Blank lipid nanoparticle-based transfection reagent, its preparation method and application

Abstract

The present invention provides a transfection reagent based on blank lipid nanoparticles, a preparation method thereof and applications, belonging to the technical field of biomedicine. 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 includes: 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 based on blank lipid nanoparticles is prepared from blank lipid nanoparticles and nucleic acids, can flexibly adjust the dosage according to the needs of users, has a stable transfection effect, can transfect a variety of cells (such as 293T, Hela, HepG2, TWO3, MCF7), has low cytotoxicity and good transfection effect.

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 highly efficient gene delivery agents.

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

[0005] The working principle of these commercially available transfection reagents generally involves using cationic polymers or cationic lipids to bind to anionic substances such as nucleic acids, transporting the nucleic acid substances into cells, and sometimes other reagents (e.g., peptides) must be added as adjuvants to achieve 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, including 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 achieve effective delivery of 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 prepare 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. Compared with commercially available transfection reagents, it has better transfection effects. 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, a preparation method thereof and an 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 includes: 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 the blank lipid nanoparticles and nucleic acids, and the dosage can be flexibly adjusted according to the needs of users. Moreover, the transfection effect is stable, and it can transfect a variety of cells (such as 293T, Hela, HepG2, TWO3, MCF7) with low cytotoxicity and good transfection effects. In cell transfection, compared with commercially available transfection reagents, it has better transfection effects.

[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 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 lipids; in the transfection reagent, the dosage of the biologically active components is 0.1%-50% (w / w) of the total amount of the blank lipid nanoparticles and the biologically active components.

[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 microcircles, and msDNA.

[0013] In a preferred embodiment, the polypeptide is selected from polypeptides containing 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, R 1 , R 2 , R 3 are independently of each other H, C 5-40 linear or branched alkyl, C 5-40 linear or branched alkenyl, C 5-40 linear or branched alkynyl, 3-6 membered saturated or partially unsaturated cycloalkyl group having 1-3 side chains, or 6-10 membered aromatic group having 1-3 side chains; the side chains are independently selected from C 10-30 linear or branched alkyl, C 10-30 linear or branched alkenyl, C 10-30 linear or branched alkynyl; provided that R 1 , R 2 , R 3 are at most 1 H;

[0017] M is selected from -NR 4 R 5 , saturated or partially unsaturated 3-6 membered heterocyclic group containing at least one nitrogen atom, 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;

[0018] R 4 、R 5 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, and the C 1-6 straight-chain or branched-chain alkyl group, C 2-6 straight-chain or branched-chain alkenyl group or C 2-6 straight-chain or branched-chain alkynyl group is unsubstituted or substituted with one or more -OH, carboxyl, amino amide, amidino, guanidine or halogen groups;

[0019] G 1 、G 2 、G 3 are each independently -O-, -S-, -NR 6 -, -S-S-, -C(=O)-, -C(=S)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR 6 -, -NR 6 C(=O)-, -OC(=O)O-, -NR 6 C(=O)O-, -OC(=O)NR 6 -, -NR 6 C(=O)NR 13 -, -C(=O)S-, -C(=S)S-, -SC(=S)-, -SC(=O)-, -OC(=O)S-, -SC(=O)O-, -SC(=O)S-, -OS(=O) 2 O-, -S(=O) 2 O-, -OS(=O) 2 -, -S(=O) 2 -, -S(=O) 2 -NR 6 -, -NR 6 -S(=O) 2 -, -P(=O)(OR 6 )O-, -OP(=O)(OR 6 )-, or -OP(=O)(OR 6 )O-; where each R 6 、R 13 is independently selected from H, hydroxyl, C 1-30 a straight-chain or branched-chain alkyl or cycloalkyl group, C 2-30 a straight-chain or branched-chain alkenyl group;

[0020] L 1 is selected from -X 1 - or -(CR 7R 8 ) m -X 1 -, where each X 1 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) 2 O-, -S(=O) 2 O-, -OS(=O) 2 -, -S(=O) 2 -, -S(=O) 2 -NR 14 -, -NR 14 -S(=O) 2 -, -P(=O)(OR 14 )O-, -OP(=O)(OR 14 )-, or -OP(=O)(OR 14 )O-; where m is an integer from 2 to 6, and R 7 , R 8 are independently H, hydroxyl, halogen, a linear or branched alkyl or cycloalkyl of C 1-6 , a linear or branched alkenyl of C 2-6 , and each R 14 , R 15 are independently selected from H, a linear or branched alkyl or cycloalkyl of C 1-30 , a linear or branched alkenyl of C 2-30 ;

[0021] L 2 is -(CR 9 R 10 ) n - or -(CR 9 R 10 ) n -X 2 -(CR 11 R 12 ) k -, where X 2Selected 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) 2 O-, -S(=O) 2 O-, -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 from 1 to 6; k is an integer from 1 to 6; R 9 , R 10 , R 11 , R 12 are independently of each other H, hydroxy, halogen, a straight-chain or branched-chain alkyl or cycloalkyl of C 1-6 , a straight-chain or branched-chain alkenyl of C 2-6 , each R 16 , R 17 are independently of each other selected from H, a straight-chain or branched-chain alkyl or cycloalkyl of C 1-30 , a straight-chain or branched-chain alkenyl of C 2-30 ;

[0022] wherein the alkyl, cycloalkyl, alkenyl as described for R 4 to R 17 is unsubstituted or substituted by one or more groups selected from hydroxy, mercapto, amino, substituted amino, halogen;

[0023] The salt does not include quaternary ammonium salts.

[0024] In a preferred embodiment, the R 1 , R 2 , R 3 are independently of each other the following groups:

[0025]

[0026] Among them, Y does not exist, or is C 1-30 Straight-chain or branched-chain alkyl or cycloalkyl, C 2-20 Straight-chain or branched-chain alkenyl, C 2-20 Straight-chain or branched-chain alkynyl; R 1 ’, R 2 ’ are independently of each other H, C 1-30 Straight-chain or branched-chain alkyl, C 2-30 Straight-chain or branched-chain alkenyl, C 2-30 Straight-chain or branched-chain alkynyl, and the total carbon chain length of Y, R 1 ’ and R 2 ’ is 8 - 40.

[0027] In a preferred embodiment, the R 1 , R 2 , R 3 are independently of each other selected from the following groups:

[0028]

[0029] Among them, R 1 ’, R 2 ’ are independently of each other H, C 1-30 Straight-chain or branched-chain alkyl, C 2-30 Straight-chain or branched-chain alkenyl, C 2-30 Straight-chain or branched-chain alkynyl, and the total carbon chain length of R 1 ’ and R 2 ’ is 8 - 30.

[0030] In a preferred embodiment, the R 1 , R 2 , R 3 are independently of each other selected from any one of the following groups:

[0031]

[0032] In a preferred embodiment, G 1 , G 2 , G 3 are independently of each other -O-, -S-, -NR 6 -, -S-S-, -C(=O)-, -C(=O)O-, -CH(OH)-, -OC(=O)-, -C(=O)NR 6 -, -NR 6 C(=O)-, -OC(=O)O-, -NR 6 C(=O)O-, -OC(=O)NR 6 -, -NR6 C(=O)NR 13 -, -P(=O)(OR 6 )O-, -OP(=O)(OR 6 )- or -OP(=O)(OR 6 )O-.

[0033] In a preferred embodiment, L 1 is selected from -(CR 7 R 8 ) m -X 1 -, where X 1 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-.

[0034] In a preferred embodiment, L 2 is -(CR 9 R 10 ) n -X 2 -(CR 11 R 12 ) k -, where X 2 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-.

[0035] In a preferred embodiment, M is selected from the following structures:

[0036]

[0037] wherein m' and n' are independently integers from 0 to 6, R 1 ” and R 2 ” 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 hydroxyl, mercapto, amino, substituted amino, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 6-14 aryl.

[0038] In a preferred embodiment, the compound of formula (1) is selected from the compounds shown in formula (1A):

[0039]

[0040] In a preferred embodiment, the compound of formula (1) is selected from the compounds shown in formula (1B):

[0041]

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

[0043]

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

[0045]

[0046] In a preferred embodiment, the compound of formula (1) is selected from the compounds shown in formula (1E)

[0047]

[0048] In a preferred embodiment, the compound of formula (1) is selected from the compounds shown in formula (1F):

[0049]

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

[0051]

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

[0053]

[0054] In a preferred embodiment, M is selected from any one of the following groups:

[0055]

[0056] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1I):

[0057]

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

[0059]

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

[0061]

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

[0063]

[0064] wherein R 1 ’, R 2 ’ are independently selected from H, C 1-30 linear or branched alkyl, C 2-30 linear or branched alkenyl, C 2-30 linear or branched alkynyl, and the total carbon chain length of R 1 ’ and R 2 ’ is 8 - 40.

[0065] In a preferred embodiment, the compound of formula (1) is selected from the compounds represented by formula (1M):

[0066]

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

[0068]

[0069]

[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]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] 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):

[0146]

[0147] wherein Xa and Xb are groups containing a leaving group or a nucleophilic group, and Xa and Xb form L through a nucleophilic reaction or a condensation reaction 1 .

[0148] 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):

[0149]

[0150] wherein Xc and Xd are groups containing a leaving group or a nucleophilic group, and Xc and Xd form L through a nucleophilic reaction or a condensation reaction 2 .

[0151] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the step of reacting a compound of formula (VI):

[0152]

[0153] 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.

[0154] In a preferred embodiment, the method for preparing the first ionizable lipid comprises the steps 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):

[0155]

[0156] wherein Xg, Xh, Xi, Xj, Xk, Xl are groups containing a leaving group or a nucleophilic group, and Xg and Xj form G through a nucleophilic reaction or a condensation reaction 1 , Xh and Xk form G through a nucleophilic reaction or a condensation reaction 2 , Xi and Xl form G through a nucleophilic reaction or a condensation reaction 3 .

[0157] 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):

[0158]

[0159] The step of reacting a compound of formula (XIII) with a compound of formula (XII):

[0160]

[0161] wherein Xm is a group containing a nucleophilic group, and Xm and C═C-L of the compound of formula (XII) 2a form X through an addition reaction 1 -L 2 .

[0162] In a preferred embodiment, R 1 , R 2 , R 3 are, independently of one another, the following groups:

[0163]

[0164] wherein Y, R 1 ’, R 2 ’ have the same meanings as defined above;

[0165] which further comprises the step of forming the tail chains R 1 , R 2 , R 3 :

[0166]

[0167] wherein X is a leaving group.

[0168] In a preferred embodiment, the second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, or tautomer thereof:

[0169]

[0170] wherein N 1 is NH or O;

[0171] R a is selected from C 6 -C 24 alkyl, C 6 -C 24 alkenyl, C 6 -C 24 cycloalkyl, C 6 -C 24 alcohol, C 6 -C24 Short-chain polyoxyethylene; the C 6 -C 24 alkyl, C 6 -C 24 alkenyl, C 6 -C 24 cycloalkyl of, C 6 -C 24 alcohol, C 6 -C 24 The short-chain polyoxyethylene is a straight-chain or branched-chain structure; R b and R c are each independently selected from C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 1 -C 12 alkynyl, C 3 -C 12 cycloalkane group, C 6 -C 12 aryl group of, C 1 -C 12 alkyl alcohol, C 1 -C 12 heterocyclic group, alkylamine;

[0172] The alkylamine is wherein, R a ’ is C 1 -C 12 alkyl, the R b ’ and R b ” are each independently selected from H, C 1 -C 6 alkylamine, R c ” is selected from C 1 -C 6 alkyl which is unsubstituted or substituted by an amino group, R c ”’ is H, or -R c ’-A 1 ’-R c ”-NH 2 ;

[0173] Provided that when A 1 ’ is -CO-NH-, -NH-CO- or -CO-O-, R c ’ is C 1 -C 6 alkyl; when A 1 ’ is -CO-, R c ’ does not exist.

[0174] In a preferred embodiment, R b and R c are each independently selected from C 1 -C 12 alkyl alcohols, alkyl amines.

[0175] In a preferred embodiment, the condition is that: when R a is C 6 -C 24 alkenyl, A 1 is NH, R b and R c are each independently selected from C 1 -C 12 alkyl alcohols;

[0176] When R a is branched C 6 -C 24 alkyl, A 1 is NH, R b and R c are each independently selected from C 1 -C 12 alkyl alcohols, alkyl amines;

[0177] R a is linear C 6 -C 24 alkyl, A 1 is NH or O, R b and R c are each independently selected from alkyl amines;

[0178] When R a is linear C 6 -C 24 alkyl, A 1 is O, R b and R c are each independently selected from C 1 -C 12 alkyl alcohols;

[0179] When R a is C 6 -C 24 short chain polyoxyethylene, A 1 is NH or O, R b and R c are each independently selected from alkyl amines;

[0180] Or when R a is C 6 -C 24 alkyl alcohol, A 1 is NH or O, R b and R cEach independently selected from alkylamines.

[0181] In a preferred embodiment, R a is selected from the following compound structures:

[0182]

[0183] In a preferred embodiment, R b and R c are selected from the following compound structures:

[0184]

[0185] R d is selected from C1-C6 alkanes or cycloalkanes.

[0186] In a preferred embodiment, the compound of formula (2) is selected from at least one of the following compounds:

[0187]

[0188]

[0189]

[0190] In a preferred embodiment, the method for preparing the second ionizable lipid comprises the following reaction steps:

[0191] R a ”-NH 2 ① reacts with an α,β-unsaturated carbonyl compound ② to form an ionizable lipid compound ③:

[0192]

[0193] wherein, R a ”-NH 2 is R a -NH 2 or

[0194] R b ’ and R b ” are both H, or R b ’ and R b ” are both C 1 -C 6 amine, or R b ’ and R b ” are both -R c ’-A 1 ’-R c ”-NH 2 .

[0195] In a preferred embodiment, the method for preparing the second ionizable lipid comprises:

[0196] 1) R a ”-NH 2 ① reacts with the α,β-unsaturated carbonyl compound ④ to form the compound ⑤;

[0197] 2) The compound ⑤ reacts with the nucleophile ⑥ to form the ionizable lipid compound ③;

[0198]

[0199] wherein the nucleophile ⑥ is R b -NH 2 or R b -OH; R a ”-NH 2 is selected from R a -NH 2 or

[0200]

[0201] R b ’ and R b ” are both H, or R b ’ and R b ” are both C 1 -C 6 amine, or R b ’ and R b ” are both -R c ’-A 1 ’-R c ”-NH 2 ; Z 2 is a leaving group, and Z 2 reacts with NH 2 to obtain A 1 .

[0202] In a preferred embodiment, the method for preparing the second ionizable lipid comprises:

[0203] 1) R a ”-NH 2 ① reacts with the α,β-unsaturated carbonyl compound ② to form the compound ⑦;

[0204] 2) The compound ⑦ reacts with the α,β-unsaturated carbonyl compound ⑧ to form the compound ⑨;

[0205] 3) The compound ⑨ reacts with the nucleophile ⑩ to form the ionizable lipid compound

[0206]

[0207] Among them, R a ”-NH 2 is R a -NH 2 , Z 3 is a leaving group, A 3 reacts with Z 3 to obtain A 1 .

[0208] 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 a bifurcated structure containing a carbon-carbon bond, a carbon-oxygen bond, a carbon-nitrogen bond, a carbon-sulfur bond or a carbon-selenium bond.

[0209] 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;

[0210] The compound of Formula (2) with a terminal amino group is

[0211] Among them, R b ’ and R b ” are both H at the same time, or R b ’ and R b ” are both C 1 -C 6 amine at the same time, or R b ’ and R b ” are both -R c ’-A 1 ’-R c ”-NH 2 .

[0212] 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; ;

[0213] The compound of Formula (2) with a terminal amino group is

[0214] Among them, R b ’ and R b ” are both H at the same time, or R b ’ and R b ” are both C 1 -C 6 amine at the same time, or R b ’ and Rb ”Simultaneously for -R c ’-A 1 ’-R c ”-NH 2 。

[0215] The leaving group described above refers to the leaving part in a nucleophilic reaction or a condensation reaction, including but not limited to: H,

[0216] OH, H 2 O, halogen (such as F, Cl, Br and I), cyanate anion, inorganic acid (such as nitric acid, sulfuric acid, phosphoric acid), carboxylic acid (such as acetic acid, trifluoroacetic acid and benzoic acid, etc.), sulfonic acid (such as methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and p-nitrobenzenesulfonic acid, etc.), carbon dioxide (CO 2 )), nitrogen (N 2 ), imidazole, alkoxy (R-O-), amino group (-NHR, where R is an alkyl or aryl group from which H is removed), phenoxy group, tertiary carbocation (such as tert-butyl cation), carbocation stabilized by an unsaturated system or a heteroatom, or various protecting groups described above.

[0217] The nucleophilic group described above refers to a molecule or ion that can provide an electron pair to form a new chemical bond in a chemical reaction. Common nucleophilic groups include: hydroxide (HO - ), ammonia (NH 3 ), hydroxylamine (NH 2 OH), hydrazine (NH 2 -NH 2 ), substituted hydrazine, nucleophilic halogen (such as Cl - , Br - or I-), hydride ion (H-), azide anion (N 3 - ), cyanate anion (CN-), alcohol or alkoxide anion (such as alcohol from which the hydroxyl hydrogen is removed), amino group (including primary amine, secondary amine and tertiary amine) or amine anion, carbanion (such as the carbanion in organometallic reagents such as Grignard reagent, organolithium reagent, Gilman reagent, etc.), mercapto group or mercapto anion, thioether, enol or enolate anion, vinyl ether, enamine, carboxylic acid or carboxylate anion, alkyl or aryl phosphine (such as triphenylphosphine), heteroaromatic ring with lone pair electrons (such as pyridine), etc.

[0218] In a preferred embodiment, the raw materials used in the reaction process further contain protecting groups, and the reaction steps include protection and / or deprotection steps.

[0219] 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:

[0220]

[0221]

[0222]

[0223]

[0224]

[0225] 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-diundecanoyl-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).

[0226] 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.

[0227] 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.

[0228] 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), dipalmitoyl glycerol-polyethylene glycol (DPG-PEG), 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol (DSG-PEG), 2-[(polyethylene glycol)-2000]-N,N-tetracosanoyl acetamide (ALC-0159), dipalmitoyl phosphatidylethanolamine-polyethylene glycol (DPPE-PEG), distearoyl phosphatidylethanolamine-polyethylene glycol (PEG-DSPE), dilauroyl phosphatidylethanolamine-polyethylene glycol (PEG-DLPE), and dimyristoyl phosphatidylethanolamine-polyethylene glycol (PEG-DMPE) lipids.

[0229] 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.

[0230] 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.

[0231] In a preferred embodiment, the mixing methods include manual mixing, vortex mixing, stirring mixing, and microchannel mixing.

[0232] In a preferred embodiment, the concentration of the nucleic acid after mixing is 5-1000 ng / μl.

[0233] In a preferred embodiment, the solvent is at least one of water, an aqueous solution of an organic solvent, and a buffer salt solution.

[0234] 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%.

[0235] In a preferred embodiment, the alcohol is ethanol.

[0236] 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.

[0237] 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.

[0238] In a third aspect, the present invention provides the application of the aforementioned transfection reagent based on blank lipid nanoparticles in in vitro transfection.

[0239] In a preferred embodiment, the application in in vitro transfection is the application in the preparation of products for in vitro transfection.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] In a preferred embodiment, the survival rate of the cells is ≥80%.

[0244] Compared with the prior art, the present invention has the following beneficial effects:

[0245] 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 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.

[0246] 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 a stable transfection effect.

[0247] 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

[0248] Figure 1 is the luciferase activity of the blank lipid nanoparticle - Luciferase pDNA complex in different cells;

[0249] Figure 2 is the luciferase activity of the blank lipid nanoparticle - Luciferase mRNA complex in different cells;

[0250] Figure 3 is the transfection of the blank lipid nanoparticle - eGFP mRNA complex in 293T cells;

[0251] Figure 4 is the transfection of the blank lipid nanoparticle - eGFP mRNA complex in Hela cells;

[0252] Figure 5 is the transfection of the blank lipid nanoparticle - eGFP mRNA complex in HepG2 cells;

[0253] Figure 6 is the transfection of the blank lipid nanoparticle - siRNA - cy3 complex in different cells. DETAILED DESCRIPTION OF THE INVENTION

[0254] The synthetic 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 the compound may need to be further converted into its pharmaceutically acceptable salt. The compounds of the present invention can be prepared in various ways using commercially available starting materials, compounds known in the literature, or intermediates that are easily prepared, by employing 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 and 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.

[0255] 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 illustrated in the corresponding general synthetic routes. The variables in each general synthetic route (such as R1 , R 2 and R 3 etc.) 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.

[0256] 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.

[0257] (1) Synthesis of the first ionizable lipid in Preparation Example 1

[0258] The general synthetic route of the first ionizable lipid is as in General Synthetic Route 1-5.

[0259] General Synthetic Route 1

[0260]

[0261] Wherein, M' is M or M with a protecting group.

[0262] 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.

[0263] 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).

[0264] 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).

[0265] If the M' 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 reaction conditions for the removal of the selected protecting group.

[0266] General Synthetic Route 2

[0267]

[0268] Wherein M' is M or M with a protecting group; A is O, NH or S.

[0269] As described in the 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.

[0270] Then, compound 3 is deprotected with a tert-butoxy protecting 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)).

[0271] 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.

[0272] If the M' 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.

[0273] General synthetic route 3

[0274]

[0275] Wherein, X is a halogen, such as Cl, Br or I; R 4 ’ is R 4 or R with a protecting group 4 ; R 5 ’ is R 5 or R with a protecting group 5 .

[0276] As described in the 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.

[0277] Then, compound 3 is deprotected with a Boc protecting 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).

[0278] Next, 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 ))

[0279] Next, 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., K 2 CO 3 )) and a catalyst (KI or NaI).

[0280] If there are the above-mentioned protecting groups in the R 4 ’ and / or R 5 ’ 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.

[0281] General synthetic route 4

[0282]

[0283] Among them, X is a halogen, such as Cl, Br or I; R 4 ’ is R 4 or R 4 containing a protecting group; R 5 ’ is R 5 or R 5 containing a protecting group.

[0284] As described in the above general synthetic route 4, 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.

[0285] Next, 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).

[0286] Next, 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.

[0287] 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., K 2 CO 3 )) and a catalyst (KI or NaI).

[0288] If the R 4 ’ and / or R 5 ’ 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.

[0289] General synthetic route 5

[0290]

[0291] Wherein M pr o is M or M with a protecting group.

[0292] 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.

[0293] 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).

[0294] Subsequently, 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 )).

[0295] If the M pr o 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.

[0296] In addition, it should also be understood that any specific embodiment of the present invention within the scope of the prior art may be expressly excluded from any one or more claims. Since these embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if not expressly stated as such herein.

[0297] All cited sources, such as the references, publications, databases, database entries, and technologies cited herein, are incorporated herein by reference, even if not expressly stated in the citation. In the event of a conflict between the cited source and the statements of this application, the statements of this application shall prevail.

[0298] (1) Synthesize a compound according to formula (1), (1A), (1B), (1C), (1D), (1E), (1F), (1G), (1H), (1I), (1J), (1K), (1L), or (1M)

[0299] A. General Considerations

[0300] It should be noted that the raw materials used in the present invention are all ordinary commercially available products, and no specific limitation is imposed on their sources.

[0301] The following described process routes can be used to synthesize the compounds 1001 - 3422 of the present invention.

[0302] The following abbreviations are used herein:

[0303] THF: Tetrahydrofuran

[0304] MeCN: Acetonitrile

[0305] MeOH: Methanol

[0306] PE: Petroleum ether

[0307] EA: Ethyl acetate

[0308] DMF: N,N - Dimethylformamide

[0309] EDCl: 1 - Ethyl - (3 - dimethylaminopropyl)carbodiimide hydrochloride

[0310] LAH: Lithium aluminum hydride

[0311] DCM: Dichloromethane

[0312] DMAP: 4 - Dimethylaminopyridine

[0313] LDA: Lithium diisopropylamide

[0314] rt: Room temperature

[0315] DCE: 1,2 - Dichloroethane

[0316] n - BuLi: n - Butyllithium

[0317] i - Pr2EtN: N,N - Diisopropylethylamine

[0318] B. Intermediate Synthesis

[0319] Intermediate A:

[0320]

[0321] Intermediate A is obtained through the following synthetic process route:

[0322]

[0323] Dissolve tris(hydroxymethyl)aminomethane (50.0 g) and di-tert-butyl carbonate (Boc 2 O) (99.1 g) in a mixed solvent of methanol (300 mL) / H 2 O (30 mL), react at room temperature for 72 h, and purify by silica gel column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain the white solid compound Intermediate A (N-Boc-aminotrimethylolmethane) (75.0 g). LCMS (ESI) calcd for C 9 H 19 NO 5 , [M+H] + m / z 222.13, found 222.25.

[0324] Intermediate B:

[0325]

[0326] Intermediate B is obtained through the following synthetic process route:

[0327]

[0328] 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 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 liquid, wash the organic phase with saturated brine, separate out the organic phase, and purify by silica gel column chromatography (PE:EA = 100:1 - 50:1) to obtain diethyl 2-ethyl-2-pentadecylmalonate (16.5 g). Add EtOH (50 mL), H 2O (50 mL), KOH (11.3 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 for liquid separation. The organic phase is washed with saturated brine, the organic phase is separated, and purified by column chromatography (PE:EA = 5:1 - 1:1) to obtain 2-ethyl-2-pentadecylmaleic acid (5.2 g). 2-ethyl-2-pentadecylmaleic acid (10.0 g) is reacted at 170 °C for 6 h under open conditions, cooled to room temperature, added with water and ethyl acetate and stirred for liquid separation. The organic phase is washed with saturated brine, the organic phase is separated, concentrated, and purified by column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain intermediate B (2-ethylheptadecanoic acid) (8.2 g). 1H NMR (400 MHz, CDCl 3 ) 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).

[0329] Intermediate C:

[0330]

[0331] Intermediate C is obtained by the following synthetic process route:

[0332]

[0333] Add capric acid (50 g) and solvent THF (500 mL) to a three-necked flask, cool the system to 0 °C and slowly add the reactant NaH (23.22 g), and 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, add the reactant iodo-nonane (88.52 g) and stir overnight at room temperature, then dilute with 1 L of DCM, and wash with saturated NH 4 Cl solution and water respectively. The organic layer is dried with anhydrous Na 2 SO 4 , filtered and 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).

[0334] Intermediate D:

[0335]

[0336] Intermediate D is obtained through the following synthetic process route:

[0337]

[0338] 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). After stirring the reaction solution at 0 °C under nitrogen protection for 1 h, slowly drop LDA (317.87 g) into the reaction system; continue to stir the reaction solution at 0 °C under nitrogen protection for 1 h. After dropping the reactant n-hexyl iodide (94.39), raise the temperature to room temperature and stir overnight. Then dilute the reaction solution with 1 L of DCM and wash it with saturated NH 4 Cl solution and water respectively. Dry the organic layer with anhydrous Na 2 SO 4 , filter and concentrate. Perform 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).

[0339] Intermediate E:

[0340]

[0341] Intermediate E is obtained through the following synthetic process route:

[0342]

[0343] 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 pulping, filter to obtain the white solid compound Intermediate E (11.5 g). 1HNMR (400 MHz, CDCl 3 ) 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).

[0344] Intermediate F:

[0345]

[0346] The intermediate F is obtained by the following synthetic process route:

[0347]

[0348] Add (2-aminoethyl)carbamic acid tert-butyl ester (2.7 g), MeCN (90 mL), benzyl 2-bromoethyl ether (7.99 g), and K 2 CO 3 (11.65 g) into a single-necked flask. 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 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, that is, intermediate F (6 g). LCMS(ESI) calcd for C 20 H 28 N 2 O 2 , [M + H] + m / z 329.22, found 329.24.

[0349] Intermediate G:

[0350]

[0351] Intermediate G is obtained by the following synthetic process route:

[0352]

[0353] Add compound 1 (3.0 g), MeCN (90 mL), 3-benzyloxybromopropane (9.45 g), and K 2 CO 3 (12.94 g) into a single-necked flask. 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). Add Dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL) to compound 2 (6.9 g), stir at room temperature for 3 h, then concentrate under reduced pressure to obtain intermediate G (7.5 g). LCMS(ESI) calcd for C 22 H 32 N 2 O 2 , [M + H]+ m / z 357.25, found 357.51。

[0354] Intermediate H:

[0355]

[0356] Intermediate H is obtained by the following synthetic process route:

[0357]

[0358] Into a single-necked flask, add compound 1 (1.5 g), MeCN (90 mL), 4-bromobutyl benzyl ether (5.02 g), K 2 CO 3 (6.47 g), react at 80 °C overnight, then add water and ethyl acetate and stir for liquid separation. The organic phase is washed with saturated brine, the organic phase is separated, concentrated, and purified by column chromatography (PE:EA = 20:1 - 10:1) to obtain compound 2 (4.1 g). Add Dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL) to compound 2 (4.1 g.), stir at room temperature for 3 h, then concentrate under reduced pressure to obtain intermediate H (4.9 g). LCMS (ESI) calcd for C 24 H 36 N 2 O 2 , [M + H] + m / z 385.28, found 385.56。

[0359] Intermediate I:

[0360]

[0361] Intermediate I is obtained by the following synthetic process route:

[0362]

[0363] Into a single-necked flask, add compound 1 (3.9 g), MeCN (90 mL), benzyl 2-bromoethyl ether (5.78 g), K 2 CO 3(15.46 g), after reacting overnight at 80 °C, water and ethyl acetate were added and stirred for liquid separation. The organic phase was washed with saturated brine, the organic phase was separated, concentrated, and purified by column chromatography (PE:EA = 20:1 - 10:1) to obtain compound 2 (6.8 g). To compound 2 (6.8 g), Dioxane (30 mL) and hydrochloric acid dioxane solution (30 mL) were added. After stirring at room temperature for 3 h, it was concentrated under reduced pressure to obtain intermediate I (6.5 g). LCMS (ESI) calcd for C 12 H 20 N 2 O, [M+H] + m / z 209.16, found 209.31.

[0364] Intermediate J:

[0365]

[0366] Intermediate J was obtained by the following synthetic route:

[0367]

[0368] To a single-necked flask, butyric acid (5.0 g), THF (100 mL) were added. At 0 °C, NaH (2.73 g) was added, and LDA (56.8 mL) was slowly added dropwise. After reacting at room temperature for 30 min, 1-bromotridecane was added. After continuing to react overnight at room temperature, ice water and ethyl acetate were added and stirred for liquid separation. The organic phase was washed with saturated brine, the organic phase was separated, concentrated, and purified by column chromatography (PE:EA = 10:1 - 5:1) to obtain intermediate J (5.0 g). LCMS (ESI) calcd for C 17 H 34 O 2 , [M+H] + m / z 271.26, found 271.46.

[0369] Intermediate K:

[0370]

[0371] Intermediate K was obtained by the following synthetic route:

[0372]

[0373] Add reactant 1 (24 g), Imidazole (19.00 g) and solvent DCM (200 mL) to a three-necked flask. After cooling to 0 °C, slowly add reactant TBDMSCI (38.57 g). After stirring at 25 °C for 4 h, dilute with 300 mL of DCM. Wash the organic phase twice with 1 L of water and then dry with anhydrous Na 2 SO 4 After drying, filtering and concentrating, add silica gel and column chromatograph with 1:20

[0374] DCM:MeOH to collect the sample and concentrate to obtain intermediate K (28.00 g). 1H NMR (400 MHz, CDCl 3 ) 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).

[0375] Intermediate L:

[0376]

[0377] Intermediate L is obtained by the following synthetic process route:

[0378]

[0379] Add compound DMSO (3.58 g) and anhydrous solvent DCM (30 mL) to a three-necked flask. After cooling the system to -78 °C, slowly add oxalyl chloride (2.91 g). After stirring the reaction solution at -78 °C under N 2 protection for 10 minutes, slowly dropwise add compound 1 (3 g). Under N 2 protection at -78 °C, continue stirring for 1 h. Finally, dropwise add TEA (9.28 g). Under N 2 protection at -78 °C, continue stirring for 0.5 h, then dilute with 100 mL of DCM and wash with saturated NH 4 Cl solution and water respectively. Dry the organic layer with anhydrous Na 2 SO 4 After drying, filtering and concentrating, obtain intermediate L (3.0 g). LCMS (ESI) calcd for C 11 H 14 O 3 , [M + H] + m / z 195.09, found 195.23.

[0380] Intermediate M:

[0381]

[0382] The intermediate M is obtained through the following synthetic process route:

[0383]

[0384] Add intermediate K (2.0 g), compound 1 (1.79 g) and solvent ACN (20 mL) into a sealed tube, add reactant K 2 CO 3 (3.81 g) and KI (1.52 g) and stir overnight at 70 °C under N 2 protection, then dilute with 100 mL of EA, and wash with saturated NH4Cl solution and water respectively. The organic layer is dried with anhydrous Na 2 SO 4 and filtered and concentrated, 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, displace 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 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 NO 2 Si, [M + H] + m / z 288.52, found 288.23.

[0385] C, compound 1003, is synthesized according to the general synthetic route 1

[0386] Structural formula:

[0387] Chemical formula: C 63 H 122 N 2 O 7

[0388] Molecular weight: 1019.68

[0389] Step 1: Synthesize compound 3 in the general synthetic route 1

[0390]

[0391] Compound 1 (Intermediate A) (1.0 g), DCM (20 mL), DMAP (2.2 g), and EDCI (3.4 g) were added to Compound 2 (Intermediate B) (4.3 g). After stirring at room temperature for 12 h under nitrogen protection, water and dichloromethane 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 silica gel column chromatography (PE:EA = 40:1 - 30:1) to obtain Compound 3 (2.6 g).

[0392] Step 2: Synthesis of Compound 4 in General Synthetic Route 1

[0393]

[0394] DCM (15 mL) and TFA (5 mL) were added to Compound 3 (2.6 g). After stirring at room temperature for 3 h, it was concentrated and purified by silica gel column chromatography (PE:EA = 10:1 - 5:1) to obtain Compound 4 (3.2 g).

[0395] Step 3: Synthesis of Compound 6 in General Synthetic Route 1

[0396]

[0397] 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 for 12 h under nitrogen protection, 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).

[0398] Step 4: Compound 1003

[0399]

[0400] DCM (15 mL) and TFA (5 mL) were added to Compound 6 (2.2 g). After stirring at room temperature for 12 h, it was concentrated and purified by column chromatography (DCM:MeOH = 30:1 - 20:1) to obtain Compound 1003 (1.3 g). 1 H NMR (400 MHz, CDCl 3 ) 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).

[0401] D. Compound 1002, synthesized according to General Synthetic Route 1

[0402] Structural formula:

[0403] Chemical formula: C 60 H 116 N 2 O 7

[0404] Molecular weight: 977.60

[0405] Synthesized according to General Synthetic Route 1, with a synthetic process similar to that of Compound 1003, except that Intermediate C is used as Compound 2 in General Synthetic Route 1. 1 H NMR(400MHz,CDCl3)δ4.48(s,6H),3.27(s,2H),2.37(tt,J=8.5,5.5Hz,3H),1.64(d,J=6.9Hz,6H),1.50 - 1.42(m,6H),1.27(s,72H),0.90(t,J=6.7Hz,18H).

[0406] E. Compound 1004, synthesized according to General Synthetic Route 1

[0407] Structural formula:

[0408] Chemical formula: C 54 H 104 N 2 O 7

[0409] Molecular weight: 893.43

[0410] Synthesized according to General Synthetic Route 1, with a synthetic process similar to that of Compound 1003, except that palmitic acid is used as Compound 2 in General Synthetic Route 1. 1H NMR(400MHz,CDCl3)δ7.55(s,1H),4.45(s,6H),3.74(s,2H),2.35(t,J=7.6Hz,6H),1.60(p,J=6.9Hz,6H),1.27(s,72H),0.90(t,J=6.7Hz,9H).

[0411] F. Compound 1001, synthesized according to General Synthetic Route 1

[0412] Structural formula:

[0413] Chemical formula: C 59 H 108 N 2 O 7

[0414] Molecular weight: 957.52

[0415] Synthesized according to General Synthetic Route 1, similar to the synthesis process of Compound 1003, except that (9Z)-9-hexadecenoic acid is used as Compound 2 in General Synthetic Route 1, and 5-(N,N-dimethylamino)pentanoic acid is used as Compound 5 in General Synthetic Route 1. 1H NMR (400 MHz, CDCl 3 ) 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).

[0416] G. Compound 1014, synthesized according to General Synthetic Route 2

[0417] Structural formula:

[0418] Chemical formula: C 70 H 137 N 3 O 9

[0419] Molecular weight: 1164.88

[0420] Step 1: Synthesize Intermediate 3 of General Synthetic Route 2

[0421]

[0422] Synthesize Intermediate 3 according to the method shown in Step 1 of General Synthetic Route 1, except that Intermediate E is used as Compound 1 in General Synthetic Route 1.

[0423] Step 2: Synthesize Compound 4 of General Synthetic Route 2

[0424]

[0425] 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, followed by liquid separation. The organic phase was washed with saturated brine, separated, concentrated, and purified by silica gel column chromatography (DCM:MeOH = 20:1 - 10:1) to obtain compound 4 (3.3 g).

[0426] Step 3: Synthesis of compound 6 of general synthetic route 2

[0427]

[0428] To compound 5 (1.9 g), 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. 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).

[0429] Step 4: Compound 1014

[0430]

[0431] To compound 6 (4.0 g), MeOH (30 mL), DCM (10 mL), and Pd / C (4.73 g (10%)) were added. Under hydrogen gas, after stirring at room temperature overnight, it was filtered through diatomaceous earth and concentrated, and purified by silica gel column chromatography (DCM:MeOH 30:1 - 15:1) to obtain compound 1014 (2 g). 1 H(400MHz,CDCl 3 )δ7.38(t,J=5.8Hz,1H),4.14 - 4.05(m,6H),3.58(t,J=4.9Hz,4H),3.31(q,J=5.7Hz,2H),2.89(t,J=6.3Hz,2H),2.64(q,J=5.2Hz,6H),2.29(dq,J=10.8,4.4Hz,6H),1.66 - 1.37(m,12H),1.24(d,J=4.3Hz,78H),0.87(t,J=7.1Hz,18H).

[0432] H. Compound 1015, synthesized according to general synthetic route 2

[0433] Structural formula:

[0434] Chemical formula: C 72H 141 N 3 O 9

[0435] Molecular weight: 1192.93

[0436] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, except that Intermediate G is used as Compound 5 in General Synthetic Route 2. 1H (400 MHz, CDCl 3 ) 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).

[0437] I. Compound 1020, synthesized according to General Synthetic Route 2

[0438] Structural formula:

[0439] Chemical formula: C 74 H 145 N 3 O 9

[0440] Molecular weight: 1220.99

[0441] 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.

[0442] J. Compound 1025, synthesized according to General Synthetic Route 2

[0443] Structural formula:

[0444] Chemical formula: C 69 H 135 N 3 O 8

[0445] Molecular weight: 1134.85

[0446] 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).

[0447] K. Compound 1005, synthesized according to General Synthetic Route 2

[0448] Structural formula:

[0449] Chemical formula: C 68 H 133 N 3 O 7

[0450] Molecular weight: 1104.83

[0451] 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 (400 MHz, CDCl 3 ) δ 4.19 - 4.07 (m, 6H), 3.40 (d, J = 5.7 Hz, 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 = 5.4 Hz, 78H), 0.89 (td, J = 7.0, 2.8 Hz, 18H).

[0452] L. Compound 1006, synthesized according to General Synthetic Route 2

[0453] Structural formula:

[0454] Chemical formula: C 53 H 103 N 3 O 7

[0455] Molecular weight: 894.42

[0456] 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,CDCl 3 )δ7.99(t,J=5.8Hz,1H),4.13(s,6H),3.60(q,J=5.3

[0457] Hz,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).

[0458] M. Compound 1007, synthesized according to General Synthetic Route 2

[0459] Structural formula:

[0460] Chemical formula: C 68 H 133 N 3 O 7

[0461] Molecular weight: 1104.83

[0462] 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=14.2,6.7Hz,6H),1.27(d,J=3.4Hz,78H),0.89(td,J=6.7,2.5Hz,18H).

[0463] N. Compound 1008, synthesized according to General Synthetic Route 2

[0464] Structural formula:

[0465] Chemical formula: C68 H 133 N 3 O 7

[0466] Molecular weight: 1104.01

[0467] 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. 1 HNMR(400MHz,Chloroform-d)δ4.13(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).

[0468] O. Compound 1009, synthesized according to General Synthetic Route 2

[0469] Structural formula:

[0470] Chemical formula: C 62 H 121 N 3 O 7

[0471] Molecular weight: 1020.66

[0472] 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,CDCl 3 )δ7.04(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).

[0473] P. Compound 1011, synthesized according to General Synthetic Route 2

[0474] Structural formula:

[0475] Chemical formula: C 70 H 137 N 3 O 7

[0476] Molecular weight: 1132.88

[0477] Synthesized according to General Synthesis Route 2, similar to the synthesis process of Compound 1014, except that N,N - diethylethylenediamine is used as Compound 5 in General Synthesis Route 2. 1 H NMR(400MHz,CDCl 3 ) δ4.19 - 4.08(m,6H),3.31(q,J=5.8Hz,2H),2.92(t,2H),2.57(t,J=6.9Hz,6H),2.37 - 2.26(m,5H),1.62(ddd,J=18.3,9.0,4.3Hz,12H),1.27(d,J=4.7Hz,78H),1.04(t,J=7.1Hz,6H),0.94 - 0.86(m,18H).

[0478] Q. Compound 1012, synthesized according to General Synthesis Route 2

[0479] Structural formula:

[0480] Chemical formula: C 72 H 141 N 3 O 7

[0481] Molecular weight: 1160.93

[0482] Synthesized according to General Synthesis Route 2, similar to the synthesis process of Compound 1014, except that N,N - diethylethylenediamine is used as Compound 5 in General Synthesis Route 2. 1 H NMR(400MHz,CDCl 3 ) δ6.59(s,1H),4.18 - 4.07(m,6H),3.29(q,J=5.7Hz,2H),2.91(t,J=6.2Hz,2H),2.54(t,J=6.1Hz,2H),2.40(t,J=7.5Hz,4H),2.35 - 2.27(m,5H),1.63 - 1.42(m,16H),1.27(m,J=4.5Hz,78H),0.93 - 0.85(m,24H).

[0483] Compound 1013, synthesized according to General Synthetic Route 2

[0484] Structural formula:

[0485] Chemical formula: C 74 H 145 N 3 O 7

[0486] Molecular weight: 1188.99

[0487] Synthesized according to General Synthetic Route 2, with a synthetic process similar to that of Compound 1014, except that N,N - diethylethylenediamine is used as Compound 5 in General Synthetic Route 2. 1H NMR (400 MHz, CDCl 3 ) δ4.10 (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 (m, 5H), 1.60 (d, J = 11.8 Hz, 16H), 1.24 (d, J = 4.5 Hz, 82H), 0.96 - 0.79 (m, 24H).

[0488] Compound 1029, synthesized according to General Synthetic Route 2

[0489] Structural formula:

[0490] Chemical formula: C 68 H 132 N 2 O 8

[0491] Molecular weight: 1105.81

[0492] Synthesized according to General Synthetic Route 2, with a synthetic process similar to that of Compound 1014, except that N,N - diethyl - 2 - hydroxyethylamine is used as Compound 5 in General Synthetic Route 2. 1 1H NMR (400 MHz, CDCl 3 ) δ4.16 (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).

[0493] Compound 1111, synthesized according to General Synthetic Route 2

[0494] Structural formula:

[0495] Chemical formula: C 70 H 136 N 4 O 7

[0496] Molecular weight: 1145.88

[0497] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, with the difference 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).

[0498] U. Compound 1118, synthesized according to General Synthetic Route 2

[0499] Structural formula:

[0500] Chemical formula: C 71 H 139 N 3 O 8

[0501] Molecular weight: 1162.91

[0502] Synthesized according to General Synthetic Route 2, similar to the synthesis process of Compound 1014, with the difference 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 1H 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).

[0503] V. Compound 1010, synthesized according to General Synthetic Route 3

[0504] Structural formula:

[0505] Chemical formula: C 67 H 133 N 3 O 6

[0506] Molecular weight: 1076.82

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

[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 3

[0511]

[0512] Add Compound 4 (600 mg), chloroacetaldehyde (Compound 5, 242 mg (40%)), and NaBH(OAc) 3 (394 mg) to DCE (60 mL). After reacting overnight at room temperature, add water and dichloromethane and stir for liquid separation. Wash the organic phase with saturated brine, separate the organic phase, concentrate it, and perform silica gel column chromatography (DCM:MeOH = 30:1 - 15:1) to obtain Compound 6 (270 mg).

[0513] Step 4: Synthesize Compound 1010

[0514]

[0515] Add MeCN (20 mL), N,N - dimethylethylenediamine (Compound 7, 229 mg), KI (43 mg), and K 2 CO 3 (179 mg) to Compound 6 (270 mg). React overnight at 70 °C under nitrogen protection, and then perform silica gel column chromatography to obtain Compound 1010 (16 mg). 1 H (400 MHz, CDCl 3)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).

[0516] W. Compound 1059, synthesized according to General Synthetic Route 3

[0517] Structural formula:

[0518] Chemical formula: C 67 H 132 N 2 O 7

[0519] Molecular weight: 1077.80

[0520] Synthesized according to General Synthetic Route 3, similar to the synthesis process of Compound 1010, except that Intermediate C and Intermediate L are used as Compound 2 and Compound 5 in General Synthetic Route 3 respectively. In addition, the functional group transformation of Compound 6 in General Synthetic Route 3 needs to be carried out through the following process steps:

[0521]

[0522] Add solvent MeOH (5 mL) and THF (5 mL) to Intermediate 6a (1.5 g), add reactants Pd(OH) 2 / C (10%) (0.46 g) and Pd / C (10%) (0.35 g), react overnight at 25 °C under H 2 condition, 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). Add DCM (5 mL) to Intermediate 6b (470 mg), dropwise add SOCl 2 (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). Add dimethylamine (143.39 mg), DMF (2 mL), potassium carbonate (43.96 mg) and potassium iodide (26.39 mg) to Intermediate 6c (170 mg), stir the reaction solution at 70 °C and 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) δ 4.11 (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).

[0523] X. Compound 1112 was synthesized according to General Synthetic Route 3

[0524] Structural formula:

[0525] Chemical formula: C 69 H 136 N 2 O 6

[0526] Molecular weight: 1089.85

[0527] Synthesized according to General Synthetic Route 3, similar to the synthesis process of Compound 1010, except that intermediate C and 6-bromohexanal were used instead of Compound 2 and Compound 5 in General Synthetic Route 3, respectively. 1 H NMR (400 MHz, Chloroform-d) δ 4.11 (s, 6H), 3.11 (s, 1H), 2.57 (t, J = 6.8 Hz, 2H), 2.44 - 2.19 (m, 12H), 2.04 (q, J = 6.5 Hz, 2H), 1.53 - 1.38 (m, 13H), 1.27 (s, 92H), 0.90 (t, J = 6.7 Hz, 20H).

[0528] Y. Compound 1113 was synthesized according to General Synthetic Route 4

[0529] Structural formula:

[0530] Chemical formula: C 71 H 138 N 2 O 8

[0531] Molecular weight: 1147.89

[0532] Steps 1 and 2: Synthesize Compound 3 and Compound 4 of General Synthetic Route 4

[0533]

[0534] The procedures of Step 1 and Step 2 in General Synthetic Route 1 are the same as those in the synthesis process of Compound 1003.

[0535] Step 3: Synthesize Compound 6 in General Synthetic Route 4

[0536]

[0537] Add DIEA (0.30 g) and solvent DCM (20 mL) to Compound 4. Under nitrogen protection, cool the temperature to 0 °C, then add 5-bromovaleryl chloride (Compound 5, 0.27 g). Stir at 25 °C for 2 h. After diluting with 100 mL of DCM, wash the organic phase twice with 100 mL of water. The organic phase is dried with anhydrous Na 2 SO 4 After drying, filtering, and concentrating, perform silica gel column chromatography (PE:EA 20:1) to obtain Compound 6 (800 mg).

[0538] Step 4: Synthesize Compound 8 in General Synthetic Route 4

[0539]

[0540] Add Compound 6 (750.00 mg), Intermediate K (Compound 7, 217.31 mg), and solvent DMF (20 mL) to a three-necked flask. Add reactant K 2 CO 3 (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; the organic phase is dried with anhydrous Na 2 SO 4 After drying, filtering, and concentrating, add silica gel and elute through a column with 20:1 DCM:MeOH. Collect the sample and concentrate to obtain Compound 8 (700.00 mg).

[0541] Step 5: Synthesize Compound 1113

[0542]

[0543] Add Compound 8 (700.00 mg) and solvent THF (5 mL) to a three-necked flask. Add hydrochloric acid dioxane solution (1 M) (5.55 mL). Stir the reaction solution at 25 °C for 2 h, then dilute with 50 mL of EA, and wash the organic phase with saturated NaHCO 3 solution and water. After that, the organic phase is dried with anhydrous Na 2 SO 4After drying, filtration and concentration, silica gel column chromatography (MeOH:DCM 1:20) gave compound 1113 (201.1 mg, yield 31.59%). 1 H NMR(400MHz,CDCl 3 )δ4.50 - 4.39(m,6H),3.59(t,J=4.6Hz,2H),2.49(s,3H),2.35 - 2.26(m,6H),2.14(t,J=7.5Hz,2H),1.75 - 1.69(m,4H),1.66 - 1.43(m,17H),1.27(d,J=3.7Hz,78H),0.94 - 0.87(m,18H).

[0544] Z. Compound 1115 was synthesized according to the general synthetic route 4

[0545] Structural formula:

[0546] Chemical formula: C 67 H 131 N 3 O 7

[0547] Molecular weight: 1090.80

[0548] Synthesized according to the general synthetic route 4, similar to the synthesis process of compound 1010, except that 2 - bromoacetyl chloride and N,N - dimethylethylenediamine were used for compound 5 and compound 7 in the general synthetic route 4 respectively. 1 HNMR(400MHz,CDCl 3 )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(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).

[0549] AA. Compound 1114 was synthesized according to the general synthetic route 5

[0550] Structural formula:

[0551] Chemical formula: C 70 H 138 N 2 O 7

[0552] Molecular weight: 1119.88

[0553] Steps 1 and 2: Synthesize Compounds 3 and 4 of General Synthetic Route 5

[0554]

[0555] According to Steps 1 and 2 of General Synthetic Route 1, the synthesis process is the same as that of Compound 1003.

[0556] Step 3: Synthesize Compound 6 of General Synthetic Route 5

[0557]

[0558] 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. The reaction solution is stirred at N 2 , 25 °C for 16 h. Then add 100 mL of water to the reaction solution, extract twice with EA (100 mL), combine the organic phases, concentrate, add silica gel, and column chromatograph with 1:20 DCM:MeOH to collect the sample and concentrate to obtain Compound 6 (500 mg).

[0559] Step 4: Synthesize Compound 1114

[0560]

[0561] Add Compound 6 (450 mg) and solvent THF (5 mL) to a three-necked flask, and add hydrochloric acid dioxane solution (1 M) (3.65 mL). After stirring at 25 °C for 2 h, dilute with 50 mL of EA, and then wash the organic phase with 50 mL of saturated NaHCO 3 solution and water. The organic layer is dried with anhydrous Na 2 SO 4 , filtered and concentrated, and then column chromatographed on silica gel (MeOH:DCM 1:20) to obtain Compound 1114 (107 mg, yield 24.84%). 1 1H NMR (400 MHz, CDCl 3 ) δ 4.16 - 4.07 (m, 6H), 3.60 (t, J = 4.8 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).

[0562] Z. Compound 1120, synthesized according to General Synthetic Route 5

[0563] Structural formula:

[0564] Chemical formula: C 72 H 142 N 2 O 7

[0565] Molecular weight: 1147.90

[0566] Synthesized according to the general synthetic route 5, similar to the synthesis process of compound 1114, except that intermediate C and 6-bromohexanal are 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).

[0567] (2) Synthesis of the second ionizable lipid in Preparation Example 2

[0568] 1. Preparation of compound 6001

[0569] Structural formula:

[0570] Molecular weight: 499.43

[0571] 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 0.8 g of hydroxyethyl acrylamide, and continue the reaction for 16 h, then pass through a reverse column to obtain compound 6002.

[0572] Add 0.7 g of compound 6002, 0.4 g of palladium-carbon and 28 mL of tetrahydrofuran to a reaction flask and stir. Replace with hydrogen and keep the pressure with a hydrogen balloon. After 16 h, filter and pass through a normal column (methanol - DCM system) to obtain 200 mg of compound 6001. 1 H NMR (400 MHz, CDCl 3 ) δ 7.54 (t, J = 5.5 Hz, 2H), 3.79 - 3.68 (m, 4H), 3.42 (dd, J = 10.0, 5.4 Hz, 4H), 2.89 (t, J = 6.0 Hz, 4H), 2.55 (dt, J = 11.8, 6.8 Hz, 6H), 1.54 (s, 2H), 1.27 (d, J = 11.2 Hz, 32H), 0.91 (t, J = 6.8 Hz, 3H).

[0573] 2. Preparation of Compound 6002

[0574] Structural formula:

[0575] Molecular weight: 497.42

[0576] 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 400 mg of Compound 6002 in total. 1 H NMR(400MHz,CDCl 3 )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).

[0577] 3. Preparation of Compound 6003

[0578] Structural formula:

[0579] Molecular weight: 445.34

[0580] Add 4.00 g of tetradecylamine, 6.53 g of 2 - hydroxyethyl acrylate and 80 mL of tert - butanol to a reaction flask, stir and heat to 70 °C. After 29 h, concentrate the organic solvent, pass through a reverse column, and lyophilize to obtain 1.5 g of Compound 6003. 1 H NMR(400MHz,CDCl 3 )δ4.29(t,J=7.5Hz,4H),3.89 - 3.75(m,4H),2.81(t,J=6.2Hz,4H),2.50(ddd,J=23.2,14.1,7.1Hz,6H),1.46(s,2H),1.28(s,24H),0.91(t,J=6.3Hz,3H).

[0581] 4. Preparation of Compound 6004

[0582] Structural formula:

[0583] Molecular weight: 553.48

[0584] Add 0.8 g of oleylamine, 1.5 g of n-butyl acrylate and 5.6 mL of n-butanol to a reaction flask, stir and heat up to 100 °C. After 4 h, add 1 mL of n-butyl acrylate. Continue the reaction for 1 h, then pass through a normal-phase column (petroleum ether-ethyl acetate system). Add 0.4 g of sodium hydroxide, 1 mL of water and 10 mL of methanol, stir and hydrolyze for 30 min. Add 1 mL of concentrated hydrochloric acid and 10 mL of methanol to the mixed solution to adjust the pH to neutral. Rotavapor to dryness, dissolve in DCM, dry with anhydrous magnesium sulfate, filter, rotavapor to remove the solvent. Add 10 mL of DCM, 1.06 g of 4-amino-1-butanol, 0.81 g of HOBT and 2.3 g of EDCI, react at room temperature for 18 h. After concentrating the organic solvent, pass through a reverse-phase column and lyophilize to obtain 240 mg of compound 6004. 1 HNMR(400MHz,CDCl 3 )δ7.58(d,J=16.6Hz,2H),5.44-5.31(m,2H),3.72(d,J=16.5Hz,4H),3.31(d,J=5.6Hz,4H),3.17(s,4H),2.84(s,2H),2.69(s,4H),2.09-2.01(m,4H),1.67(d,J=2.7Hz,10H),1.37-1.26(m,24H),0.91(t,J=6.8Hz,3H).

[0585] 5. Preparation of compound 6005

[0586] Structural formula:

[0587] Molecular weight: 597.54

[0588] Add 25.00 g of 11-heneicosanone, 62.05 g of ammonium acetate and 500 mL of methanol to a reaction flask, 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.

[0589] 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, stir and heat up to 100 °C. Add 10 mL of n-butyl acrylate. After 16 h, pass through a column (petroleum ether-ethyl acetate system) to obtain 31.00 g of compound 6005-B.

[0590] 31.00 g of compound 6005-B, 6.55 g of sodium hydroxide, 310 mL of methanol, and 31 mL of water were mixed to form a solution, which was added to a reaction flask and stirred. After 20 min, 150 mL of THF was added, and the mixture was stirred and heated to 50 °C. After 30 min, a solution prepared from 16.13 g of concentrated hydrochloric acid and 160 mL of methanol was added and stirred. After 30 min, the solvent was evaporated, 300 mL of DCM was added for dissolution, dried over anhydrous magnesium sulfate, filtered, and the solvent was evaporated to obtain 27.00 g of an oily compound 6005-C.

[0591] 27.00 g of compound 6005-C, 25.00 g of 4-amino-1-butanol, 41.85 g of EDCI, 14.74 g of HOBT, and 310 mL of DCM were added to a reaction flask and stirred. After 18 h, the organic solvent was concentrated, and column chromatography (methanol-DCM system) was carried out to remove the excess 4-amino-1-butanol compound, obtaining 6.80 g of compound 6005. 1 HNMR(400MHz,CDCl 3 )δ3.70(t,J=5.6Hz,4H),3.28(t,J=5.6Hz,4H),2.74(t,J=6.0Hz,4H),2.40-2.45(m,1H),2.34(t,J=6.4Hz,4H),1.63-1.65(m,8H),1.25-1.35(m,36H),0.91(t,J=6.4Hz,6H).

[0592] 6. Preparation of compound 6006

[0593] Structural formula:

[0594] Molecular weight: 553.48

[0595] 1.00 g of oleylamine, 0.47 g of hydroxyethyl acrylamide, and 8 mL of ethanol were added to a reaction flask and stirred and heated to 70 °C. After 13 h, the solvent was evaporated and column chromatography (methanol-DCM system) was carried out to obtain 800 mg of intermediate 1. 1.5 mL of n-butyl acrylate and 5 mL of n-butanol were added and stirred and heated to 100 °C. After 3 h, the solvent was evaporated and column chromatography (methanol-DCM system) was carried out. A solution prepared from 0.4 g of sodium hydroxide, 10 mL of methanol, and 1 mL of water was added and stirred for 1 h. A solution prepared from 1 mL of concentrated hydrochloric acid and 8 mL of methanol was added and stirred for 30 min. The solvent was evaporated, 100 mL of DCM was added for dissolution, 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 column chromatography (methanol-DCM system) was carried out to obtain 300 mg of compound 6006. 1 H NMR(400MHz,CDCl 3)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).

[0596] Compound 6007

[0597] Structural formula:

[0598] Molecular weight: 953.78

[0599]

[0600] 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).

[0601] 8. Compounds 6008 - 6018

[0602] Synthesis of Compound 6009

[0603] Structural formula:

[0604] At room temperature, a methanol (20 mL) solution of freshly recrystallized hexadecylamine (0.03 mol) 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 evaporated to dryness at room temperature. 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 it was separated by column chromatography to obtain a colorless oil. 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).

[0605] Then, the solution of the above colorless oil (11.05 g) in methanol (20 ml) was added to the solution of 1,2-diaminoethane (75 g) in methanol (100 ml) that was vigorously stirred 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 finally obtain a white solid. 1H-NMR (300 MHz, CDCl 3 ): 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).

[0606] The preparation methods of 6008 and 6009 are only different in that: equimolar amounts of tetradecylamine are used to replace hexadecylamine. For 6010 - 6017, refer to the preparation method of 6009 and use equimolar amounts of the corresponding R a -NH 2 to replace hexadecylamine for preparation.

[0607] For 6018, refer to the preparation method of 6007 and use equimolar amounts of the corresponding R a -NH 2 to replace octadecylamine for preparation.

[0608] 9. Compound 6019

[0609] Structural formula:

[0610] Molecular weight: 753.66

[0611]

[0612] 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, compound 18807-71-1 (2.5 g), DCC (2.7 g), and DMAP (1.6 g) were added, and the mixture was stirred and mixed. The reaction was carried out 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 replaced, and 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 evaporated to dryness 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. The reaction was carried out overnight at room temperature; water (500 mL) and EA (500 mL) were added, and liquid-liquid extraction was performed. The organic phase was dried with Na 2 SO 4 dried, 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. The reaction was carried out at room temperature for 2 h; 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).

[0613] 10. Compound 6020

[0614] Structural formula:

[0615] Molecular weight: 810.19

[0616]

[0617] 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 (4M) (2 mL) to a single-necked flask and react at room temperature for 1 h. Stop the reaction, concentrate and purify the reaction solution to obtain compound 6020. 1H NMR (400 MHz, Methanol-d4) δ 3.90 (t, J = 6.4 Hz, 2H), 3.57 - 3.34 (m, 10H), 3.24 (dt, J = 16.1, 8.0 Hz, 8H), 2.77 (t, J = 6.5 Hz, 4H), 2.03 - 1.58 (m, 10H), 1.31 (s, 30H), 1.01 - 0.82 (m, 3H).

[0618] 11. Compound 6021

[0619] Structural formula:

[0620] Molecular weight: 1182.79

[0621]

[0622] At room temperature, add compound 2 (3.5 g, the synthesis process is the same as product 2 in compound 6020) to a single-necked flask. After adding MeOH (20 mL) and stirring to mix, add TREN (23.0 g), stir to mix, heat 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) to a single-necked flask, cool to 0 °C, and slowly dropwise add (Boc) 2O (71.7 g), after dropping, it was restored to room temperature, reacted for 3 h, quenched with water (200 mL), then added DCM (100 mL) for liquid-liquid extraction, the organic phase was dried with sodium sulfate, concentrated under reduced pressure, purified by column chromatography, and obtained 4 (5.8 g) of yellow oil. At room temperature, in a single-necked flask, add yellow oil 4 (1.4 g), slowly add HCl / Dioxane (4 M) (15 mL) under an ice-water bath and stir to mix. After adding, it was restored to room temperature and reacted for 0.5 h, concentrated under reduced pressure, and obtained compound 5 (800 mg). At room temperature, in a single-necked flask, add compound 5 (1.0 g), (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid (2.6 g), EDCI (1.4 g), HOBt (1.0 g), DIEA (1.0 g) and solvent DMF (10 mL), reacted overnight at room temperature, added 100 mL of water and 100 mL of EA, performed liquid-liquid extraction, the EA phase was washed once with 100 mL of saturated brine, concentrated under reduced pressure, and purified by column chromatography (DCM:MeOH = 10:1) to obtain compound 6 (1.1 g). At room temperature, in a single-necked flask, add compound 6 (1.2 g), HCl / Dioxane (4 M) (10 mL) and solvent DCM (10 mL), reacted overnight at room temperature, concentrated under reduced pressure, and purified 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).

[0623] 12. Compound 6023

[0624] Structural formula:

[0625] Molecular weight: 670

[0626]

[0627] At room temperature, add dodecylamine (10 g) to a three-necked flask. After adding MeOH (100 mL) and stirring to mix, cool the mixture to 5 °C in an ice-water bath. Slowly add methyl acrylate (10.2 g) dropwise, stir to mix, and then return to room temperature. Stop the reaction after the reaction solution has reacted at room temperature for 4 h. Concentrate under reduced pressure and purify by column chromatography (PE:EA = 20:1 - 10:1) to obtain product 2 (19 g). At room temperature, add product 2 (19.0 g), MeOH (200 mL), and ethylenediamine (127.7 g) to a single-necked flask. Heat the mixture to 60 °C and maintain this temperature to react overnight. Concentrate under reduced pressure to obtain compound 3 (20 g). Take 500 mg for purification and lyophilize to obtain compound 6023-1 (218 mg). At room temperature, add starting material 6023-1 (1.4 g) to a single-necked flask. After adding DCM (20 mL) and stirring to dissolve, add (S)-2,6-di-tert-butoxycarbonylaminohexanoic acid (3.4 g), EDCI (1.9 g), and DMAP (1.2 g), stir to mix, maintain at room temperature, and react overnight. Separate by liquid-liquid extraction, dry, and concentrate under reduced pressure to obtain compound 4 (3.0 g). At room temperature, add compound 4 (2.8 g) and HCl / 1,4-Dioxane (4 M) (20 mL) to a single-necked flask and react at room temperature for 2 h; concentrate under reduced pressure, purify, and lyophilize to obtain compound 6023 (123 mg). 1H NMR (400 MHz, Methanol-d4) δ 3.88 (t, J = 6.6 Hz, 2H), 3.55 - 3.36 (m, 8H), 3.36 - 3.33 (m, 2H), 3.31 - 3.14 (m, 4H), 3.04 - 2.93 (m, 4H), 2.77 (t, J = 6.5 Hz, 4H), 2.00 - 1.82 (m, 4H), 1.82 - 1.66 (m, 6H), 1.50 (qd, J = 8.2, 7.8, 4.2 Hz, 4H), 1.45 - 1.25 (m, 18H), 0.96 - 0.87 (m, 3H).

[0628] Compound 6024

[0629] Structural formula:

[0630] Molecular weight: 870.24

[0631]

[0632] 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 the mixture 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). 1H NMR (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).

[0633] 14. Compound 6026

[0634] Structural formula:

[0635] Molecular weight: 443.67

[0636]

[0637] Compound 6026 was synthesized by referring to the synthetic route of compound 6019, with the only difference being that equimolar amounts of compound 1 were used to replace the raw material 5 of 6019 and equimolar amounts of N-(tert-butoxycarbonyl)ethanolamine were used to replace intermediate 4 of compound 6019. 1H NMR (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).

[0638] Compounds 6025 - 6034 were obtained by referring to the synthetic routes of the aforementioned compounds, with the difference being that hydroxyethylamine was used to replace the corresponding ethylenediamine, and the amino group of hydroxyethylamine was first protected with a protecting group such as Fmoc or Boc, and the protecting group was removed by a conventional method after the reaction was completed.

[0639] (3) Exemplary Compounds of the First Ionizable Lipid and Their Properties

[0640] 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.

[0641] The calculated c-pKa (molnetwork) and LogP (cLogP driver) of the exemplary compounds of the present invention, and the c-pKa and cLogP values are generated by the ChemDraw module of Chemoffice.

[0642] Properties of Exemplary Compounds of the First Ionizable Lipid in Table 1

[0643]

[0644]

[0645]

[0646]

[0647]

[0648]

[0649]

[0650]

[0651]

[0652]

[0653]

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665]

[0666]

[0667]

[0668]

[0669]

[0670] 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, particularly suitable for preparing lipid nanoparticles for use as drug delivery carriers.

[0671] (4) Preparation of transfection reagent based on blank lipid nanoparticles

[0672] 4.1 Preparation of blank lipid nanoparticles

[0673]

[0674]

[0675] Prepare blank lipid nanoparticles according to the above molar ratios respectively, and the specific method is as follows:

[0676] 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 with a 50 mM citrate buffer (pH 4.0) solution in a volume ratio of 1:3 in a nanoparticle preparation device, ultrafiltration is carried out, and the sample is collected to obtain a lipid composition with a lipid concentration of 2 mg / mL.

[0677] 4.2 Preparation of transfection reagent based on blank lipid nanoparticles

[0678]

[0679] 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 volumes and vortex for 2 - 3 s to obtain the transfection reagent based on blank lipid nanoparticles.

[0680] (5) Determination of particle size and Zeta potential of transfection reagent

[0681] 1. Particle size and polydispersity index (PDI) determination: The average particle size and PDI of the nanoparticle sample solution in the examples were determined by dynamic light scattering using a Malvern ZetaSizer Nano ZS90. The measurement angle was 90°, the refractive index of the dispersant was 1.330, and the test temperature was 25 °C.

[0682] 2. The encapsulation efficiency test method is as follows:

[0683] According to the manufacturer's instructions, the encapsulation efficiency of mRNA in the transfection reagent was determined using the Quant-it Ribogreen RNA quantification assay kit (ThermoFisher Scientific, UK).

[0684] According to the manufacturer's instructions, the encapsulation efficiency of pDNA in the transfection reagent was determined using the dsDNA HS kit (Vazyme / EQ121).

[0685] According to the manufacturer's instructions, the microRNA Reagent kit was used to determine the encapsulation efficiency of siRNA in the transfection reagent.

[0686] The average particle size, PDI, and encapsulation efficiency data of the transfection reagent prepared in the examples are shown in Table 2.

[0687] Table 2 Summary of particle size, PDI, and encapsulation efficiency of the transfection reagent

[0688] Example number Particle size nm PDI Entrapment 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

[0689] As can be seen from Table 2, the transfection reagent provided by the present invention has a small and uniform particle size and a high encapsulation efficiency (>80%).

[0690] (6) Evaluation of the in vitro cell transfection effect of the transfection reagent based on blank lipid nanoparticles

[0691] 6.1 Blank lipid nanoparticle - Luciferase pDNA complex

[0692] The in vitro transfection efficiency of the blank lipid nanoparticle-Luciferase pDNA complexes in 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, the cells 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 nanoparticle-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. Transfection with the luciferase plasmid using Lipofectamine 2000 was used as a positive control (PC). After incubation in a 37 °C, 5% CO 2 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 3, Figure 1 .

[0693] Table 3 Luciferase activity (RLU, dose: 100 ng) expression data of the blank lipid nanoparticle-Luciferase pDNA complexes in different cells

[0694]

[0695] The results showed that the blank lipid nanoparticle-Luciferase pDNA complexes 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.

[0696] 6.2 Blank lipid nanoparticle-Luciferase mRNA complex

[0697] The in vitro transfection efficiency of the blank lipid nanoparticle-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, the cells 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 nanoparticle-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. Transfection with the luciferase mRNA using Lipofectamine 2000 was used as a positive control (PC). After incubation in a 37 °C, 5% CO 2 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 .

[0698] Table 4 Expression data of luciferase activity (RLU, dose: 100 ng) of lipid blank lipid nanoparticles - Luciferase mRNA complex in different cells

[0699]

[0700] The results show that the blank lipid nanoparticles - Luciferase mRNA 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 by Lipofectamine 2000.

[0701] The in vitro transfection efficiency of the blank lipid nanoparticles - eGFP mRNA complexes in Examples 3, 9, and 11 was evaluated using different cells.

[0702] 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 in 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 reagent Lipofectamine MessengerMAX TM 、JetMESSENGER TM were used as control preparations. After incubation in an incubator at 37°C and 5% CO 2 for 24 h, cell fluorescence photographs were taken 24 hours after transfection. The results are shown in Figures 3 - 5 .

[0703] The results show that the blank lipid nanoparticles - eGFP mRNA complexes provided by the present invention have high eGFP protein expression efficiency in different cells, and are better than commercially available transfection reagents.

[0704] 6.3 Blank lipid nanoparticles - siRNA complex

[0705] The in vitro transfection efficiency of the blank lipid nanoparticle-siRNA-cy3 complex of 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 complexes of Example 6 were 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% CO 2 incubator, and take fluorescence photos of the cells 6 hours after transfection. The results Figure 6 .

[0706] 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.

[0707] The solution of the present invention is not limited to the technical means disclosed by the above technical means, but also includes technical solutions composed of any combination of the above technical features. The above is the specific implementation manner of the present invention. It should be noted that for those of ordinary skill in the art of the present invention, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches are also regarded as the protection scope of the present invention.

Claims

1. A transfection reagent based on blank lipid nanoparticles, characterized in that, include: (1) Blank lipid nanoparticles; (2) Biologically active ingredients; The blank lipid nanoparticles are composed of: 5-70 mol% of a first ionizable lipid, 0-30 mol% of a second ionizable lipid, 5-50 mol% of a phospholipid, 10-70 mol% of cholesterol, and 0-15 mol% of a polyethylene glycol-conjugated lipid, wherein the second ionizable lipid is not 0; in the transfection reagent, the amount 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; The first ionizable lipid is selected from a compound containing the general formula (1E) or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof; Wherein, R1, R2, and R3 are independently H, C 5-40 Straight or branched alkyl, C 5-40 Straight-chain or branched alkenyl, C 5-40 A straight or branched alkynyl group, a 3-6 membered saturated or partially unsaturated cyclic hydrocarbon group containing 1-3 side chains, or a 6-10 membered aromatic group containing 1-3 side chains; the side chains are independently selected from C 10-30 Straight or branched alkyl, C 10-30 Straight or branched alkenyl, C 10-30 Straight or branched alkynyl; 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, wherein the heterocyclic group and the heteroaryl group are unsubstituted or substituted by one or more -OH, carboxyl, amine, oxo or halogen; R4 and R5 are independently H, C 1-6 Straight or branched alkyl, C 2-6 Straight or branched alkenyl or C 2-6 A straight chain or branched chain alkynyl group, the C 1-6 Straight or branched alkyl, C 2-6 Straight or branched alkenyl or C 2-6 The straight-chain or branched alkynyl group is unsubstituted or substituted with one or more -OH, carboxyl, amine, amide, amidino, guanidino or halogen; G1, G2, and G3 are independently -O-, -S-, -NR6-, -SS-, -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-; where each R6, R 13 are independently selected from H, hydroxyl, C 1-30 Straight or branched alkyl or cycloalkyl, C 2-30 Straight-chain or branched alkenyl; R 9、 R 10 Independently of each other are H; X2 is selected from -O-, -S-, NR 16 -, -SS-, -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 Independently of each other are H, C 1-6 A straight or branched alkyl or cycloalkyl group; Each R 16 , R 17 are independently selected from H, C 1-30 Straight or branched alkyl or cycloalkyl, C 2-30 Straight-chain or branched alkenyl; Among them, R4, R5, R6, R 11 , R 12 , R 13 , R 16 , R 17 The alkyl, cycloalkyl, alkenyl described in is unsubstituted or substituted by one or more groups selected from hydroxyl, thiol, amine, substituted amine, and halogen; n is an integer from 2 to 6; k is an integer from 1 to 6; The salts do not include quaternary ammonium salts; The second ionizable lipid is a compound of formula (2), or a salt, stereoisomer, or tautomer thereof: Among them, A1 is NH; R a Selected from C6-C 24 Alkyl, C6-C 24 Alkenyl, C6-C 24 Alkyl alcohol, C6-C 24 The cycloalkyl group of C6-C 24 The alkyl group is a branched structure; the C6-C 24 Alkenyl, C6-C 24 Alkyl alcohol is a straight chain or branched chain structure; R b and R c Each independently selected from C1-C 12 Alkyl alcohol.

2. The transfection reagent according to claim 1, characterized in that The biologically active component 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 minicircle, and msDNA.

4. The transfection reagent according to claim 2, characterized in that The polypeptide is selected from polypeptides comprising 2-50 amino acids.

5. The transfection reagent according to claim 1, characterized in that The R1, R2, and R3 are independently the following groups: Where Y does not exist or is C 1-30 Straight or branched alkyl or cycloalkyl, C 2-20 Straight or branched alkenyl, C 2-20 Straight or branched alkynyl; R1', R2' are independently H, C 1-30 Straight or branched alkyl, C 2-30 Straight or branched alkenyl, C 2-30 A straight chain 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, characterized in that The R1, R2, and R3 are independently selected from the following groups: Wherein, R1' and R2' are independently H, C 1-30 Straight or branched alkyl, C 2-30 Straight or branched alkenyl, C 2-30 The total carbon chain length of R1' and R2' is 8-30.

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

8. The transfection reagent according to claim 1, characterized in that G1, G2, and G3 are independently -O-, -S-, -NR6-, -SS-, -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, characterized in that M is selected from the following structures: wherein m' and n' are independently integers of 0-6, R1" and R2" are independently H, C 1-6 Alkyl, C 2-6 The alkenyl, guanidino, amidino, amide, fatty amine, 3-10 membered nitrogen-containing heterocyclic ring; the nitrogen-containing heterocyclic ring is selected from pyrrole, imidazole, pyridine, pyrazole, triazole, oxazole, isoxazole, thiophene, isothiazole, pyridazine, pyrazine, piperazine, indole, benzimidazole, carbazole, quinoline, isoquinoline, purine and pyrimidine and tautomeric forms thereof, which are unsubstituted or optionally substituted with one or more selected from hydroxyl, thiol, amine, substituted amine, halogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 6-14 The aromatic group is substituted with an organic group.

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

11. The transfection reagent according to claim 10, characterized in that The compound of formula (1E) is selected from the compound represented by formula (1G):

12. The transfection reagent according to claim 10, characterized in that The compound of formula (1E) is selected from the compound represented by formula (1H):

13. The transfection reagent according to claim 1, characterized in that The compound of formula (1E) is selected from:

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

15. The transfection reagent according to any one of claims 1 to 14, characterized in that The transfection reagent further comprises 0-60 mol% of other ionizable lipids, wherein the other ionizable lipids are selected from at least one of the following compounds:

16. The transfection reagent according to claim 1, characterized in that The phospholipids include 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-diondecanoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-di-0 -Octadecenyl-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-diamidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diphyton Alkanoyl-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) At least one of sodium salt, dipalmitoylphosphatidylglycerol, palmitoyloleoylphosphatidylethanolamine, distearoyl-phosphatidyl-ethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphoethanolamine, 1-stearoyl-2-oleoyl-stearoylethanolamine, 1-stearoyl-2-oleoyl-phosphatidylcholine, sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine.

17. The transfection reagent according to claim 1, characterized in that The polyethylene glycol-conjugated lipid includes at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

18. The transfection reagent according to claim 17, characterized in that The polyethylene glycol conjugated lipid includes at least one of PEG-distearyloxypropyl, PEG-c-DOMG, PEG-DPPC, polyethylene glycol dimethacrylate, 1,2-dimethylstyrene-rac-glycerol-3-methoxypolyethylene glycol, dipalmitoylglycerol-polyethylene glycol, 1,2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol, 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide, dipalmitoylphosphatidylethanolamine-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, dilauroylphosphatidylethanolamine-polyethylene glycol, and dimyristoylphosphatidylethanolamine-polyethylene glycol lipids.

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

20. The method for preparing a transfection reagent according to any one of claims 1 to 19, characterized in that: The method comprises the steps of mixing blank lipid nanoparticles and nucleic acid in a solvent.

21. The preparation method according to claim 20, characterized in that: The concentration of the nucleic acid after mixing is 5-1000 ng / μl.

22. The preparation method according to claim 20, characterized in that: The solvent is at least one of water, an aqueous solution of an organic solvent, and a buffered saline solution.

23. The preparation method according to claim 22, characterized in that: 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 of less than 50%.

24. The preparation method according to claim 22, characterized in that: The buffered saline solution is selected from at least one of a citrate solution, an acetate solution, a tartrate solution, a phosphate solution, a carbonate solution, a Tris-HCl solution, and a sodium chloride solution.

25. The preparation method according to claim 20, characterized in that: The transfection reagent also includes culture medium, sugar, glycerol, DMSO, salts, antibiotics and surfactants.

26. Use of the transfection reagent according to any one of claims 1 to 19 in in vitro transfection.

27. An in vitro transfection method, characterized in that: The transfection reagent according to any one of claims 1 to 19 is mixed with cells for transfection.

28. The in vitro transfection method according to claim 27, characterized in that 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, algae cells, and fungal cells.

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