyl acrylate (2.5 g) and stir to mix. Heat to 60 °C and react overnight; stop the reaction, concentrate the reaction solution under reduced pressure, and purify by column chromatography (DCM:MeOH = 50:1 - 20:1) to obtain compound 4 (2.5 g). At room temperature, add compound 4 (2.3 g) and ethylenediamine (20 mL) to a single-necked flask and react overnight at room temperature; stop the reaction and directly concentrate under reduced pressure to obtain compound 6007 (24 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.87 (t, J = 6.6 Hz, 2H), 3.55 - 3.36 (m, 8H), 3.33 (s, 8H), 3.27 - 3.16 (m, 2H), 3.06 - 2.90 (m, 4H), 2.77 (t, J = 6.5 Hz, 4H), 1.90 (dtdd, J = 21.2, 14.0, 9.5, 6.9 Hz, 4H), 1.73 (p, J = 7.7 Hz, 4H), 1.49 (tdd, J = 9.2, 7.9, 7.1, 4.0 Hz, 4H), 1.31 (s, 28H), 0.92 (t, J = 6.8 Hz, 3H).

[0557] 8. Compounds 6008 - 6018

[0558] Synthesis of Compound 6009

[0559] Structural formula:

[0560] At room temperature, a solution of freshly recrystallized hexadecylamine (0.03 mol) in methanol (20 mL) was added dropwise, under a nitrogen atmosphere, to a stirred solution of methyl methacrylate (6 mL) in methanol (20 mL). The reaction was carried out overnight. The reaction mixture was rotary evaporated at room temperature, and the residue was dissolved in chloroform and washed twice with 0.1 M NaOH solution. The chloroform solution was collected and dried over anhydrous calcium chloride. Then, a colorless oil was obtained by column chromatography. 1H-NMR (300 MHz, CDCl3): 0.78 (t, 3H), 1.16 (s, 30H), 2.38 (m, 6H), 2.71 (t, 4H), 3.57 (m, 6H).

[0561] Then, a solution of the above-mentioned colorless oil (11.05 g) in methanol (20 mL) was added to a vigorously stirred solution of 1,2-diaminoethane (75 g) in methanol (100 mL) at room temperature. After complete addition, the mixture was stirred at room temperature for another 24 hours. The solvent was removed under reduced pressure, keeping the temperature not higher than 40 °C. The excess 1,2-diaminoethane was removed with an azeotropic mixture of toluene and methanol (9:1). The remaining toluene was removed by azeotropic distillation with methanol. Finally, a white powder (10.5 g) was obtained, which was recrystallized repeatedly from chloroform and cyclohexane to give a white solid. 1H-NMR (300 MHz, CDCl3): 0.88 (t, 3H), 1.25 (s, 30H), 1.84 (s, 4H), 2.38 (m, 6H), 2.73 (m, 4H), 2.82 (m, 4H), 3.29 (m, 4H), 7.47 (s, 2H).

[0562] The preparation methods of 6008 and 6009 are only different in that: equimolar amounts of tetradecylamine are used instead of hexadecylamine. For 6010 - 6017, referring to the preparation method of 6009, they are prepared by using equimolar amounts of the corresponding R a -NH2 to replace hexadecylamine.

[0563] For 6018, referring to the preparation method of 6007, it is prepared by using equimolar amounts of the corresponding R a -NH2 to replace octadecylamine.

[0564] 9. Compound 6019

[0565] Structural formula:

[0566] Molecular weight: 753.66

[0567]

[0568] At room temperature, compound 2483-46-7 (3 g) was added to a single-necked flask. After adding DCM (50 mL) and stirring to mix, 18807-71-1 (2.5 g), DCC (2.7 g), and DMAP (1.6 g) were added, and the mixture was stirred and mixed, and reacted overnight at room temperature; concentrated under reduced pressure and purified by column chromatography (DCM:MeOH = 5:1) to obtain product 3 (4 g). At room temperature, product 3 (5.0 g), MeOH (50 mL), and Pd / C (1.0 g) were added to a three-necked flask, the hydrogen was displaced, the temperature was raised to 50 °C, and stirred for 1 h; the reaction was stopped, the reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated by rotation to obtain product 4 (3.5 g). At room temperature, raw material 5 (1.2 g) was added to a single-necked flask. After adding DMF (50 mL) and stirring to dissolve, then product 4 (3.3 g), HATU (3.2 g), and TEA (0.9 g) were added and stirred and mixed, and reacted overnight at room temperature; water (500 mL) and EA (500 mL) were added, and liquid-liquid extraction was carried out. The organic phase was dried over Na2SO4, concentrated under reduced pressure, and the crude product was purified by column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain solid 6 (380 mg). At room temperature, solid 6 (380 mg) and HCl / Dioxane (4 M) (1.6 mL) were added to a single-necked flask, and reacted for 2 h at room temperature; concentrated under reduced pressure and purified to obtain compound 6019 (220 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.87 (t, J = 6.6 Hz, 2H), 3.55 - 3.36 (m, 8H), 3.33 (s, 8H), 3.27 - 3.16 (m, 2H), 3.06 - 2.90 (m, 4H), 2.77 (t, J = 6.5 Hz, 4H), 1.90 (dtdd, J = 21.2, 14.0, 9.5, 6.9 Hz, 4H), 1.73 (p, J = 7.7 Hz, 4H), 1.49 (tdd, J = 9.2, 7.9, 7.1, 4.0 Hz, 4H), 1.31 (s, 28H), 0.92 (t, J = 6.8 Hz, 3H).

[0569] 10. Compound 6020

[0570] Structural formula:

[0571] Molecular weight: 810.19

[0572]

[0573] At room temperature, add octadecylamine (50 g) to a three-necked flask. After adding MeOH (200 mL) and stirring to mix, cool the mixture to 5 °C in an ice-water bath. Then, dropwise add methyl acrylate (31.5 g), stir to mix, restore to room temperature, and react for 4 h. Stop the reaction, directly concentrate under reduced pressure, and purify by column chromatography (PE:EA = 20:1 - 10:1) to obtain product 2 (68 g). At room temperature, add product 2 (15.0 g), MeOH (100 mL), and ethylenediamine (1.0 g) to a single-necked flask, heat to 60 °C, and maintain this temperature to react overnight. Stop the reaction, concentrate the reaction solution under reduced pressure, add 30 mL of toluene, heat to 60 °C to dissolve, slowly cool to room temperature, filter, and evaporate to dryness under reduced pressure to obtain compound 3 (15 g). At room temperature, add compound 3 (1.3 g) to a single-necked flask. After adding DMF (20 mL) and stirring to mix, add 35897 - 34 - 8 (3.6 g), EDCI (3.0 g), HOBt (2.1 g), and DIEA (2.0 g), stir to mix, and react overnight at room temperature. Add water (200 mL) and directly lyophilize under reduced pressure to obtain compound 4 (4 g). At room temperature, add compound 4 (100 mg) and HCl / Dioxane (4 M) (2 mL) to a single-necked flask and react for 1 h at room temperature. Stop the reaction, concentrate and purify the reaction solution under reduced pressure to obtain compound 6020. 1H NMR (400 MHz, Methanol-d4) δ 3.90 (t, J = 6.4 Hz, 2H), 3.57 - 3.34 (m, 10H), 3.24 (dt, J = 16.1, 8.0 Hz, 8H), 2.77 (t, J = 6.5 Hz, 4H), 2.03 - 1.58 (m, 10H), 1.31 (s, 30H), 1.01 - 0.82 (m, 3H).

[0574] 11. Compound 6021

[0575] Structural formula:

[0576] Molecular weight: 1182.79

[0577]

[0578] At room temperature, add compound 2 (3.5 g, the synthesis process is the same as product 2 in compound 6020) into a single-necked flask. After adding MeOH (20 mL) and stirring to mix, add TREN (23.0 g), stir to mix, heat up to 60 °C, stir overnight, concentrate under reduced pressure, and lyophilize the obtained crude product to obtain product 3 (25 g); at room temperature, add product 3 (22.0 g) and DCM (200 mL) into a single-necked flask, cool down to 0 °C, slowly add (Boc)2O (71.7 g) dropwise, after dropping, restore to room temperature, react for 3 h, add water (200 mL) to quench, then add DCM (100 mL) for liquid-liquid extraction, dry the organic phase with sodium sulfate, concentrate under reduced pressure, and purify by column chromatography to obtain yellow oil 4 (5.8 g). At room temperature, add yellow oil 4 (1.4 g) into a single-necked flask, slowly add HCl / Dioxane (4M) (15 mL) under an ice-water bath and stir to mix. After adding, restore to room temperature, react for 0.5 h, concentrate under reduced pressure to obtain compound 5 (800 mg). At room temperature, add compound 5 (1.0 g), (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid (2.6 g), EDCI (1.4 g), HOBt (1.0 g), DIEA (1.0 g) and solvent DMF (10 mL) into a single-necked flask, react overnight at room temperature, add 100 mL of water and 100 mL of EA, perform liquid-liquid extraction, wash the EA phase once with 100 mL of saturated brine, concentrate under reduced pressure, and purify by column chromatography (DCM:MeOH = 10:1) to obtain compound 6 (1.1 g). At room temperature, add compound 6 (1.2 g), HCl / Dioxane (4M) (10 mL) and solvent DCM (10 mL) into a single-necked flask, react overnight at room temperature, concentrate under reduced pressure, and purify to obtain compound 6021 (220 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.96 (t, J = 6.6 Hz, 4H), 3.75 (dt, J = 14.0, 6.6 Hz, 4H), 3.62 - 3.45 (m, 12H), 3.36 - 3.33 (m, 14H), 3.25 - 3.17 (m, 2H), 3.04 - 2.90 (m, 8H), 2.83 (t, J = 6.9 Hz, 4H), 2.05 - 1.82 (m, 8H), 1.73 (p, J = 7.7 Hz, 10H), 1.53 (qd, J = 8.3, 7.7, 4.3 Hz, 8H), 1.30 (s, 28H), 0.97 - 0.87 (m, 3H).

[0579] 12. Compound 6023

[0580] Structural formula:

[0581] Molecular weight: 670

[0582]

[0583] At room temperature, add dodecylamine (10 g) to a three-necked flask. After adding MeOH (100 mL) and stirring to mix, cool the mixture to 5 °C in an ice-water bath, slowly dropwise add methyl acrylate (10.2 g), stir to mix, and then restore to room temperature. After the reaction solution reacts at room temperature for 4 h, stop the reaction, concentrate under reduced pressure, and purify by column chromatography (PE:EA = 20:1 - 10:1) to obtain product 2 (19 g). At room temperature, add product 2 (19.0 g), MeOH (200 mL), and ethylenediamine (127.7 g) to a single-necked flask, heat to 60 °C, and maintain this temperature to react overnight. Concentrate under reduced pressure to obtain compound 3 (20 g). Take 500 mg for purification and lyophilize to obtain compound 6023-1 (218 mg). At room temperature, add starting material 6023-1 (1.4 g) to a single-necked flask. After adding DCM (20 mL) and stirring to dissolve, then add (S)-2,6-di-tert-butoxycarbonylaminocaproic acid (3.4 g), EDCI (1.9 g), and DMAP (1.2 g), stir to mix, maintain at room temperature, and react overnight. Separate and extract, dry, and concentrate under reduced pressure. Obtain compound 4 (3.0 g). At room temperature, add compound 4 (2.8 g) and HCl / 1,4-Dioxane (4M) (20 mL), and react at room temperature for 2 h; concentrate under reduced pressure, purify, and lyophilize to obtain compound 6023 (123 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.88 (t, J = 6.6 Hz, 2H), 3.55 - 3.36 (m, 8H), 3.36 - 3.33 (m, 2H), 3.31 - 3.14 (m, 4H), 3.04 - 2.93 (m, 4H), 2.77 (t, J = 6.5 Hz, 4H), 2.00 - 1.82 (m, 4H), 1.82 - 1.66 (m, 6H), 1.50 (qd, J = 8.2, 7.8, 4.2 Hz, 4H), 1.45 - 1.25 (m, 18H), 0.96 - 0.87 (m, 3H).

[0584] Compound 6024

[0585] Structural formula:

[0586] Molecular weight: 870.24

[0587]

[0588] At room temperature, to a single-necked flask, add product 6023-1 (1.4 g, synthesized in the same method as 6023-1 in compound 6023). After adding MeOH (20 mL) and stirring until dissolved, slowly add methyl acrylate (2.8 g) and stir to mix. Heat up to 60 °C and react overnight at this temperature. Concentrate under reduced pressure to obtain compound 4 (2.0 g). At room temperature, add compound 4 (2.4 g) and ethylenediamine (20 mL) to a single-necked flask and react overnight at room temperature. Concentrate under reduced pressure and purify to obtain compound 6024 (191 mg). 1HNMR (400 MHz, Methanol-d4) δ 3.69 (t, J = 6.0 Hz, 4H), 3.60 - 3.48 (m, 20H), 3.42 (t, J = 6.1 Hz, 4H), 3.25 - 3.19 (m, 2H), 3.12 (t, J = 5.8 Hz, 8H), 2.83 (t, J = 6.5 Hz, 12H), 1.78 (tt, J = 11.0, 6.4 Hz, 2H), 1.51 - 1.18 (m, 20H), 0.91 (t, J = 6.7 Hz, 3H).

[0589] 14. Compound 6026

[0590] Structural formula:

[0591] Molecular weight: 443.67

[0592]

[0593] Compound 6026 was synthesized with reference to the synthetic route of compound 6019, except that: equimolar amounts of compound 1 were used to replace the raw material 5 of 6019, and equimolar amounts of N-(tert-butoxycarbonyl) ethanolamine were used to replace the intermediate 4 of compound 6019. 1HNMR (300 MHz, DMSO) δ: 4.51 (t, J = 7.3 Hz, 4H), 3.76 (t, J = 6.1 Hz, 4H), 3.18 (t, J = 6.7 Hz, 4H), 3.01 (t, J = 5.8 Hz, 2H), 2.49 (t, J = 7.1 Hz, 4H), 1.36 - 1.26 (m, 24H), 0.89 (t, J = 6.2 Hz, 3H).

[0594] Compounds 6025 - 6034 were obtained by referring to the synthetic routes of the aforementioned compounds, with the difference that hydroxyethylamine was used to replace the corresponding ethylenediamine, and the amino group of hydroxyethylamine was first protected with protecting groups such as Fmoc or Boc, and the protecting group was removed by conventional methods after the reaction was completed.

[0595] (3) Exemplary Compounds of the First Ionizable Lipids and Their Properties

[0596] Exemplary compounds of the present invention and their properties are listed in Table 1, which are synthesized according to the process routes in Preparation Example 1 respectively.

[0597] The calculated c-pKa (molnetwork) and LogP (cLogP driver) of the exemplary compounds of the present invention, as well as the c-pKa and cLogP values, are generated by the ChemDraw module of Chemoffice.

[0598] Properties of exemplary compounds of the first ionizable lipid of the present invention

[0599]

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608]

[0609]

[0610] The above properties of the exemplary compounds of the first ionizable lipid of the present invention indicate that they can be used as excellent surfactants and are particularly suitable for preparing lipid nanoparticles for use as drug delivery carriers.

[0611] (4) Preparation of transfection reagent based on blank lipid nanoparticles

[0612] 4.1 Preparation of blank lipid nanoparticles

[0613] Prescription Number Prescription Composition Prescription Dosage Molar Percentage % 1 Compound 1008 / DSPC / Chol / ALC-0159 40 / 20 / 39 / 1 2 Compound 6019 / Compound 1005 / DSPC / Chol / ALC-0159 5 / 25 / 15 / 54.5 / 0.5 3 Compound 6002 / Compound 1113 / DSPC / Chol / PEG-DMG 10 / 30 / 10 / 47 / 3 4 Compound 1001 / ALC-0315 / DSPC / Chol / ALC-0159 10 / 40 / 10 / 38.5 / 1.5 5 Compound 6008 / Compound 1009 / DSPC / Chol / PEG-DMG 25 / 25 / 10 / 39 / 1 6 Compound 1114 / DOPE / Chol / ALC-0159 50 / 10 / 38.5 / 1.5 7 Compound 1112 / DOPC / Chol / PEG-DMG 45 / 10 / 40 / 5 8 Compound 6021 / Compound 1009 / DOPE / Chol / ALC-0159 2.5 / 30 / 30 / 37 / 0.5 9 Compound 6018 / Compound 1115 / DOPE / Chol / PEG-DMG 5 / 45 / 10 / 39 / 1 10 Compound 1118 / POPC / Chol / PEG-DMG 40 / 20 / 38 / 2 11 Compound 1004 / ALC-315 / DSPC / Chol / ALC-0159 5 / 40 / 10 / 43.5 / 1.5 12 Compound 1006 / SM102 / DSPC / Chol / ALC-0159 20 / 30 / 10 / 38.5 / 1.5 13 Compound 6023 / Compound 1007 / DLin-KC2-DMA / DOPE / Chol / ALC-0159 2 / 5 / 30 / 10 / 53 / 2 14 Compound 1111 / cKK-E12 / DSPC / Chol / ALC-0159 5 / 30 / 15 / 49 / 1 15 Compound 1011 / DOPC / Chol / ALC-0159 35 / 20 / 44 / 1 16 Compound 6024 / Compound 1112 / POPC / Chol / ALC-0159 3 / 30 / 15 / 50 / 2

[0614] Blank lipid nanoparticles are prepared respectively according to the above molar ratios, and the specific method is as follows:

[0615] Dissolve the lipid raw materials in ethanol to obtain a lipid ethanol solution. The total concentration of all lipid raw materials in ethanol is 8 mg / mL. After mixing the lipid ethanol solution and 50 mM citrate buffer (pH 4.0) solution in a volume ratio of 1:3 in a nano-preparation device, ultrafiltration is carried out, and the sample is collected to obtain a lipid composition with a lipid concentration of 2 mg / mL.

[0616] 4.2 Preparation of transfection reagent based on blank lipid nanoparticles

[0617]

[0618] Using nucleic acid as the active ingredient, configure the nucleic acid into a solution with a concentration twice that of the nucleic acid in the transfection reagent in each example using nuclease-free water as Solution 1; dilute the blank lipid nanoparticles according to the prescription ratio of each example using nuclease-free water as Solution 2; mix Solution 1 and Solution 2 in equal volume and vortex for 2 - 3 s to obtain a transfection reagent based on blank lipid nanoparticles.

[0619] (5) Determination of particle size and Zeta potential of transfection reagent

[0620] 1. Determination of particle size and polydispersity index (PDI): Use a Malvern ZetaSizer Nano ZS90 to measure the average particle size and PDI of the nanoparticle sample solution in the examples by dynamic light scattering. The measurement angle is 90°, the refractive index of the dispersant is 1.330, and the test temperature is 25 °C.

[0621] 2. The encapsulation efficiency test method is as follows:

[0622] According to the manufacturer's instructions, use the Quant-it Ribogreen RNA quantification assay kit (ThermoFisher Scientific, UK) to determine the encapsulation efficiency of mRNA in the transfection reagent.

[0623] According to the manufacturer's instructions, use the dsDNA HS kit (Novizan / EQ121) to determine the encapsulation efficiency of pDNA in the transfection reagent.

[0624] According to the manufacturer's instructions, use the microRNA Reagent kit to determine the encapsulation efficiency of siRNA in the transfection reagent.

[0625] The average particle size, PDI, and encapsulation efficiency data of the transfection reagent prepared in the examples are shown in Table 2.

[0626] Table 2 Summary of particle size, PDI, and encapsulation efficiency of transfection reagent

[0627] Example Number Particle Size nm PDI Encapsulation Efficiency % 1 203.9 0.175 86.5 2 184.5 0.113 82.5 3 169.3 0.117 85.7 4 137.2 0.108 98.6 5 166.8 0.096 85.6 6 113.5 0.076 98.8 7 101.3 0.117 98.9 8 120.5 0.156 95.6 9 162.0 0.154 90.4 10 328.1 0.177 86.7 11 504.9 0.485 80.5 12 127.8 0.070 92.6 13 651.5 0.378 82.6 14 229.8 0.167 89.5 15 174.7 0.164 91.6 16 206.2 0.201 85.7 17 168.4 0.154 90.2 18 756.2 0.325 80.3 19 760.4 0.256 81.3 20 115.6 0.156 86.5

[0628] As can be seen from Table 2, the transfection reagent provided by the present invention has small and uniform particle size and high encapsulation efficiency (>80%).

[0629] (6) Evaluation of in vitro cell transfection effect of transfection reagent based on blank lipid nanoparticles

[0630] 6.1 Blank lipid nanoparticle-Luciferase pDNA complex

[0631] The in vitro transfection efficiency of the blank lipid nanoparticle-Luciferase pDNA complexes of Examples 1, 2, 4, 14, and 15 was evaluated using different cells (293T, Hela). The cells were routinely cultured in DMEM + 10% FBS medium to ensure that the cells were in the logarithmic growth phase; one day before transfection, they were seeded onto a 96-well culture plate at an appropriate cell density and grown overnight. At the time of transfection, the cell confluence should reach 70-90%; the blank lipid nanoparticle-nucleic acid complexes of each example were diluted with DMEM and added to the 96-well cell culture plate so that the dose per well reached 100 ng, and Lipofectamine 2000 transfected luciferase plasmid was used as the positive control (PC). After incubation in a 37 °C, 5% CO2 incubator for 24 h, a fluorescence substrate was added to the wells, and the luciferase activity was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The results are shown in Table 3. Figure 1 。

[0632] Table 3 Expression data of luciferase activity (RLU, dose: 100 ng) of blank lipid nanoparticle-Luciferase pDNA complexes in different cells

[0633] Cell Type Lipofectamine2000 Example 1 Example 2 Example 4 Example 14 Example 15 HEK293 1.55E+04 4.02E+06 4.09E+06 1.71E+06 3.55E+06 4.76E+06 Hela 3.92E+03 6.98E+05 4.72E+05 1.00E+05 4.70E+05 4.88E+05

[0634] The results show that the blank lipid nanoparticle-Luciferase pDNA complexes provided by the present invention have high luciferase expression efficiency in different cells, and the expression efficiency is significantly higher than that of the luciferase plasmid transfected with Lipofectamine 2000.

[0635] 6.2 Blank lipid nanoparticle-Luciferase mRNA complex

[0636] The in vitro transfection efficiency of the blank lipid nanoparticles-Luciferase mRNA complexes in Examples 5, 10, 16, 17, and 18 was evaluated using different cells (293T, Hela). The cells were routinely cultured in DMEM + 10% FBS medium to ensure that the cells were in the logarithmic growth phase; one day before transfection, they were seeded onto 96-well culture plates at an appropriate cell density and grown overnight. At the time of transfection, the cell confluence should reach 70-90%; the blank lipid nanoparticles-nucleic acid complexes of each example were diluted with DMEM and added to the 96-well cell culture plates so that the dose per well reached 100 ng, and the transfection of luciferase mRNA with Lipofectamine 2000 was used as a positive control (PC). After incubation in a 37 °C, 5% CO2 incubator for 24 h, a fluorescent substrate was added to the wells, and the luciferase activity was measured using a microplate reader. The results are shown in Table 4, Figure 2 。

[0637] Table 4 Expression data of luciferase activity (RLU, dose: 100 ng) of the blank lipid nanoparticles-Luciferase mRNA complexes in different cells

[0638]

[0639]

[0640] The results showed that the blank lipid nanoparticles-Luciferase mRNA provided by the present invention had a high luciferase expression efficiency in different cells, and the expression efficiency was significantly higher than that of the luciferase plasmid transfected with Lipofectamine 2000.

[0641] The in vitro transfection efficiency of the blank lipid nanoparticles-eGFP mRNA complexes in Examples 3, 9, and 11 was evaluated using different cells.

[0642] The cells (293T, Hela, HepG2) were routinely cultured in DMEM + 10% FBS medium to ensure that the cells were in the logarithmic growth phase; one day before transfection, they were seeded onto 96-well culture plates at an appropriate cell density and grown overnight. At the time of transfection, the cell confluence should reach 70-90%; the blank lipid nanoparticles-nucleic acid complexes of Examples 3, 9, and 11 were diluted with buffer and added to the 96-well cell culture plates so that the dose per well reached 100 ng, and the commercially available transfection reagents Lipofectamine MessengerMAX TM 、JetMESSENGER TM were used as control preparations. After incubation in a 37 °C, 5% CO2 incubator for 24 h, fluorescence photographs of the cells were taken 24 hours after transfection. The results are shown in Figures 3 - 5 。

[0643] The results show that the blank lipid nanoparticle - eGFP mRNA complex provided by the present invention has a high eGFP protein expression efficiency in different cells, and is better than the commercially available transfection reagent.

[0644] 6.3 Blank lipid nanoparticle - siRNA complex

[0645] The in vitro transfection efficiency of the blank lipid nanoparticle - siRNA - cy3 complex in Example 6 was evaluated using different cells (293T, TWO3, MCF7, Hela). The cells were routinely cultured in DMEM + 10% FBS medium to ensure that the cells were in the logarithmic growth phase; one day before transfection, they were seeded onto a 96 - well culture plate at an appropriate cell density and grown overnight. At the time of transfection, the cell confluence should reach 70 - 90%; the blank lipid nanoparticle - siRNA - cy3 complex in Example 6 was diluted with buffer and added to the 96 - well cell culture plate so that the dose per well reached 2.5 pmol, and Lipofectamine 2000 transfected siRNA - cy3 was used as a positive control (PC). Incubate in a 37°C, 5% CO2 incubator, and take fluorescence photos of the cells 6 hours after transfection. The results Figure 6 。

[0646] The results show that the blank lipid nanoparticle - siRNA - cy3 complex provided by the present invention has a high transfection efficiency in different cells, and the transfection efficiency is significantly higher than that of siRNA - cy3 transfected by Lipofectamine 2000.

[0647] The technical solution of the present invention is not limited to the technical means disclosed above, but also includes technical solutions composed of any combination of the above technical features. The above is the specific implementation manner of the present invention. It should be noted that for those of ordinary skill in the technical field of the present invention, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

Claims

1. A transfection reagent based on blank lipid nanoparticles, characterized in that, Comprising: (1) Blank lipid nanoparticles; (2) Biologically active ingredients; The composition of the blank lipid nanoparticles includes: 5 - 70 mol% of a first ionizable lipid, 0 - 30 mol% of a second ionizable lipid, 5 - 50 mol% of phospholipids, 10 - 70 mol% of cholesterol, and 0 - 15 mol% of polyethylene glycol-conjugated lipid; in the transfection reagent, the dosage of the biologically active ingredient is 0.1% - 50% (w / w) of the total amount of the blank lipid nanoparticles and the biologically active ingredient; The first ionizable lipid is selected from compounds having the general formula (1E) or pharmaceutically acceptable salts, stereoisomers, tautomers thereof; wherein, R1, R2, and R3 are each independently H, C 5-40 a straight-chain or branched-chain alkyl group, C 5-40 a straight-chain or branched-chain alkenyl group, C 5-40 a straight-chain or branched-chain alkynyl group, a 3- to 6-membered saturated or partially unsaturated cycloalkyl group having 1 to 3 side chains, or a 6- to 10-membered aromatic group having 1 to 3 side chains; the side chains are independently selected from C 10-30 a straight-chain or branched-chain alkyl group, C 10-30 a straight-chain or branched-chain alkenyl group, C 10-30 a straight-chain or branched-chain alkynyl group; provided that at most one of R1, R2, and R3 is H; M is selected from -NR4R5, a saturated or partially unsaturated 3 - 6-membered heterocyclic group containing at least one nitrogen atom, a 6 - 10-membered heteroaryl group containing at least one nitrogen atom, and the heterocyclic group and heteroaryl group are unsubstituted or substituted by one or more -OH, carboxyl, amino, oxo, or halogen; R4 and R5 are each independently H, C 1-6 a straight-chain or branched-chain alkyl group, C 2-6 a straight-chain or branched-chain alkenyl group or C 2-6 a straight-chain or branched-chain alkynyl group, wherein the C 1-6 a straight-chain or branched-chain alkyl group, C 2-6 a straight-chain or branched-chain alkenyl group or C 2-6 a straight-chain or branched-chain alkynyl group is unsubstituted or substituted by one or more -OH, carboxyl, amino, amide, amidino, guanidine or halogen groups; G1, G2, G3 are each independently -O-, -S-, -NR6-, -S-S-, -C(=O)-, -C(=S)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O)2O-, -S(=O)2O-, -OS(=O)2-, -S(=O)2-, -S(=O)2-NR6-, -NR6-S(=O)2-, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-; wherein each R6, R 13 is independently selected from H, hydroxy, C 1-30 a straight-chain or branched-chain alkyl or cycloalkyl, C 2-30 a straight-chain or branched-chain alkenyl; R9, R 10 each independently is H; X2 is selected from -O-, -S-, NR 16 -, -S-S-, -C(=O)-, -C(=O)O-, -OC(=O)-, -C(=O)NR 16 -, -NR 16 C(=O)-, -OC(=O)O-, -NR 16 C(=O)O-, -OC(=O)NR 16 -, -NR 16 C(=O)NR 17 -, -P(=O)(OR 16 )O-, -OP(=O)(OR 16 )-, or -OP(=O)(OR 16 )O-; R 11、 R 12 each independently is H, C 1-6 a linear or branched alkyl or cycloalkyl group; Each R 16 , R 17 are each independently selected from H, C 1-30 a linear or branched alkyl or cycloalkyl, C 2-30 a linear or branched alkenyl; Among them, the alkyl, cycloalkyl, and alkenyl groups described for R4, R5, R6, R 11 , R 12 , R 13 , R 16 , R 17 are unsubstituted or substituted by one or more groups selected from hydroxyl, mercapto, amino, substituted amino, and halogen; n is an integer from 2 to 6; k is an integer from 1 to 6; The salt does not include quaternary ammonium salts; The second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, tautomer thereof: wherein A1 is NH or O; R a selected from C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 cycloalkyl, C6-C 24 alkyl alcohol; the C6-C 24 alkyl, C6-C 24 alkenyl, C6-C 24 cycloalkyl, C6-C 24 alkyl alcohol is a straight-chain or branched-chain structure; R b and R c are each independently selected from C1-C 12 alkyl, C1-C 12 alkyl alcohol, alkylamine.

2. The transfection reagent according to claim 1, characterized in that, The biologically active ingredient includes at least one of nucleic acid and polypeptide.

3. The transfection reagent according to claim 2, characterized in that, The nucleic acid is DNA and / or RNA, including at least one of siRNA, miRNA, saRNA, sgRNA, dsRNA, shRNA, smRNA, ssRNA, mRNA, circRNA, snRNA, crRNA, IncRNA, snoRNA, piRNA, pDNA, ssDNA, circular or linear DNA, DNA microcircles, msDNA.

4. The transfection reagent according to claim 2, wherein The polypeptide is selected from polypeptides containing 2 - 50 amino acids.

5. The transfection reagent according to claim 1, wherein R1, R2, and R3 are independently of each other the following groups: wherein, Y does not exist or is C 1-30 a linear or branched alkyl or cycloalkyl group, C 2-20 a linear or branched alkenyl group, C 2-20 a linear or branched alkynyl group; R1' and R2' are each independently H, C 1-30 a linear or branched alkyl group, C 2-30 a linear or branched alkenyl group, C 2-30 a linear or branched alkynyl group, and the total carbon chain length of Y, R1' and R2' is 8 - 40.

6. The transfection reagent according to claim 1, wherein R1, R2, and R3 are independently of each other selected from the following groups: wherein, R1’ and R2’ are each independently H, C 1-30 a linear or branched alkyl group, C 2-30 a linear or branched alkenyl group, C 2-30 a linear or branched alkynyl group, and the total carbon chain length of R1’ and R2’ is 8 - 30.

7. The transfection reagent according to claim 1, wherein R1, R2, and R3 are independently of each other selected from any one of the following groups:

8. The transfection reagent according to claim 1, characterized in that, G1, G2, G3 are each independently -O-, -S-, -NR6-, -S-S-, -C(=O)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR6-, -NR6C(=O)-, -OC(=O)O-, -NR6C(=O)O-, -OC(=O)NR6-, -NR6C(=O)NR 13 -, -P(=O)(OR6)O-, -OP(=O)(OR6)- or -OP(=O)(OR6)O-.

9. The transfection reagent according to claim 1, wherein M is selected from the following structures: wherein, m' and n' are independently integers from 0 to 6, and R1" and R2" are independently H, C 1-6 alkyl, C 2-6 alkenyl, guanidyl, amidino, amido, aliphatic amine group, 3- to 10-membered nitrogen-containing heterocycle; the nitrogen-containing heterocycle is selected from pyrrole, imidazole, pyridine, pyrazole, triazole, oxazole, isoxazole, thiophene, isothiazole, pyridazine, pyrazine, piperazine, indole, benzimidazole, carbazole, quinoline, isoquinoline, purine, and pyrimidine and their tautomeric forms, which are unsubstituted or optionally substituted by one or more organic groups selected from hydroxyl, mercapto, amino, substituted amino, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 6-14 aryl.

10. The transfection reagent according to claim 1, characterized in that, The compound of formula (1E) is selected from the compounds shown in formula (1F):

11. The transfection reagent according to claim 1, wherein The compound of formula (1E) is selected from the compounds shown in formula (1G):

12. The transfection reagent according to claim 1, wherein The compound of formula (1E) is selected from the compounds shown in formula (1H):

13. The transfection reagent according to claim 1, wherein The compound of formula (1) is selected from:

14. The transfection reagent according to claim 1, characterized in that, In the formula (2), the alkylamine is wherein R a ’ is a C1-C 12 alkyl group, and the R b ’ and R b ” are each independently selected from H, C1-C6 alkylamines, R c ” is selected from a C1-C6 alkyl group which is unsubstituted or substituted by an amino group, R c ”’ is H, or -R c ’-A1’-R c ”-NH2; Provided that when A1’ is -CO-NH-, -NH-CO- or -CO-O-, R c ’ is a C1-C6 alkyl; when A1’ is -CO-, R c ’ does not exist.

15. The transfection reagent according to claim 1, wherein The compound of formula (2) is selected from at least one of the following compounds:

16. The transfection reagent according to any one of claims 1-15, characterized in that, The transfection reagent further includes 0 - 60 mol% of other ionizable lipids, and the other ionizable lipids are selected from at least one of the following compounds:

17. The transfection reagent according to claim 1, wherein The phospholipids include at least one of 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-distearoyl-sn-glycero-3-phosphocholine, 1,2-dicumyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-0-octadecenoyl-sn-glycero-3-phosphocholine, 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt, dipalmitoyl phosphatidylglycerol, palmitoyl oleoyl phosphatidylethanolamine, distearoyl-phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dimyristoyl phosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine.

18. The transfection reagent according to claim 1, wherein The polyethylene glycol-conjugated lipids include at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.

19. The transfection reagent according to claim 18, wherein, The PEG-conjugated lipids include at least one of PEG-distearyloxypropyl, PEG-c-DOMG, PEG-DPPC, polyethylene glycol dimethacrylate, 1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol, dipalmitin-polyethylene glycol, 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol, 2-[(polyethylene glycol)-2000]-N,N-tetracosanoylacetamide, dipalmitoylphosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, dilauroylphosphatidylethanolamine-polyethylene glycol, and dimyristoylphosphatidylethanolamine-polyethylene glycol lipids.

20. The transfection reagent according to claim 1, characterized in that, In the transfection reagent, the nucleic acid molecule is encapsulated inside the lipid nanoparticles and / or adsorbed on the surface of the lipid nanoparticles to form a complex.

21. The preparation method of the transfection reagent according to any one of claims 1-20, characterized in that, It includes the step of mixing blank lipid nanoparticles and nucleic acid in a solvent.

22. The preparation method according to claim 21, characterized in that, After mixing, the concentration of the nucleic acid is 5-1000 ng / μl.

23. The preparation method according to claim 21, characterized in that, The solvent is at least one of water, an aqueous solution of an organic solvent, and a buffer salt solution.

24. The preparation method according to claim 23, wherein The buffer salt solution has a pH of 1-9, the buffer salt concentration is 0.1-200 mM, and the aqueous solution of the organic solvent is an alcohol solution with a volume concentration <50%.

25. The preparation method according to claim 24, characterized in that, The buffer salt solution is selected from at least one of citrate solution, acetate solution, tartrate solution, phosphate solution, carbonate solution, Tris-HCl solution, and sodium chloride solution.

26. The preparation method according to claim 1, characterized in that, At least one of a culture medium, sugar, glycerol, DMSO, salt, antibiotic, and surfactant is further added to the transfection reagent.

27. Use of the transfection reagent according to any one of claims 1-20 in in vitro transfection.

28. An in vitro transfection method, characterized in that, Mix the transfection reagent according to any one of claims 1-20 with cells for transfection.

29. The in vitro transfection method according to claim 28, wherein The cells include eukaryotic cells and / or prokaryotic cells; the prokaryotic cells are bacterial cells; the eukaryotic cells are at least one of animal cells, plant cells, algal cells, and fungal cells.

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