Ionizable lipid and use thereof

Abstract

The present invention relates to the field of biomedicine, and specifically relates to an ionizable lipid and the use thereof. The ionizable lipid has the structure as represented by formula (I). Lipid nanoparticles constructed by using the ionizable lipid of the present invention can realize the safe and efficient delivery of nucleic acid drugs, small molecule drugs, peptide drugs and protein drugs.

Description

Ionizable lipids and their applications

[0001] This application claims priority to Chinese patent application CN2024119548859, filed on December 27, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedicine, and more specifically, to an ionizable lipid and its applications. Background Technology

[0003] In recent years, messenger RNA (mRNA) drugs have become a key treatment for the prevention and treatment of infectious diseases and tumors. The technology of mRNA drugs is recognized in the industry due to its short development cycle, low risk of insertional mutagenesis, and the diversity of encoded proteins; mRNA is particularly suitable for the development of vaccines or therapeutic drugs. However, mRNA itself is very unstable and easily degraded by ubiquitous RNases. Furthermore, due to the inherent negative charge and large molecular weight of mRNA (usually greater than 10⁻⁶), it is also susceptible to degradation. 6 This restriction (Da) limits the entry of mRNA molecules into the cell. Therefore, developing suitable delivery vectors to protect fragile mRNA molecules and deliver them into the cytoplasm is of great importance.

[0004] Various mRNA delivery vectors have been developed, including lipid nanoparticles (LNPs), inorganic nanoparticles, polymer nanoparticles, viral vectors, and exosomes. LNPs are currently widely used as drug delivery vectors, and their main components include ionizable lipids, phospholipids, cholesterol, and lipids containing polyethylene glycol. The most important component of LNPs is the ionizable lipid. Early permanently positively charged cationic lipids exhibited low in vivo circulation time, high toxicity, and severe allergic reactions. This is because their inherent positive charge causes them to adsorb proteins during circulation, making them easily captured and cleared by the reticuloendothelial system; their inherent positive charge interacts with negatively charged cell membranes, leading to cell membrane instability and severe toxicity; and permanently positively charged cationic lipids activate the complement system, causing allergic reactions. Ionizable lipids are uncharged under physiological pH conditions; therefore, LNPs prepared from ionizable lipids have relatively high safety. Ionizable lipids endow LNPs with the ability to escape from lysosomes. Through the proton sponge effect and membrane fusion mechanism, LNPs escape and release mRNA into the cytoplasm, where they bind to the ribosomes that encode the protein for translation.

[0005] In short, the development of suitable ionizable lipids is one of the keys to developing LNPs with high safety and high lysosomal escape efficiency.

[0006] Therefore, it is of great significance to develop an ionizable lipid with low toxicity and high delivery efficiency. Summary of the Invention

[0007] This invention provides an ionizable lipid with low toxicity and high delivery efficiency.

[0008] In one aspect of the invention, an ionizable lipid, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, is provided, wherein the ionizable lipid is a compound of formula (I):

[0009] in,

[0010] R1 and R2 are each independently -(CH2) n -, n is an integer selected from 1 to 14;

[0011] L1 is selected from: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-;

[0012] L2 is selected from: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-;

[0013] R3, R4, R5, and R6 are each independently selected from: H, C2-C 20 hydrocarbon group;

[0014] R7 is selected from C1-C5 hydrocarbon groups.

[0015] In some implementations, n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14.

[0016] In some implementations, n is selected from an integer between 5 and 8.

[0017] In some implementations, n is selected from 5, 6, 7, and 8.

[0018] In some implementations, L1 is selected from: -(C=O)O-, -O(C=O)-, -(C=O)NH-, -NH(C=O)-.

[0019] In some implementations, L2 is selected from: -(C=O)O-, -O(C=O)-, -(C=O)NH-, -NH(C=O)-.

[0020] In some implementations, R7 is a C2-C4 hydrocarbon group.

[0021] In some embodiments, the ionizable lipid is selected from the structures in Table 1:

[0022] Table 1

[0023] In some embodiments, the ionizable lipid is selected from the structures in Table 2:

[0024] Table 2

[0025] In some embodiments, the ionizable lipid has the following structure:

[0026] In another aspect of the invention, a method is provided for preparing the aforementioned ionizable lipid, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, the method comprising the following steps:

[0027] (S1) Under the protection of an inert gas, compounds A1 and A2 react to give compound A3;

[0028] (S2) Under inert gas protection, A3 undergoes the removal of the tert-butyloxycarbonyl group (Boc) to obtain A4;

[0029] (S3) Under the protection of an inert gas, A4 reacts with A5 or A6 to give the compound shown in formula (I);

[0030] Where R1 and R2 are each independently -(CH2). n - where n is selected from an integer from 1 to 14, preferably from an integer from 5 to 8;

[0031] G1 and G2 are each independently selected from aldehyde and halogen groups;

[0032] L1 is selected from: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-, preferably from -(C=O)O-, -O(C=O)-, -(C=O)NH-, -NH(C=O)-;

[0033] L2 is selected from: -O-, -(C=O)O-, -O(C=O)-, -(SS)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; preferably from -(C=O)O-, -O(C=O)-, -(C=O)NH-, -NH(C=O)-;

[0034] R3, R4, R5, and R6 are each independently selected from H, C2-C 20 hydrocarbon group;

[0035] R7 is selected from C1-C5 hydrocarbon groups, preferably from C2-C4 hydrocarbon groups.

[0036] In another aspect of the invention, a lipid nanoparticle is provided, the lipid nanoparticle comprising the aforementioned ionizable lipid, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0037] In another aspect of the invention, a lipid nanoparticle is provided, the lipid nanoparticle comprising the aforementioned ionizable lipid, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, or an ionizable lipid prepared as described above, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

[0038] In some embodiments, the lipid nanoparticles also contain auxiliary lipids.

[0039] In some embodiments, the ionizable lipids in the lipid nanoparticles are present in a molar ratio of 30-65% of the total lipid content.

[0040] In some embodiments, the assisting lipids include assisting phospholipids, sterols, polymer-conjugated lipids, or combinations thereof.

[0041] In some embodiments, the assisting lipid is a combination of assisting phospholipids, sterols, and lipids conjugated with various polymers.

[0042] In some embodiments, the auxiliary phospholipid is selected from: 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), and 1,2-myristoyl-sn-glycerol-3-phosphoethanolamine (DMP). E), 1,2-dioleoyl-sn-glycerol-3-phosphoyl-rac-(1-glycerol) sodium salt (DOPG-Na), 1,2-dipalmitoylphosphatidylglycerol (DPPG), 1-palmitoyl-2-oleoyllecithin (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), distearate phosphatidylethanolamine (DSPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), 1-stearoyl-2-oleoyl-SN-glycerol-3-phosphatidylethanolamine (SOPE), or combinations thereof.

[0043] In some embodiments, the sterol includes cholesterol or cholesterol derivatives.

[0044] In some embodiments, the polymer-conjugated lipid is a polyethylene glycol (PEG) lipid.

[0045] In some embodiments, the polyethylene glycolated (PEG) lipid is a maleimide-modified polyethylene glycolated (PEG) lipid.

[0046] In some embodiments, the maleimide-modified polyethylene glycol (PEG) lipid is further coupled with other active substances. These other active substances are coupled via maleimide.

[0047] In some embodiments, the other active substances are selected from polypeptides, proteins, amino acids, vitamins, mannose, or combinations thereof.

[0048] In some embodiments, the PEGylated lipid is selected from: DMG-PEG2000, DMG-PEG5000, DSPE-PEG2000, DSPE-PEG5000, DSG-PEG2000, DMG-PEG2000-MAL, DSPE-PEG2000-MAL, DMG-PEG5000-MAL, DSPE-PEG5000-MAL, DMG-PEG2000-MAL- / DSPE-PEG2000-MAL- / DMG-PEG5000-MAL- / DSPE-PEG5000-MAL- conjugates (the conjugates are polypeptides, proteins, amino acids, vitamins, mannose, or other active substances), or combinations thereof.

[0049] In some embodiments, the PEGylated lipid is selected from: DMG-PEG2000, DSPE-PEG2000, DSPE-PEG5000-MAL, DSPE-PEG5000-MAL-protein (such as antibody), or combinations thereof.

[0050] In some embodiments, the lipid nanoparticles comprise ionizable lipids, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of ionizable lipids:DSPC:cholesterol:DMG-PEG2000 is (30-65):(10-30):(20-50):(1-5).

[0051] In some implementations, the molar ratio of ionizable lipids:DSPC:cholesterol:DMG-PEG2000 is (40-60):(10-20):(25-45):(1-2).

[0052] In some embodiments, the lipid nanoparticles comprise ionizable lipids, DSPC, cholesterol, and DMG-PEG2000, wherein the molar ratio of ionizable lipids:DSPC:cholesterol:DMG-PEG2000 is (40-60):(10-20):(28.5-38.5):1.5.

[0053] In some embodiments, the lipid nanoparticles are lipid nanoparticles modified with a targeting substance.

[0054] In some embodiments, the lipid nanoparticles modified with the targeting substance comprise ionizable lipids, DSPC, cholesterol, DSPE-PEG2000 or DMG-PEG2000, and DSPE-PEG5000-MAL-conjugate (or targeting substance) in a molar ratio of (30-65):(10-30):(20-50):(0.5-5):(0.1-2), preferably (40-60):(10-20):(25-45):(0.5-2):(0.1-2).

[0055] In some embodiments, the lipid nanoparticles modified with the targeting substance comprise ionizable lipids, DSPC, cholesterol, DSPE-PEG2000 or DMG-PEG2000, and DSPE-PEG5000-MAL-conjugate (or targeting substance) in a molar ratio of (40-60):(10-20):(28.5-38.5):(0.5-1.5):(0.1-1).

[0056] In some embodiments, the targeting substance is selected from at least one of the following: ligand, receptor, antibody, antigen-binding fragment of antibody, aptamer, and polypeptide.

[0057] In some embodiments, the targeting substance includes an antibody or an antigen-binding fragment thereof that targets a target protein.

[0058] In some implementations, the antibody includes monoclonal antibodies and polyclonal antibodies.

[0059] In some embodiments, the antibody or its antigen-binding fragment is selected from: intact antibody, nanobody (VHH), Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), double scFv, (scFv)2.

[0060] In some embodiments, the modification includes covalent coupling, non-covalent mixing, and / or other chemical bonding.

[0061] In some implementations, the target protein is an immune cell surface protein.

[0062] In some implementations, the immune cells include T cells, B cells, NK cells, or combinations thereof.

[0063] In some embodiments, the T cells are selected from: human primary T cells, JM cells, Jurkat cells, or combinations thereof.

[0064] In some implementations, the primary human T cells include helper T cells, cytotoxic T cells, immunoregulatory T cells, and memory T cells.

[0065] In some embodiments, the target protein is a cell surface protein selected from the group consisting of: CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD1 53, CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR1, CCR2, CCR4, CCR6, CCR7, or combinations thereof.

[0066] In some embodiments, the target protein is a cell surface protein selected from the group consisting of CD3, CD4, CD5, CD8, CD7, CD28, CD45, CD2, or a combination thereof.

[0067] In some embodiments, the lipid nanoparticles further comprise bioactive substances encapsulated within the lipid nanoparticles.

[0068] In some embodiments, the lipid nanoparticles have a particle size of 80-170 nm.

[0069] In some embodiments, the lipid nanoparticles have a particle size of 85-150 nm.

[0070] In some embodiments, the lipid nanoparticles have a particle size of 90-135 nm.

[0071] In some embodiments, the lipid nanoparticles have a particle size of 95-125 nm.

[0072] In some embodiments, the lipid nanoparticles have a particle size of 88.68-132.78 nm.

[0073] In some embodiments, the lipid nanoparticles have a particle size of 88.08-99.84 nm.

[0074] In some embodiments, the PDI (Polydispersity Index) of the lipid nanoparticles is ≤0.25.

[0075] In some embodiments, the PDI of the lipid nanoparticles is ≤0.22.

[0076] In some embodiments, the PDI of the lipid nanoparticles is 0.04-0.22.

[0077] In some embodiments, the PDI of the lipid nanoparticles is 0.05-0.20.

[0078] In some embodiments, the PDI of the lipid nanoparticles is 0.07-0.16.

[0079] In some embodiments, the PDI of the lipid nanoparticles is 0.073-0.136.

[0080] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is ≥60%.

[0081] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is ≥80%.

[0082] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is ≥85%.

[0083] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 70-98%.

[0084] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 85-98%.

[0085] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 90-98%.

[0086] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 66.53-96.28%.

[0087] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 90.33%-97.81%.

[0088] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 93.0-95.77%.

[0089] In some embodiments, the cell viability of the lipid nanoparticles is ≥50%.

[0090] In some embodiments, the cell viability of the lipid nanoparticles is ≥70%.

[0091] In some embodiments, the cell viability of the lipid nanoparticles is ≥85%.

[0092] In some embodiments, the cell viability of the lipid nanoparticles is ≥90%.

[0093] In some embodiments, the cell viability of the lipid nanoparticles is 88-99%.

[0094] In some embodiments, the cell viability of the lipid nanoparticles is 93-99%.

[0095] In some embodiments, the cell viability of the lipid nanoparticles is 92.26% to 98.31%.

[0096] In some embodiments, the cell viability of the lipid nanoparticles is 97.86%-99.38%.

[0097] In some embodiments, the transfection efficiency of the lipid nanoparticles is >74.54%.

[0098] In some embodiments, the transfection efficiency of the lipid nanoparticles is 75-99%.

[0099] In some embodiments, the transfection efficiency of the lipid nanoparticles is 75-89%.

[0100] In some embodiments, the expression level of the lipid nanoparticles is >6338.1 RFU.

[0101] In some embodiments, the expression level of the lipid nanoparticles is 6500-12400 RFU.

[0102] In some embodiments, the expression level of the lipid nanoparticles is 6600-9100 RFU.

[0103] In some embodiments, the bioactive substance is selected from: nucleic acids, proteins, polypeptides, small molecules, or combinations thereof.

[0104] In some embodiments, the nucleic acid includes DNA, plasmid, messenger RNA, small interfering RNA, antisense oligonucleotide, ribosomal RNA, self-replicating RNA, circular RNA, transfer RNA, and preferably mRNA.

[0105] In some embodiments, the nucleic acid includes DNA, plasmid, messenger RNA, small interfering RNA, antisense oligonucleotide, ribosomal RNA, self-replicating RNA, circular RNA, transfer RNA, or combinations thereof, preferably mRNA.

[0106] In another aspect of the invention, a lipid nanoparticle pharmaceutical formulation is provided, the lipid nanoparticle pharmaceutical formulation comprising:

[0107] i) The aforementioned lipid nanoparticles;

[0108] ii) Bioactive substances encapsulated in the lipid nanoparticles; and

[0109] iii) Pharmaceutically acceptable carriers.

[0110] In some embodiments, the bioactive substance is selected from: nucleic acids, proteins, polypeptides, small molecules, or combinations thereof.

[0111] In some embodiments, the nucleic acid includes DNA, plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide, ribosomal RNA, self-replicating RNA, circular RNA, microRNA, transfer RNA, preferably mRNA.

[0112] In some embodiments, the nucleic acid includes DNA, plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide, ribosomal RNA, self-replicating RNA, circular RNA, microRNA, transfer RNA, or combinations thereof, preferably mRNA.

[0113] In some embodiments, the bioactive substance is a nucleic acid, and in the lipid nanoparticle drug, the molar ratio of ionizable N atoms in the ionizable lipid molecule to phosphate groups in the nucleic acid molecule is (2-10):1.

[0114] In some embodiments, in the lipid nanoparticle drug, the molar ratio of ionizable N atoms in the ionizable lipid molecule to phosphate groups in the nucleic acid molecule is (4–8):1.

[0115] In some embodiments, the molar ratio of ionizable N atoms in the ionizable lipid nanoparticle drug to phosphate groups in the nucleic acid molecule is 3:1, 4:1, 5:1, or 6:1.

[0116] In some embodiments, the hydrated particle size of the lipid nanoparticle drug is 50-200 nm.

[0117] In some embodiments, the hydrated particle size of the lipid nanoparticle drug is 70-150 nm.

[0118] In some embodiments, the hydrated particle size of the lipid nanoparticle drug is 75-110 nm.

[0119] In some embodiments, the lipid nanoparticles have a particle size of 80-170 nm.

[0120] In some embodiments, the lipid nanoparticles have a particle size of 85-150 nm.

[0121] In some embodiments, the lipid nanoparticles have a particle size of 90-135 nm.

[0122] In some embodiments, the lipid nanoparticles have a particle size of 95-125 nm.

[0123] In some embodiments, the lipid nanoparticles have a particle size of 88.68-132.78 nm.

[0124] In some embodiments, the lipid nanoparticles have a particle size of 88.08-99.84 nm.

[0125] In some embodiments, the PDI of the lipid nanoparticles is ≤0.25.

[0126] In some embodiments, the PDI of the lipid nanoparticles is ≤0.22.

[0127] In some embodiments, the PDI of the lipid nanoparticles is 0.04-0.22.

[0128] In some embodiments, the PDI of the lipid nanoparticles is 0.05-0.20.

[0129] In some embodiments, the PDI of the lipid nanoparticles is 0.07-0.16.

[0130] In some embodiments, the PDI of the lipid nanoparticles is 0.073-0.136.

[0131] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is ≥60%.

[0132] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is ≥80%.

[0133] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is ≥85%.

[0134] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 70-98%.

[0135] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 85-98%.

[0136] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 90-98%.

[0137] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 66.53-96.28%.

[0138] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 90.33%-97.81%.

[0139] In some embodiments, the encapsulation efficiency of the lipid nanoparticles is 93.0-95.77%.

[0140] In some embodiments, the cell viability of the lipid nanoparticles is ≥50%.

[0141] In some embodiments, the cell viability of the lipid nanoparticles is ≥70%.

[0142] In some embodiments, the cell viability of the lipid nanoparticles is ≥85%.

[0143] In some embodiments, the cell viability of the lipid nanoparticles is ≥90%.

[0144] In some embodiments, the cell viability of the lipid nanoparticles is 88-99%.

[0145] In some embodiments, the cell viability of the lipid nanoparticles is 93-99%.

[0146] In some embodiments, the cell viability of the lipid nanoparticles is 92.26% to 98.31%.

[0147] In some embodiments, the cell viability of the lipid nanoparticles is 97.86%-99.38%.

[0148] In some embodiments, the transfection efficiency of the lipid nanoparticles is >74.54%.

[0149] In some embodiments, the transfection efficiency of the lipid nanoparticles is 75-99%.

[0150] In some embodiments, the transfection efficiency of the lipid nanoparticles is 75-89%.

[0151] In some embodiments, the expression level of the lipid nanoparticles is >6338.1 RFU.

[0152] In some embodiments, the expression level of the lipid nanoparticles is 6500-12400 RFU.

[0153] In some embodiments, the expression level of the lipid nanoparticles is 6600-9100 RFU.

[0154] In some embodiments, the lipid nanoparticle drug formulation can be used for the treatment and / or prevention of tumors, infectious diseases, and rare diseases.

[0155] In some embodiments, the dosage form of the lipid nanoparticle drug formulation is selected from: injection, lyophilized formulation, nebulized inhalation formulation, and topical formulation.

[0156] In some embodiments, the lipid nanoparticle drug formulation is administered by injection, i.e., intravenous, intramuscular, intradermal, subcutaneous, intrathecal, duodenal, or intraperitoneal injection.

[0157] In some embodiments, the lipid nanoparticle drug formulation is administered by inhalation, such as intranasal administration.

[0158] In some embodiments, the lipid nanoparticle drug formulation is administered transdermally, such as by topical application or electrode delivery.

[0159] In another aspect of the present invention, a method for preparing the aforementioned lipid nanoparticle drug formulation is provided, wherein the method comprises:

[0160] (a) The aforementioned ionizable lipid, or its pharmaceutically acceptable salt, tautomer or stereoisomer, and optionally an auxiliary lipid are mixed with an organic solvent to obtain a lipid organic phase;

[0161] (b) The bioactive substance is mixed with an aqueous solvent to obtain an aqueous phase containing the bioactive substance;

[0162] (c) The lipid organic phase from step (a) is mixed with the aqueous phase from step (b) to obtain the lipid nanoparticle drug.

[0163] In another aspect of the present invention, a method for preparing the aforementioned lipid nanoparticle drug formulation is provided, wherein the method comprises:

[0164] (a) The aforementioned ionizable lipid, or a pharmaceutically acceptable salt thereof, tautomer or stereoisomer, or an ionizable lipid prepared as described above, or a pharmaceutically acceptable salt thereof, tautomer or stereoisomer, and optionally an auxiliary lipid, are mixed with an organic solvent to obtain a lipid organic phase.

[0165] (b) The bioactive substance is mixed with an aqueous solvent to obtain an aqueous phase containing the bioactive substance;

[0166] (c) The lipid organic phase from step (a) is mixed with the aqueous phase from step (b) to obtain the lipid nanoparticle drug.

[0167] In some embodiments, the organic solvent includes ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran, or combinations thereof, with ethanol being the preferred solvent.

[0168] In some implementations, the aqueous solvent is a buffer solution.

[0169] In some embodiments, the aqueous solvent is a buffer solution with a pH range of 3-7.

[0170] In some embodiments, the acidic buffer solution is a sodium acetate buffer solution with a pH of 4.0.

[0171] In some embodiments, the volume ratio of the lipid organic phase to the aqueous phase containing bioactive substances is 1:(2-5), preferably 1:(3-4).

[0172] In some implementations, in step (c), the lipid organic phase and the aqueous phase are mixed via a microfluidic chip.

[0173] In some embodiments, the method further includes step (d): purifying, concentrating, and filtering the lipid nanoparticle drug obtained in step (c) to remove bacteria.

[0174] In another aspect of the invention, there is provided the use of the aforementioned ionizable lipid, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, for the preparation of a drug delivery system.

[0175] In another aspect of the invention, there is provided the use of the aforementioned ionizable lipid, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, or an ionizable lipid prepared by the aforementioned method, or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, in the preparation of a drug delivery system.

[0176] In some embodiments, the delivery system is selected from lipid nanoparticles, liposomes, and polymer nanoparticles, and is preferably used for preparing lipid nanoparticles.

[0177] In some implementations, the drug delivery system is used to deliver drugs for the treatment and / or prevention of cancer, infectious diseases, and rare diseases.

[0178] In some embodiments, the drug delivery system is used to deliver drugs for the treatment and / or prevention of cancer, infectious diseases, preventive vaccines, autoimmune diseases, and rare diseases.

[0179] In some embodiments, the drug delivery system is a lipid nanoparticle.

[0180] In some implementations, the drug delivery system contains a targeting substance modification.

[0181] In some embodiments, the target substance is one or more of a ligand, receptor, antibody and / or its antigen-binding fragment, aptamer, or polypeptide.

[0182] In some embodiments, the targeting substance includes an antibody or an antigen-binding fragment thereof that targets a target protein.

[0183] In some implementations, the antibody includes monoclonal antibodies and polyclonal antibodies.

[0184] In some embodiments, the antibody or its antigen-binding fragment is selected from: intact antibody, nanobody (VHH), Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), double scFv, (scFv)2.

[0185] In some embodiments, the modification includes covalent coupling, non-covalent mixing, and / or other chemical bonding.

[0186] In some embodiments, the target protein is a cell surface protein selected from the group consisting of: CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD1 53, CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR1, CCR2, CCR4, CCR6, CCR7, or combinations thereof.

[0187] In some embodiments, the target protein is a cell surface protein selected from the group consisting of CD3, CD4, CD5, CD8, CD7, CD28, CD45, CD2, or a combination thereof.

[0188] In another aspect of the invention, the use of the aforementioned ionizable lipids, or pharmaceutically acceptable salts, tautomers, or stereoisomers thereof, is provided for the preparation of lipid nanoparticle pharmaceutical formulations for the delivery of bioactive substances to cells.

[0189] In another aspect of the invention, the use of the aforementioned ionizable lipids, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, or ionizable lipids prepared by the aforementioned method, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof, for the preparation of lipid nanoparticle pharmaceutical formulations for the delivery of bioactive substances to cells is provided.

[0190] In some embodiments, the bioactive substance is selected from: nucleic acids, proteins, polypeptides, small molecules, or combinations thereof.

[0191] In some embodiments, the nucleic acid includes DNA, plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide, ribosomal RNA, self-replicating RNA, circular RNA, microRNA, and transfer RNA, preferably mRNA.

[0192] In some embodiments, the nucleic acid includes DNA, plasmid, messenger RNA (mRNA), small interfering RNA (siRNA), antisense oligonucleotide, ribosomal RNA, self-replicating RNA, circular RNA, microRNA, and transfer RNA, or combinations thereof, preferably mRNA.

[0193] In some implementations, the bioactive substance is mRNA.

[0194] In some embodiments, the mRNA is transfected into cells and expressed in the cells.

[0195] In some embodiments, the cells are in vivo cells or in vitro cells.

[0196] In some implementations, the cells are immune cells.

[0197] In some implementations, the cells are selected from T cells, B cells, NK cells, or combinations thereof.

[0198] In some embodiments, the T cells are selected from: human primary T cells, JM cells, Jurkat cells, or combinations thereof.

[0199] In some implementations, the primary human T cells are selected from helper T cells, cytotoxic T cells, immunomodulatory T cells, and memory T cells.

[0200] In some embodiments, the lipid nanoparticle drug formulation also contains targeting substance modification.

[0201] In some embodiments, the target substance is one or more of a ligand, receptor, antibody and / or its antigen-binding fragment, aptamer, or polypeptide.

[0202] In some embodiments, the targeting substance includes an antibody or an antigen-binding fragment thereof that targets a target protein.

[0203] In some implementations, the antibody includes monoclonal antibodies and polyclonal antibodies.

[0204] In some embodiments, the antibody or its antigen-binding fragment is selected from: intact antibody, nanobody (VHH), Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), double scFv, (scFv)2.

[0205] In some embodiments, the targeting material modifies lipid nanoparticles by covalent coupling, non-covalent mixing, and / or other chemical bonding.

[0206] In some embodiments, the target protein is a cell surface protein selected from the group consisting of: CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD1 53, CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR1, CCR2, CCR4, CCR6, CCR7, or combinations thereof.

[0207] In some embodiments, the target protein is a cell surface protein selected from the group consisting of CD3, CD4, CD5, CD8, CD7, CD28, CD45, CD2, or a combination thereof.

[0208] In another aspect of the invention, the use of the aforementioned lipid nanoparticles in the preparation of vaccines, medicaments for the treatment and / or prevention of tumors, infectious diseases, autoimmune diseases, and rare diseases is provided.

[0209] In another aspect of the invention, a method for administering a preventive vaccine, treating tumors, infectious diseases, autoimmune diseases, and rare diseases is provided, comprising the step of administering the aforementioned lipid nanoparticles to a patient in need.

[0210] In another aspect of the invention, the aforementioned lipid nanoparticles are provided for use in vaccine prevention, treatment and / or prevention of tumors, infectious diseases, autoimmune diseases and rare diseases.

[0211] In another aspect of the invention, the aforementioned lipid nanoparticles are provided for use in vaccine prevention, treatment and / or prevention of tumors, infectious diseases, autoimmune diseases, and rare diseases.

[0212] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0213] Figure 1 shows the 1H NMR spectrum of AL08-001.

[0214] Figure 2 shows the 1H NMR spectrum of AL08-003.

[0215] Figure 3 shows the 1H NMR spectrum of AL08-004.

[0216] Figure 4 shows the 1H NMR spectrum of AL08-005.

[0217] Figure 5 shows the 1H NMR spectrum of AL08-006.

[0218] Figure 6 shows the 1H NMR spectrum of AL08-007.

[0219] Figure 7 shows the 1H NMR spectrum of AL08-008.

[0220] Figure 8 shows the 1H NMR spectrum of AL08-009.

[0221] Figure 9 shows the 1H NMR spectrum of AL08-010.

[0222] Figure 10 shows the 1H NMR spectrum of AL08-011.

[0223] Figure 11 shows the 1H NMR spectrum of AL08-012.

[0224] Figure 12 shows the 1H NMR spectrum of AL08-014.

[0225] Figure 13 shows the 1H NMR spectrum of AL08-015.

[0226] Figure 14 shows the 1H NMR spectrum of AL08-018.

[0227] Figure 15 shows the 1H NMR spectrum of AL08-019.

[0228] Figure 16 shows the 1H NMR spectrum of AL08-020.

[0229] Figure 17 shows the 1H NMR spectrum of AL08-021.

[0230] Figure 18 shows the 1H NMR spectrum of AL08-022.

[0231] Figure 19 shows the 1H NMR spectrum of AL08-023.

[0232] Figure 20 shows the 1H NMR spectrum of AL08-024.

[0233] Figure 21 shows the 1H NMR spectrum of AL08-025.

[0234] Figure 22 shows the 1H NMR spectrum of AL08-026.

[0235] Figure 23 shows the 1H NMR spectrum of AL08-027.

[0236] Figure 24 shows the results of flow cytometry demonstration of LNP-mRNA (formulations ALP801, ALP08-005-1, ALP08-012-1, ALP08-019-1, ALP08-022-1, ALP08-025-2, ALP08-025-2, ALP08-016-1 and ALP08-017-2 encapsulated mCherry) transfection into human primary T cells.

[0237] Figure 25 shows the results of Ab-LNP transfection of hPBMCs; other cell types include B cells, NK cells, and monocytes. Detailed Implementation

[0238] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time an ionizable lipid. This ionizable lipid possesses advantages such as stable physicochemical properties and low toxicity. Drug delivery systems using this ionizable lipid to encapsulate drug payloads (e.g., mRNA) exhibit high delivery efficiency and low toxicity. While efficiently delivering drug payloads and increasing their expression levels, the safety of the drug delivery system is also improved, resulting in more prominent preventative and therapeutic effects. Furthermore, when the ionizable lipid is modified with a targeting substance (such as an antibody), it can be specifically delivered to the target cells or tissues of the antibody during delivery. Based on these findings, this invention was completed.

[0239] the term

[0240] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0241] The term "alkyl" refers to a saturated carbon chain having 1 to 20 carbon atoms, which may be straight or branched or a combination thereof, unless otherwise defined. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl and tert-butyl, pentyl, hexyl, heptyl, octyl, etc.

[0242] Unless otherwise specified in the specification, alkyl groups may be optionally substituted. The term "unsaturated hydrocarbon group" means that the group contains at least one C=C double bond (alkenyl) or at least one C≡C triple bond (alkynyl). "Alkenyl", "alkenyl" and "alkynyl" are collectively referred to as "hydrocarbon group".

[0243] In the claims of this invention, when describing "C2-C" 20 When referring to a "hydrocarbon group," it means that the group can be an alkane group, olefin group, or alkyne group with 1-30 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms). "C1-C5 hydrocarbon group," "C2-C4 hydrocarbon group," and "C2-C3 hydrocarbon group" have similar meanings.

[0244] When describing "alkyl," it refers to a saturated hydrocarbon group having a given number of carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms). It can be straight-chain or branched, and is typically a chain group without cyclic structures. Alkyl groups satisfying the aforementioned number of carbon atoms are all within the scope of this term.

[0245] When describing "alkenyl," it refers to an olefinic group having a given number of carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms), which can be linear or branched, and is generally a chain group without a cyclic structure. Alkenyl groups satisfying the aforementioned number of carbon atoms are all within the scope of this term. In different embodiments of the invention, the alkenyl group can be a group formed from a mono-olefin or a polyolefin (e.g., a diene).

[0246] When describing "C2-C" 20 When referring to "a straight-chain or branched olefin group," it means that the group can be an olefin group having a given number of carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms). It can be straight-chain or branched, and is generally a chain group without a cyclic structure. Olefin groups satisfying the aforementioned number of carbon atoms are all within the scope of this term. In different embodiments of the present invention, the olefin group can be a monoolefin or a polyolefin (e.g., a diene).

[0247] In this application, when the definition of a divalent group includes "none", it means that the adjacent structural segments of the divalent group are directly connected by chemical bonds.

[0248] Ionizable lipids

[0249] As used herein, the terms "ionizable lipids of the present invention" and "ionizable cationic lipids of the present invention" are used interchangeably and both refer to lipid compounds having the structure of Formula I, or pharmaceutically acceptable salts, tautomers or stereoisomers thereof.

[0250] Ionizable lipids protonate and transform into cationic lipids at low pH values, while at normal physiological pH values ​​they transform into helper phospholipids. Helper phospholipids interact less with the anionic cell membranes of blood cells, improving the biocompatibility of lipid nanoparticles. When lipid nanoparticles are endocytosed by cells, the pH value within the endosomes is low, causing the lipids to protonate and become positively charged. This reduces or even disrupts the membrane structure, facilitating the escape of lipid nanoparticle endosomes. In summary, the pH-sensitive nature of lipids is beneficial for the in vivo delivery of lipid nanoparticles carrying bioactive components (such as mRNA molecules).

[0251] assist lipids

[0252] As used herein, the term "accessory lipid" refers to other types of lipids in lipid nanoparticles besides ionizable lipids, including accessory phospholipids, sterols, polymer-conjugated lipids, or combinations thereof. Accessory lipids are primarily used to improve the properties of lipid nanoparticles, such as stability, delivery efficiency, tolerability, and biocompatibility.

[0253] In some embodiments, the auxiliary phospholipids include (but are not limited to) 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), and 1,2-myristoyl-sn-glycerol-3-phosphoethanolamine. Amine (DMPE), sodium 1,2-dioleoyl-sn-glycerol-3-phosphoyl-rac-(1-glycerol) (DOPG-Na), 1,2-dipalmitoylphosphatidylglycerol (DPPG), 1-palmitoyl-2-oleoyllecithin (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), distearate phosphatidylethanolamine (DSPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or combinations thereof.

[0254] In a preferred embodiment of the present invention, the auxiliary phospholipid is DSPC. DSPC is a commonly used phosphatidylcholine. The tail group of DSPC is a saturated alkane chain, with a melting point of -54°C and a cylindrical shape. It forms a layered structure in lipid nanoparticles, making the structure of lipid nanoparticles more stable.

[0255] In a preferred embodiment of the present invention, the auxiliary phospholipid is DOPE. DOPE is a commonly used phosphatidylethanolamine. The tail group of DOPE consists of two unsaturated alkane chains, has a melting point of -30°C, and is conical in shape. In lipid nanoparticles, it easily forms an inverted hexagon, causing instability in the endosome membrane and facilitating the escape of lipid nanoparticle endosomes.

[0256] In some embodiments, the sterols include (but are not limited to) cholesterol or cholesterol derivatives. Cholesterol can modulate the integrity and stiffness of lipid membranes, enhancing the stability of lipid nanoparticles, while the morphology of cholesterol derivatives can affect the delivery efficiency and biodistribution of lipid nanoparticles, such as the chain length of the hydrophobic tail group of cholesterol analogs, the flexibility of the sterol ring, and the polarity of the hydroxyl group. Cholesterol also affects the morphology of lipid nanoparticles; cholesterol derivatives result in lipid nanoparticles with a multilayered polyhedral structure defined by lipids rather than a spherical shape. Simultaneously, cholesterol affects the selectivity of lipid nanoparticles for their target sites: lipid nanoparticles containing cholesterol oleate are more selective for hepatic endothelial cells than for hepatocytes; when containing cholesterol with oxidized tail groups, the content of lipid nanoparticles in hepatic endothelial cells and Kupffer cells is higher than that in hepatocytes.

[0257] In some embodiments, the polymer-conjugated lipids are polyethylene glycol (PEG)-conjugated lipids, also known as PEGylated lipids or PEGylated lipids. PEGylated lipids have multiple effects on the properties of lipid nanoparticles: the amount of PEGylated lipids affects the particle size and potential of lipid nanoparticles; it reduces particle aggregation and improves the stability of lipid nanoparticles; it reduces the renal and mononuclear phagocyte system (MPS)-mediated particle clearance rate and prolongs particle circulation time; the surface functional groups can be modified with ligands to improve targeted delivery capability. Molar mass and lipid length affect the properties of PEGylated lipids. DMG-PEG2000 and DSG-PEG2000 are both neutral phospholipids with saturated alkyl chain lengths of C14, C16, or C18, respectively. However, DMG-PEG2000 can separate from lipid nanoparticles more quickly, facilitating cellular uptake of nanoparticles and endosome escape. Therefore, the delivery efficiency of DMG-PEG2000 is superior to that of DSG-PEG2000.

[0258] In a preferred embodiment of the invention, the auxiliary lipid is a combination of DSPC, cholesterol, and DMG-PEG2000.

[0259] In a preferred embodiment of the invention, the auxiliary lipid is a combination of DSPC, cholesterol, DSPE-PEG2000 and DSPE-PEG5000-MAL.

[0260] Lipid nanoparticles (LNPs)

[0261] As used herein, the terms "lipid nanoparticles," "lipid nanoparticles," or "LNP" refer to particles with a diameter of approximately 5 to 500 nm. In some embodiments, the lipid nanoparticles contain one or more active agents (bioactive substances). In some embodiments, the lipid nanoparticles include nucleic acids. In some embodiments, the nucleic acids are condensed within the nanoparticles with cationic lipids, polymers, or multivalent small molecules, and an external lipid coating that interacts with the biological environment. Nucleic acids are naturally rigid polymers and tend to have elongated configurations due to the repulsive forces between phosphate groups. In cells, to cope with volume constraints, DNA can package itself under appropriate solution conditions with the help of ions and other molecules. Typically, DNA condensation is defined as the collapse of an elongated DNA strand into a compact, ordered particle containing only one or a few molecules. By binding to phosphate groups, cationic lipids can concentrate DNA and cause it to pack tightly together by neutralizing the phosphate charge.

[0262] In some embodiments, the bioactive substance is encapsulated in an LNP. In some embodiments, the bioactive substance can be anionic compounds, including but not limited to DNA, RNA (messenger RNA, transfer RNA, ribosomal RNA, microRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA, and small interfering RNA), nucleoproteins, peptides, nucleic acids, ribozymes, DNA-containing nucleoproteins such as intact or partially deproteinized viral particles (viral particles), oligomeric and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins), or combinations thereof. In some embodiments, the bioactive substance can be mixed with an adjuvant.

[0263] In LNP vaccine products, the bioactive substance is typically contained within the LNP itself. In some embodiments, the bioactive substance includes nucleic acids. Typically, water-soluble nucleic acids are condensed within the particle with cationic lipids or polycationic polymers, and the particle surface is enriched with accessory phospholipids or PEG lipid derivatives. Additional ionizable cationic lipids may also be located on the surface; upon entering the lysosome, these ionizable cationic lipids become positively charged due to the acidic environment of the lysosome, interacting with the lysosomal membrane and facilitating endosome escape.

[0264] Regarding LNPs, ionizable lipids can possess different properties or functions. Due to the pKa of the amino group, when the external pH is lower than the pKa of the lipid molecule, it can be protonated and become positively charged. Under these conditions, the lipid molecule can electrostatically bind to the phosphate group of nucleic acid, which allows for LNP formation and nucleic acid encapsulation. Furthermore, the surface charge of LNPs in biological fluids (e.g., blood) at physiological pH is essentially neutral. High LNP surface charge is associated with toxicity, rapid clearance of circulating LNPs by fixed and free macrophages, hemolytic toxicity, and immune activation.

[0265] In some embodiments, the pKa can be high enough that ionizable cationic lipids can take on a positively charged form at acidic endosomal pH. This allows the cationic lipids to bind to endogenous endosomal anionic lipids to promote membrane lysis of non-bilayer structures, such as the hexagonal HII phase, resulting in more efficient intracellular delivery. In some embodiments, the pKa ranges from 6.2 to 6.5. For example, the pKa can be about 6.2, about 6.3, about 6.4, or about 6.5. An unsaturated tail also contributes to the lipids' ability to take on non-bilayer structures.

[0266] The release of nucleic acids in LNP formulations, as well as other characteristics such as liposome clearance and circulating half-life, can be altered by the presence of polyethylene glycol and / or sterols (e.g., cholesterol) or other potential additives in the LNP, and by the overall chemical structure (including the pKa of any ionizable cationic lipids that are part of the formulation).

[0267] In one aspect of the invention, a lipid nanoparticle is provided, the lipid nanoparticle comprising the ionizable lipid described in the first aspect of the invention, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof. Further, the lipid nanoparticle also comprises one or more auxiliary lipids, including auxiliary phospholipids, steroids, and polymer-conjugated lipids. Further, the lipid nanoparticle also comprises targeting modifications.

[0268] In some embodiments, the targeting substance is one or more of a ligand, receptor, antibody and / or its antigen-binding fragment, aptamer, or polypeptide. In some embodiments, the targeting substance comprises an antibody or its antigen-binding fragment targeting a target protein. In some embodiments, the antibody comprises monoclonal antibodies and polyclonal antibodies. In some embodiments, the antibody or its antigen-binding fragment is selected from: intact antibodies, nanobodies (VHH), Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single-chain Fv antibodies (“scFv”), bisscFv, (scFv)2. In some embodiments, the targeting substance modifies lipid nanoparticles by covalent coupling, non-covalent mixing, and / or other chemical bonding. In some embodiments, the target protein is a cell surface protein selected from the group consisting of: CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD1 53, CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR1, CCR2, CCR4, CCR6, CCR7, or combinations thereof.

[0269] Lipid nanoparticle drug formulation

[0270] In another aspect of the invention, a lipid nanoparticle pharmaceutical formulation (or lipid nanoparticle pharmaceutical combination or LNP composition) is provided, the lipid nanoparticle pharmaceutical formulation comprising lipid nanoparticles as described in the third aspect of the invention, a bioactive substance encapsulated within the lipid nanoparticles, a pharmaceutically acceptable carrier, and optionally a targeting substance modifying the lipid nanoparticles. The lipid nanoparticle pharmaceutical formulation is used to deliver the bioactive substance to cells or target tissues. The cells include cells in vitro or cells in vivo of a desired subject.

[0271] In some embodiments, the targeting substance is one or more of a ligand, receptor, antibody and / or its antigen-binding fragment, aptamer, or polypeptide. In some embodiments, the targeting substance comprises an antibody or its antigen-binding fragment targeting a target protein. In some embodiments, the antibody comprises monoclonal antibodies and polyclonal antibodies. In some embodiments, the antibody or its antigen-binding fragment is selected from: intact antibodies, nanobodies (VHH), Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single-chain Fv antibodies (“scFv”), bisscFv, (scFv)2. In some embodiments, the targeting substance modifies lipid nanoparticles by covalent coupling, non-covalent mixing, and / or other chemical bonding. In some embodiments, the target protein is a cell surface protein selected from the group consisting of: CD1, CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD25, CD26, CD27, CD28, CD30, CD38, CD39, CD40L, CD44, CD45, CD62L, CD69, CD73, CD80, CD83, CD86, CD95, CD103, CD119, CD126, CD150, CD1 53, CD154, CD161, CD183, CD223, CD254, CD275, CD45RA, CXCR3, CXCR5, FasL, IL18R1, CTLA-4, OX40, GITR, LAG3, ICOS, PD-1, leu-12, TCR, TLR1, TLR2, TLR3, TLR4, TLR6, NKG2D, CCR1, CCR2, CCR4, CCR6, CCR7, or combinations thereof.

[0272] In some embodiments, the bioactive substance is encapsulated in an LNP. In some embodiments, the bioactive substance can be anionic compounds, including but not limited to DNA, RNA (messenger RNA, transfer RNA, ribosomal RNA, microRNA, etc.), natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA, and small interfering RNA), nucleoproteins, peptides, nucleic acids, ribozymes, DNA-containing nucleoproteins such as intact or partially deproteinized viral particles (viral particles), and oligomeric and polymeric anionic compounds other than DNA (e.g., acidic polysaccharides and glycoproteins). In some embodiments, the bioactive substance can be mixed with an adjuvant.

[0273] In some embodiments, the LNP composition comprises: nucleic acid, an ionizable cationic lipid having the structure shown in formula (I), an accessory phospholipid (e.g., DSPC, DOPE, DOPC or combinations thereof), a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, DSPE-PEG5000-MAL, DMG-PEG2000-MAL or DMG-PEG5000-MAL, etc.). In some embodiments, the LNP composition comprises: nucleic acid; an ionizable cationic lipid having the structure shown in Formula I, comprising 30-65% (molar percentage, the same below) of the total lipids of the composition; an accessory phospholipid (e.g., DSPC, DOPE, DOPC or combinations thereof) comprising 5-30% of the total lipids of the composition; a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol) comprising 20-50% of the total lipids of the composition; and a polymer-conjugated lipid (e.g., DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, DSPE-PEG5000-MAL, DMG-PEG2000-MAL or DMG-PEG5000-MAL, etc.) comprising 0.15-5% of the total lipids of the composition. Furthermore, in the LNP composition, the molar ratio (i.e., N / P molar ratio) of ionizable N atoms in the ionizable lipid molecule to phosphate groups in the nucleic acid molecule is (2-10):1, and more preferably (4-8):1.

[0274] In a preferred embodiment of the invention, the LNP composition comprises: nucleic acid, an ionizable cationic lipid having the structure shown in formula (I), a cofactor phospholipid (e.g., DSPC, DOPE, DOPC, etc., or combinations thereof), a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, DSPE-PEG5000-MAL, DMG-PEG2000-MAL, or DMG-PEG5000-MAL, etc.). In a more preferred embodiment of the invention, the LNP composition comprises: nucleic acid, an ionizable cationic lipid having the structure shown in formula (I), a cofactor phospholipid (e.g., DSPC, DOPE, DOPC, etc., or combinations thereof), a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol), and a polymer-conjugated lipid (e.g., DMG-PEG2000, etc.).

[0275] As used herein, the terms “encapsulation” and “encapsulated” refer to mRNA, DNA, siRNA or other nucleic acid drugs, or proteins (including immunoglobulins) being contained within or bound to lipid nanoparticles. As used herein, the term “encapsulation” refers to complete or partial encapsulation. For example, mRNA may be selected to treat and / or prevent associated diseases when administered to a subject in need of a lipid nanoparticle composition comprising mRNA.

[0276] As used herein, the term “pharmaceuticalally acceptable carrier” includes, but is not limited to, any adjuvant, carrier, excipient, scintillation agent, sweetener, diluent, preservative, dye / coloring agent, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier approved by the Food and Drug Administration for use in humans or livestock.

[0277] Preparation method of lipid nanoparticle drug formulation

[0278] In another aspect of the present invention, a method for preparing a lipid nanoparticle drug formulation is provided, the method comprising: (a) mixing the ionizable lipid and optionally an auxiliary lipid described in the first aspect of the present invention with an organic solvent to obtain a lipid organic phase; (b) mixing a bioactive substance with an aqueous solvent to obtain an aqueous phase containing the bioactive substance; and (c) mixing the lipid organic phase from step (a) with the aqueous phase from step (b) to obtain the lipid nanoparticle drug. Further, the method further comprises step (d): purifying, concentrating, and sterilizing the lipid nanoparticle drug obtained in step (c).

[0279] In some embodiments, the organic solvent includes (but is not limited to) ethanol, methanol, isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, or tetrahydrofuran, or combinations thereof. In some embodiments, the lipid organic phase includes a small percentage of water or a pH buffer. The lipid organic phase may contain up to 60% by volume of water, for example, up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% by volume of water. In one embodiment, the lipid organic phase contains between about 0.05% and 60% by volume of water, for example, between about 0.05% and 50%, between about 0.05% and 40%, or between about 5% and 20% by volume of water.

[0280] In some embodiments, the lipid organic phase comprises a single type of lipid, such as an ionizable cationic lipid, a cofactor phospholipid, a sterol, or a polymer-conjugated lipid. In some embodiments, the lipid organic phase comprises multiple lipids. In one embodiment of the invention, the lipid organic phase comprises an ionizable cationic lipid of Formula I, a cofactor phospholipid (e.g., DSPC, DOPE, DOPC, or combinations thereof), a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol), and a polymer-conjugated lipid (with or without targeting modification) (e.g., DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, DSPE-PEG5000-MAL, DMG-PEG2000-MAL, DMG-PEG5000-MAL, or DSPE-PEG5000-MAL conjugate (the conjugate is a polypeptide, protein, amino acid, vitamin, mannose, or other active substance) etc.). In a preferred embodiment of the present invention, the lipid organic phase comprises an ionizable cationic lipid having the structure shown in formula (I), an accessory phospholipid (e.g., DSPC, DOPE, DOPC or combinations thereof), a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol), and a polymer-conjugated lipid (with or without targeting modification) (e.g., DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, DSPE-PEG5000-MAL, DMG-PEG2000-MAL, DMG-PEG5000-MAL or DSPE-PEG5000-MAL-conjugate (the conjugate is a polypeptide, protein, amino acid, vitamin, mannose or other active substance) etc.). In a more preferred embodiment of the present invention, the lipid organic phase comprises an ionizable cationic lipid having the structure shown in Formula (I), an accessory phospholipid (e.g., DSPC, DOPE, DOPC, or combinations thereof), a sterol (e.g., cholesterol or cholesterol derivatives, or phytosterols such as β-sitosterol), and polymer-conjugated lipids (with or without targeting modifications) (e.g., DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, DSPE-PEG5000-MAL, DMG-PEG2000-MAL, DMG-PEG5000-MAL, or DSPE-PEG5000-MAL-conjugates (the conjugates are polypeptides, proteins, amino acids, vitamins, mannose, or other active substances). In a specific embodiment of the present invention, the lipid organic phase comprises an ionizable cationic lipid having the structure shown in Formula (I), DSPC, cholesterol, and DMG-PEG2000.In one specific embodiment of the present invention, the lipid organic phase includes ionizable cationic lipids having the structure shown in formula (I), DSPC, cholesterol, DSPE-PEG2000 or DMG-PEG2000, DSPE-PEG5000-MAL-conjugates (the conjugates are polypeptides, proteins, amino acids, vitamins, mannose, or other active substances). In a preferred embodiment, the conjugate is a targeting substance (such as an antibody or its antigen-binding fragment).

[0281] In some embodiments, the aqueous solvent is water. In some embodiments, the aqueous solvent is an aqueous buffer solution with a pH between 3 and 8 (e.g., pH of about 3, about 4, about 5, or about 6, etc.). A bioactive substance, such as a nucleic acid (e.g., mRNA), is dissolved in the aqueous solvent to obtain an aqueous phase containing the bioactive substance. The aqueous phase may contain a small percentage of a water-miscible organic solvent. The aqueous phase may contain up to 60% by volume of at least one water-miscible organic solvent, such as up to about 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or any volume percentage between both of an organic solvent (e.g., a water-miscible organic solvent). In one embodiment, the aqueous phase comprises between about 0.05% and 60% by volume an organic solvent, such as an organic solution (e.g., a water-miscible organic solvent) between about 0.05% and 50%, about 0.05% and 40%, or about 5% and 20% by volume. The aqueous buffer may be a sodium acetate buffer, citrate buffer, Tris-HCl buffer, PBS buffer, or a combination thereof. In some embodiments, the aqueous buffer is a sodium acetate buffer with a pH between 4 and 6 (e.g., a pH of about 4, about 5, or about 6). In one embodiment, the aqueous buffer is a sodium acetate buffer with a pH of about 4.

[0282] In some embodiments, a solution comprising a mixture of a lipid organic phase and an aqueous phase containing a bioactive substance, including an LNP suspension, may be diluted. In some embodiments, the pH of the solution comprising the lipid organic phase and the aqueous phase containing the bioactive substance of the LNP suspension may be adjusted. The pH of the LNP suspension may be diluted or adjusted by adding water, acid, base, or an aqueous buffer. In some embodiments, the pH of the LNP suspension is not diluted or adjusted. In some embodiments, the pH of the LNP suspension is diluted and adjusted.

[0283] In some embodiments, excess reagents, solvents, and unencapsulated nucleic acids can be removed from the LNP suspension by tangential flow filtration (TFF) (e.g., percolation). Organic solvents (e.g., ethanol) and buffers can also be removed from the LNP suspension by TFF. In some embodiments, the LNP suspension is dialyzed. In some embodiments, the LNP suspension is subjected to TFF. In some embodiments, the LNP suspension is subjected to both dialyzed and TFF.

[0284] The main advantages of this invention include:

[0285] (a) The ionizable lipids in this invention can form stable nanoparticles with other components, such as cofactor phospholipids (DSPC, DOPE, DOPC, DOPS, etc.), sterols (such as cholesterol or cholesterol derivatives), PEG derivatives (such as DMG-PEG2000, DSPE-PEG2000, DSPE-PEG2000-MAL, DSPE-PEG5000-MAL, DMG-PEG2000-MAL, DMG-PEG5000-MAL or DSPE-PEG5000-MAL-couplings (or targeting substances), or DMG-PEG substances modified by other groups / or DSPE-PEG and other derivatives).

[0286] (b) The present invention can encapsulate mRNA by ionizing lipids and other components to form nanoparticles. The nanoparticles are uniform in size, have high encapsulation efficiency and good stability, which can improve the transfection efficiency of mRNA in target tissues or cells and have low toxicity, thereby making the preventive and therapeutic effects of mRNA vaccines / drugs more prominent.

[0287] (c) The present invention can ionize lipids and other components to form nanoparticles, which can achieve high T cell transfection efficiency without T cell activation treatment. After conjugation with antibodies, it can accurately deliver drugs to target cells or tissues, improve drug delivery efficiency and bioavailability, and reduce damage and toxicity to normal cells.

[0288] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0289] Example 1: Preparation of ionizable lipids

[0290] 1.1: Synthesis of common intermediates

[0291] The synthesis process of the common intermediate is as follows:

[0292] Specifically, it is divided into the following steps:

[0293] Step 1:

[0294] In a 250 mL reaction flask, under N2 protection, 1 (5 g, 0.0269 mol), 2 (3.72 g, 0.0296 mol, 1.5 equiv), Na2CO3 (5.70 g, 0.0538 mol, 2 eq), and CH3CN (24 mL) were added sequentially. The reaction was stirred overnight at 70 °C. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure. 50 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. 2 (4.8 g, 0.0209 mol) of a colorless oil was obtained.

[0295] Step Two:

[0296] In a 100 mL single-necked flask, under N2 protection, 2 (4.8 g, 0.0209 mol) of 10 mL of ethyl acetate was added sequentially to dissolve the ethyl acetate (30 mL, 4 M) while stirring. The reaction was carried out at 20 °C for 30 minutes. The reaction was confirmed to be complete by LCMS. The reaction solution was concentrated to obtain 3 (2.8 g, 0.02154 mol) of a light yellow oily substance.

[0297] The above describes the synthesis of product 3, which has an S-configuration amino head. Since the synthesis steps for the R-configuration amino head are similar, they will not be described in detail.

[0298] 1.2: Synthesis of AL-08-001~027 molecules

[0299] 1.2.1: Synthesis of AL-08-005 molecule

[0300] Step 1:

[0301] Dissolve 150 mL of DCM (8 g, 0.03112 mol) in a 250 mL three-necked flask, purge with nitrogen three times, and cool the reaction mixture to 0°C using an ice-water bath. Add 11.88 g of COCl2 (0.09360 mol) and 0.5 mL of DMF dropwise with stirring. After the addition is complete, bring the mixture to room temperature and stir for 2 hours. After the reaction is complete, add 2 drops of the reaction solution to 0.5 mL of methanol and spot the sample onto a TLC plate (PE:EtOAc = 10:1R). f=0.6) detection showed that the raw materials were completely consumed and a new spot was formed. The reaction solution was poured into a 250mL single-necked flask and evaporated to dryness, then washed twice with DCM (100mL) and evaporated to dryness. 2 (8.2g, 0.0299mol) was obtained as a yellow liquid.

[0302] Step 2:

[0303] 2A (2.56 g, 0.0175 mol) was dissolved in 100 mL of DCM in a 250 mL three-necked flask, and nitrogen gas was purged three times. The reaction was cooled to 0°C in an ice-water bath. TEA (4.43 g, 0.0438 mol) was added dropwise with stirring, followed by 2A (4 g, 0.0146 mol). After the addition was complete, the mixture was brought to room temperature and stirred for 2 hours. After the reaction was complete, TLC (PE:EtOAc = 3:1, Rf = 0.4) showed that the starting material was completely consumed and a new spot was formed. The reaction solution was added to 100 mL of water, and the aqueous phase was extracted three times with 100 mL of DCM. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in silica gel and mixed with the solution for column chromatography. The product eluted when the mobile phase was EtOAc + PE (20%). 3A (4 g, 0.0104 mol) of a colorless oil was obtained.

[0304] Step 3:

[0305] Dissolve 100 mL of DCM in DMSO (2.12 g, 0.0312 mol) into a 250 mL three-necked flask, purging with nitrogen. Cool the reaction mixture to -70°C using a dry ice-ethanol bath. Maintaining the temperature below -60°C, add COCl2 (3.99 g, 0.0312 mol) dropwise. Stir at -60°C for 30 min. Then dissolve 3 (4 g, 0.0104 mol) in 10 mL of DCM and slowly add it dropwise to the above reaction solution. After the addition is complete, maintain the temperature below -60°C and stir for 1 h. Add TEA (5.26 g, 0.0520 mol) dropwise to the reaction solution below -60°C. Stir for 30 min after the addition is complete. Take 2 drops of the reaction solution and add dichloromethane and water. Extract and spot the organic phase onto a TLC plate. Perform TLC (PE:EtOAc = 3:1, R...) f =0.6) indicates that the raw material has been consumed and a new spot has formed. The reaction solution was poured into 100 mL of saturated ammonium chloride aqueous solution on ice, and DCM (60 mL * 3) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness, dissolved, mixed, and the product was obtained by column chromatography with EtOAc (8%) as the mobile phase. Product 4 (2.5 g, 0.0065 mol) was obtained as a light yellow oil at room temperature.

[0306] Step 4:

[0307] Add 4A (200 mg, 0.0015 mol) to a 100 mL single-necked flask, dissolve in MeOH (15 mL), add TEA (0.1 mL), and add 4 (1.30 g, 0.0034 mol) while stirring. Add 50 mg of glacial acetic acid, and add sodium cyanoborohydride (483.38 mg, 0.0077 mol) to the above reaction solution. Stir at room temperature for one hour. LCMS analysis showed no residual starting material, and the main peak was the product. The reaction solution was evaporated to dryness, and then 30 mL of water was added. A saturated sodium bicarbonate solution was added to adjust the pH to approximately 8, and the solution was extracted three times with ethyl acetate (30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness. The crude product was dissolved and mixed. The mobile phase for column chromatography was 12% MeOH:88% EtOAC. Evaporation to dryness yielded AL08-005 (400 mg, 0.00046 mol), a light yellow oily substance.

[0308] 1.2.2: Synthesis of AL-08-012 molecule

[0309] Step 1:

[0310] Dissolve 1 (8 g, 0.03112 mol) of DCM (150 mL) in a 250 mL three-necked flask, purge with nitrogen three times, and cool the reaction mixture to 0°C using an ice-water bath. Add COCl2 (11.88 g, 0.09360 mol) and 0.5 mL of LDM dropwise with stirring. After the addition is complete, bring the mixture to room temperature and stir for 2 hours. After the reaction is complete, add two drops of the reaction solution to 0.5 mL of methanol and spot the sample onto a TLC plate (PE:EtOAc = 5:1R). f =0.6) detection showed that the raw materials were completely consumed and a new spot was formed. The reaction solution was poured into a 250mL single-necked flask and evaporated to dryness, then washed twice with DCM (100mL) and evaporated to dryness. 2 (8.2g, 0.0299mol) was obtained as a yellow liquid.

[0311] Step 2:

[0312] 2A (2.31 g, 0.0175 mol) was dissolved in 100 mL of DCM in a 250 mL three-necked flask, and nitrogen gas was purged three times. The reaction was cooled to 0°C in an ice-water bath. TEA (4.43 g, 0.0438 mol) was added dropwise with stirring, followed by 2A (4 g, 0.0146 mol). After the addition was complete, the mixture was brought to room temperature and stirred for 2 hours. After the reaction was complete, TLC (PE:EtOAc = 3:1, Rf = 0.6) showed that the starting material was completely consumed and a new spot was formed. The reaction solution was added to 100 mL of water, and the aqueous phase was extracted three times with 100 mL of DCM. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in silica gel and mixed with the solution for column chromatography. The product eluted when the mobile phase was EtOAc (20%). 3A (3.5 g, 0.0095 mol) of a colorless oil was obtained.

[0313] Step 3:

[0314] Dissolve 100 mL of DCM in DMSO (1.94 g, 0.0285 mol) and transfer it to a 250 mL three-necked flask, purging with nitrogen. Cool the reaction mixture to -70°C using a dry ice-ethanol bath. Maintaining the temperature below -60°C, add COCl2 (3.65 g, 0.0285 mol) dropwise. Stir at -60°C for 30 min. Then, dissolve 3 (3.5 g, 0.0095 mol) in 10 mL of DCM and slowly add it dropwise to the above reaction solution. After the addition is complete, maintain the temperature below -60°C and stir for 1 h. Add TEA (4.81 g, 0.0475 mol) dropwise to the reaction solution below -60°C. After the addition is complete, stir at 0°C for 30 min. Take 2 drops of the reaction solution and add dichloromethane and water. Extract and spot the organic phase onto a TLC plate. Perform TLC (PE:EtOAc = 3:1, R...) f =0.6) indicates that the raw material has been consumed and a new spot has formed. The reaction solution was poured into 100 mL of saturated ammonium chloride aqueous solution on ice, and DCM (60 mL * 3) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness, dissolved, mixed, and column chromatography was performed with EtOAc (8%) as the mobile phase. Product 4 (2 g, 0.0054 mol) was obtained as a light yellow oil at room temperature.

[0315] Step 4:

[0316] Add 4A (200 mg, 0.0015 mol) to a 100 mL single-necked flask, dissolve in MeOH (15 mL), add TEA (0.1 mL), and add 4 (1.25 g, 0.0034 mol) while stirring. Add 50 mg of glacial acetic acid, and add sodium cyanoborohydride (483.38 mg, 0.0077 mol) to the above reaction solution. Stir at room temperature for one hour. LCMS analysis showed no residual starting material, and the main peak was the product. The reaction solution was evaporated to dryness, and then 30 mL of water was added. A saturated sodium bicarbonate solution was added to adjust the pH to approximately 8, and the solution was extracted three times with ethyl acetate (30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness. The crude product was dissolved and mixed. The mobile phase for column chromatography was 12% MeOH:88% EtOAC. Evaporation to dryness yielded AL08-012 (371 mg, 0.00044 mol), a pale yellow oil.

[0317] 1.2.3: Synthesis of molecules such as AL08-001 / 003 / 004 / 006 / 007 / 008 / 011 / 018 / 019 / 020 / 021 / 022 / 023

[0318] The synthesis steps of molecules such as AL08-001 / 003 / 004 / 006 / 007 / 008 / 011 / 018 / 019 / 020 / 021 / 022 / 023 are similar to those of the molecules shown in 1.2.1 to 1.2.2 above, and therefore will not be described in detail.

[0319] 1.2.4: Synthesis of AL08-024 molecule

[0320] Step 1:

[0321] Dissolve 1 (4 g, 0.01556 mol) of DCM (150 mL) in a 250 mL three-necked flask, purge with nitrogen three times, and cool the reaction mixture to 0°C using an ice-water bath. Add COCl2 (5.94 g, 0.0468 mol) and 0.5 mL of DMF dropwise with stirring. After the addition is complete, bring the mixture to room temperature and stir for 2 hours. After the reaction is complete, add 2 drops of the reaction solution to 0.5 mL of methanol and spot the sample onto a TLC plate (PE:EtOAc = 10:1R). f =0.6) detection showed that the raw materials were completely consumed and a new spot was formed. The reaction solution was poured into a 250mL single-necked flask and evaporated to dryness, and washed twice with DCM (100mL)*2 and evaporated to dryness. 2 (4.1g, 0.01495mol) of yellow liquid was obtained.

[0322] Step 2:

[0323] 2A (2.21 g, 0.0187 mol) was dissolved in DCM (80 mL) in a 250 mL three-necked flask, and nitrogen gas was purged three times. The reaction was cooled to 0°C in an ice-water bath. TEA (3.16 g, 0.0312 mol) was added dropwise with stirring, followed by 2 (4 g, 0.0156 mol). After the addition was complete, the mixture was brought to room temperature and stirred for 2 hours. After the reaction was complete, TLC (PE:EtOAc = 3:1, Rf = 0.6) showed that the starting material was completely consumed and a new spot was formed. The reaction solution was added to 200 mL of water, and the aqueous phase was extracted three times with 100 mL of DCM. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in silica gel and mixed with the solution for column chromatography. The product eluted when the mobile phase was EtOAc (20%). 3 (3 g, 0.0084 mol) of a colorless oily substance was obtained.

[0324] Step 3:

[0325] Dissolve 100 mL of DCM in DMSO (1.719 g, 0.0253 mol) into a 250 mL three-necked flask, purging with nitrogen. Cool the reaction mixture to -70°C using a dry ice-ethanol bath. Maintaining the temperature below -60°C, add COCl2 (3.160 g, 0.0253 mol) dropwise. Stir at -60°C for 30 min. Then dissolve 3 g (0.0084 mol) in 10 mL of DCM and slowly add it dropwise to the above reaction solution. After the addition is complete, maintain the temperature below -60°C and stir for 1.5 h. Add TEA (4.264 g, 0.0421 mol) dropwise to the reaction solution below -60°C. After the addition is complete, stir at 0°C for 30 min. Take 2 drops of the reaction solution and add dichloromethane and water. Extract and spot the organic phase onto a TLC plate. Perform TLC (PE:EtOAc = 3:1, R...) f =0.6) indicates that the raw material has been consumed and a new spot has formed. The reaction solution was poured into 100 mL of saturated ammonium chloride aqueous solution on ice, and DCM (60 mL * 3) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness, dissolved, mixed, and column chromatography was performed with EtOAc (8%) as the mobile phase. Product 4 (2 g, 0.0056 mol) was obtained as a light yellow oil at room temperature.

[0326] Step 4:

[0327] Dissolve 5 g (4 g, 0.01561 mol) of DCM (150 mL) in a 250 mL three-necked flask, purge with nitrogen three times, and cool the reaction mixture to 0°C using an ice-water bath. While stirring, add 11.88 g (0.02341 mol) of COCl2 and 0.5 mL of DMF dropwise. After the addition is complete, bring the mixture to room temperature and stir for 2 hours. After the reaction is complete, add 2 drops of the reaction solution to 0.5 mL of methanol and spot the sample onto a TLC plate (PE:EtOAc = 10:1R).f =0.6) detection showed that the raw materials were completely consumed and a new spot was formed. The reaction solution was poured into a 250mL single-necked flask and evaporated to dryness, and washed twice with DCM (100mL)*2 and evaporated to dryness. 6 (3.8g, 0.0139mol) of a yellow liquid was obtained.

[0328] Step 5:

[0329] 6A (1.95 g, 0.0167 mol) was dissolved in DCM (80 mL) in a 250 mL three-necked flask, and nitrogen gas was purged three times. The reaction was cooled to 0°C in an ice-water bath. TEA (4.88 g, 0.0417 mol) was added dropwise with stirring, followed by 6A (3.8 g, 0.0139 mol). After the addition was complete, the mixture was brought to room temperature and stirred for 2 hours. After the reaction was complete, TLC (PE:EtOAc = 1:1, Rf = 0.4) showed that the starting material was completely consumed and a new spot was formed. The reaction solution was added to 200 mL of water, and the aqueous phase was extracted three times with 100 mL of DCM. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in silica gel and mixed with the solution for column chromatography. The product eluted when the mobile phase was EtOAc (25%). 3A (2.8 g, 0.0079 mol) of a colorless oil was obtained.

[0330] Step 6:

[0331] Dissolve 100 mL of DCM in DMSO (1.612 g, 0.0237 mol) into a 250 mL three-necked flask, purging with nitrogen. Cool the reaction mixture to -70°C using a dry ice-ethanol bath. Maintaining the temperature below -60°C, add COCl2 (3.034 g, 0.0237 mol) dropwise. Stir at -60°C for 30 min. Then, dissolve 2.8 g (0.0079 mol) in 10 mL of DCM and slowly add it dropwise to the above reaction solution. After the addition is complete, maintain the temperature below -60°C and stir for 1.5 h. Add TEA (3.997 g, 0.0395 mol) dropwise to the reaction solution below -60°C, stirring for 30 min after the addition is complete. Take 2 drops of the reaction solution, add dichloromethane and water, extract and spot the organic phase onto a TLC plate (PE:EtOAc = 1:1, R...). f =0.6) indicates that the raw material has been consumed and a new spot has formed. The reaction solution was poured into 100 mL of saturated ammonium chloride aqueous solution on ice, and DCM (60 mL * 3) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness, dissolved, mixed, and column chromatography was performed with EtOAc (8%) as the mobile phase. Product 7 (1.7 g, 0.0048 mol) was obtained as a light yellow oil at room temperature.

[0332] Step 7:

[0333] Add 9A (200 mg, 0.0015 mol) to a 100 mL single-necked flask, dissolve in MeOH (15 mL), then add TEA (0.1 mL), and add 4 (478.32 mg, 0.0014 mol) while stirring. Add 50 mg of glacial acetic acid, and then add sodium cyanoborohydride (283.5 mg, 0.0045 mol) to the above reaction solution. Stir at room temperature for one hour. LCMS analysis showed no residue of the starting material, and the main peak was the product. The reaction solution was evaporated to dryness, and then 30 mL of water was added. The pH was adjusted to approximately 8 with saturated sodium bicarbonate solution, and extracted three times with ethyl acetate (30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 9 (600 mg, 0.0013 mol, crude product) as a colorless oil.

[0334] Step 8:

[0335] Add 9 (200 mg, 0.00043 mol) to a 100 mL single-necked flask, dissolve in MeOH (15 mL), add TEA (0.1 mL), and add 8 (151.50 mg, 0.00043 mol) while stirring. Add 50 mg of glacial acetic acid and sodium cyanoborohydride (283.5 mg, 0.0045 mol) to the above reaction solution. Stir at room temperature for one hour. LCMS analysis showed no residual starting material and the main peak was the product. The reaction solution was evaporated to dryness, and then 30 mL of water was added. The pH was adjusted to approximately 8 with saturated sodium bicarbonate solution, and extracted three times with ethyl acetate (30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness, dissolved, mixed, and the product was obtained by column chromatography with MeOH (228%) as the mobile phase. The product AL08-024 (76.9 mg, 0.000096 mol) was obtained as a pale yellow oil at room temperature.

[0336] 1.2.5: Synthesis of AL08-027 molecule

[0337] Step 1:

[0338] Dissolve 1 (5g, 0.01951mol) of DCM (150mL) in a 250mL three-necked flask, purge with nitrogen three times, and cool the reaction mixture to 0°C using an ice-water bath. Add COCl2 (5.94g, 0.03903mol) and 0.5mL DMF dropwise with stirring. After the addition is complete, bring the mixture to room temperature and stir for 2 hours. After the reaction is complete, add 2 drops of the reaction solution to 0.5mL of methanol. Spot the solution onto a TLC plate (PE:EtOAc = 10:1, Rf = 0.6) and observe the result, showing complete consumption of the reactants and formation of new spots. Pour the reaction solution into a 250mL single-necked flask and evaporate to dryness. Wash twice with 100mL DCM and evaporate to dryness again. 2 (5.1g, 0.01860mol) is obtained as a yellow liquid.

[0339] Step 2:

[0340] 2A (2.95 g, 0.02232 mol) was dissolved in DCM (80 mL) in a 250 mL three-necked flask, purged with nitrogen three times. The reaction mixture was cooled to 0°C in an ice-water bath. TEA (5.65 g, 0.0558 mol) was added dropwise with stirring, followed by 2 (5.1 g, 0.01860 mol). After the addition was complete, the mixture was brought to room temperature and stirred for 2 hours. After the reaction was complete, TLC (PE:EtOAc = 3:1, Rf = 0.6) showed complete consumption of the reactants and formation of a new spot. The reaction solution was added to 200 mL of water, and the aqueous phase was extracted three times with 100 mL of DCM. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in silica gel and mixed with the solution for column chromatography. The product eluted when the mobile phase was EtOAc (20%). 3 (5.3 g, 0.01431 mol) of a colorless oily substance was obtained.

[0341] Step 3:

[0342] Dissolve 100 mL of DCM in DMSO (2.92 g, 0.0429 mol) into a 250 mL three-necked flask and purge with nitrogen. Cool the reaction mixture to -70°C using a dry ice-ethanol bath. Maintaining the temperature below -60°C, add COCl2 (5.49 g, 0.0429 mol) dropwise. Stir at -60°C for 30 min. Then dissolve 3 (5.3 g, 0.0143 mol) in 10 mL of DCM and slowly add it dropwise to the above reaction solution. After the addition is complete, maintain the temperature below -60°C and stir for 1.5 h. Add TEA (7.23 g, 0.0715 mol) dropwise to the reaction solution below -60°C. After the addition is complete, stir at 0°C for 30 min. Take 2 drops of the reaction solution and add dichloromethane and water. Extract and spot the organic phase onto a TLC plate. Perform TLC (PE:EtOAc = 3:1, R...) f=0.6) indicates that the raw material has been consumed and a new spot has formed. The reaction solution was poured into 100 mL of saturated ammonium chloride aqueous solution on ice, and DCM (60 mL * 3) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness, dissolved, mixed, and column chromatography was performed with EtOAc (8%) as the mobile phase. Product 4 (3 g, 0.00814 mol) was obtained as a light yellow oil at room temperature.

[0343] Step 4:

[0344] Dissolve 5 g (4 g, 0.01561 mol) of DCM (150 mL) in a 250 mL three-necked flask, purge with nitrogen three times, and cool the reaction mixture to 0°C using an ice-water bath. Add COCl2 (11.88 g, 0.02341 mol) and 0.5 mL of DMF dropwise with stirring. After the addition is complete, bring the mixture to room temperature and stir for 2 hours. After the reaction is complete, add 2 drops of the reaction solution to 0.5 mL of methanol and spot the sample onto a TLC plate (PE:EtOAc = 10:1R). f =0.6) detection showed that the raw materials were completely consumed and a new spot was formed. The reaction solution was poured into a 250mL single-necked flask and evaporated to dryness, and washed twice with DCM (100mL)*2 and evaporated to dryness. 6 (3.8g, 0.0139mol) of a yellow liquid was obtained.

[0345] Step 5:

[0346] 6A (1.95 g, 0.0149 mol) was dissolved in DCM (80 mL) in a 250 mL three-necked flask, purged with nitrogen three times. The reaction was cooled to 0°C in an ice-water bath. TEA (4.88 g, 0.0417 mol) was added dropwise with stirring, followed by 6A (3.8 g, 0.0139 mol). After the addition was complete, the mixture was brought to room temperature and stirred for 2 hours. After the reaction was complete, TLC (PE:EtOAc = 1:1, Rf = 0.4) showed complete consumption of the starting material and formation of a new spot. The reaction solution was added to 200 mL of water, and the aqueous phase was extracted three times with 100 mL of DCM. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in silica gel and mixed with the solution for column chromatography. The product eluted when the mobile phase was EtOAc (25%). 7A (2.8 g, 0.0079 mol) was obtained as a colorless oil.

[0347] Step 6:

[0348] Dissolve 100 mL of DCM in DMSO (1.612 g, 0.0237 mol) into a 250 mL three-necked flask, purging with nitrogen. Cool the reaction mixture to -70°C using a dry ice-ethanol bath. Maintaining the temperature below -60°C, add COCl2 (3.034 g, 0.0237 mol) dropwise. Stir at -60°C for 30 min. Then, dissolve 2.8 g (0.0079 mol) in 10 mL of DCM and slowly add it dropwise to the above reaction solution. After the addition is complete, maintain the temperature below -60°C and stir for 1 h. Add TEA (3.997 g, 0.0395 mol) dropwise to the reaction solution below -60°C. After the addition is complete, stir at 0°C for 30 min. Take 2 drops of the reaction solution, add dichloromethane and water, extract and spot the organic phase onto a TLC plate (PE:EtOAc = 1:1, R...). f =0.6) indicates that the raw material has been consumed and a new spot has formed. The reaction solution was poured into 100 mL of saturated ammonium chloride aqueous solution on ice, and DCM (60 mL * 3) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness, dissolved, mixed, and column chromatography was performed with EtOAc (8%) as the mobile phase. Product 8 (1.7 g, 0.0048 mol) was obtained as a light yellow oil at room temperature.

[0349] Step 7:

[0350] Add 9A (200 mg, 0.0015 mol) to a 100 mL single-necked flask, dissolve in MeOH (15 mL), then add TEA (0.1 mL), and while stirring, add 4 (510.0 mg, 0.0014 mol), then add 50 mg of glacial acetic acid. Add sodium cyanoborohydride (283.5 mg, 0.0045 mol) to the above reaction solution and stir at room temperature for one hour. LCMS analysis showed no residue of the starting material, and the main peak was the product. The reaction solution was evaporated to dryness, then 30 mL of water was added, and saturated sodium bicarbonate solution was added to adjust the pH to approximately 8. Extract three times with ethyl acetate (30 mL). Combine the organic phases, dry over anhydrous sodium sulfate, and evaporate to dryness to obtain 9 (550 mg, 0.0014 mol, crude product) as a colorless oil.

[0351] Step 8:

[0352] Add 9 (550 mg, 0.0014 mol) to a 100 mL single-necked flask, dissolve in MeOH (15 mL), add TEA (0.1 mL), and add 8 (512.88 mg, 0.0014 mol) while stirring. Add 50 mg of glacial acetic acid and sodium cyanoborohydride (283.5 mg, 0.0045 mol) to the above reaction solution. Stir at room temperature for one hour. LCMS analysis showed no residual starting material and the main peak was the product. The reaction solution was evaporated to dryness, and then 30 mL of water was added. The pH was adjusted to approximately 8 with saturated sodium bicarbonate solution, and extracted three times with ethyl acetate (30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness, dissolved, mixed, and the product was obtained by column chromatography with MeOH (22%) as the mobile phase. The product AL08-027 (52.6 mg, 0.000063 mol) was obtained as a light yellow oil at room temperature.

[0353] 1.2.6: Synthesis of AL08-025 / 026 molecules

[0354] The synthesis steps for molecules AL08-025 / 026 are similar to those for molecules AL08-024 and AL08-027, and will not be described in detail here.

[0355] 1.2.7: Synthesis of AL08-009 molecule

[0356] Step 1:

[0357] Dissolve 150 mL of DCM (5 g, 0.0195 mol) in a 250 mL three-necked flask, purge with nitrogen three times, and cool the reaction mixture to 0°C using an ice-water bath. Add 5.00 g (0.0390 mol) of COCl2 and 0.5 mL of LDM dropwise with stirring. After the addition is complete, bring the mixture to room temperature and stir for 2 hours. After the reaction is complete, add 2 drops of the reaction solution to 0.5 mL of methanol and spot the sample onto a TLC plate (PE:EtOAc = 5:1R). f =0.6) detection showed that the raw materials were completely consumed and a new spot was formed. The reaction solution was poured into a 250mL single-necked flask and evaporated to dryness, and washed twice with DCM (100mL)*2 and evaporated to dryness. 2 (4g, 0.0146mol) of yellow liquid was obtained.

[0358] Step 2:

[0359] Dissolve 2A (2.31 g, 0.0175 mol) in DCM (100 mL) in a 250 mL three-necked flask, purging with nitrogen three times. Cool the reaction mixture to 0°C using an ice-water bath. While stirring, add TEA (4.43 g, 0.0438 mol) dropwise, followed by 2A (4 g, 0.0146 mol). After the addition is complete, bring the mixture to room temperature and stir for 2 hours. After the reaction is complete, perform TLC (PE:EtOAc = 3:1R). f =0.6) detection showed that the raw material was completely consumed and a new spot was formed. The reaction solution was added to 100 mL of water, and the aqueous phase was extracted three times with 100 mL of DCM. The organic phases were combined, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in silica gel and mixed with the sample, and column chromatography was performed. The product was eluted when the mobile phase was EtOAc (30%). 3 (4.5 g, 0.0122 mol) of colorless oily substance was obtained.

[0360] Step 3:

[0361] Dissolve DMSO (2.149 g, 0.0366 mol) in 100 mL of DCM in a 250 mL three-necked flask, purging with nitrogen. Cool the reaction mixture to -70°C using a dry ice-ethanol bath. Maintaining the temperature below -60°C, add COCl2 (4.68 g, 0.0366 mol) dropwise. Stir at -60°C for 30 min. Then, dissolve 3 (4.5 g, 0.0122 mol) in 10 mL of DCM and slowly add it dropwise to the above reaction solution. After the addition is complete, maintain the temperature below -60°C and stir for 1 h. Add TEA (5.26 g, 0.0520 mol) dropwise to the reaction solution below -60°C. After the addition is complete, stir at 0°C for 30 min. Take 2 drops of the reaction solution and add dichloromethane and water. Extract and spot the organic phase onto a TLC plate. Perform TLC (PE:EtOAc = 5:1, R...) f =0.6) indicates that the raw material has been consumed and a new spot has formed. The reaction solution was poured into 100 mL of saturated ammonium chloride aqueous solution on ice, and DCM (60 mL * 3) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness, dissolved, mixed, and column chromatography was performed with EtOAc (8%) as the mobile phase. Product 4 (2.5 g, 0.0068 mol) was obtained as a light yellow oil at room temperature.

[0362] Step 4:

[0363] Dissolve 5 g (0.0219 mol) of DCM (150 mL) in a 250 mL three-necked flask, purge with nitrogen three times, and cool the reaction mixture to 0°C using an ice-water bath. Add COCl2 (5.61 g, 0.0438 mol) and 0.5 mL of DMF dropwise with stirring. After the addition is complete, bring the mixture to room temperature and stir for 2 hours. After the reaction is complete, add 2 drops of the reaction solution to 0.5 mL of methanol and spot the sample onto a TLC plate (PE:EtOAc = 5:1R). f =0.6) detection showed that the raw materials were completely consumed and a new spot was formed. The reaction solution was poured into a 250mL single-necked flask and evaporated to dryness, and washed twice with DCM (100mL)*2 and evaporated to dryness. 6 (5g, 0.0203mol) of a yellow liquid was obtained.

[0364] Step 5:

[0365] Dissolve 6A (3.56 g, 0.0244 mol) in DCM (100 mL) in a 250 mL three-necked flask, purging with nitrogen three times. Cool the reaction mixture to 0°C using an ice-water bath. While stirring, add TEA (4.43 g, 0.0609 mol) dropwise, followed by 6A (5 g, 0.0203 mol). After the addition is complete, bring the mixture to room temperature and stir for 2 hours. After the reaction is complete, perform TLC (PE:EtOAc = 3:1R). f =0.5) detection showed that the raw material was completely consumed and a new spot was formed. The reaction solution was added to 100 mL of water, and the aqueous phase was extracted three times with 100 mL of DCM. The organic phases were combined, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in silica gel and mixed with the sample, and column chromatography was performed. The product was eluted when the mobile phase was EtOAc (30%). 3 (4.5 g, 0.0126 mol) of colorless oily substance was obtained.

[0366] Step 6:

[0367] Dissolve 100 mL of DCM in DMSO (2.570 g, 0.0378 mol) into a 250 mL three-necked flask, purging with nitrogen. Cool the reaction mixture to -70°C using a dry ice-ethanol bath. Maintaining the temperature below -60°C, add COCl2 (4.84 g, 0.0378 mol) dropwise. Stir at -60°C for 30 min. Then, dissolve 4.5 g (0.0126 mol) in 10 mL of DCM and slowly add it dropwise to the above reaction solution. After the addition is complete, maintain the temperature below -60°C and stir for 1 h. Add TEA (5.26 g, 0.0520 mol) dropwise to the reaction solution below -60°C. After the addition is complete, stir at 0°C for 30 min. Take 2 drops of the reaction solution and add dichloromethane and water. Extract and spot the organic phase onto a TLC plate. Perform TLC (PE:EtOAc = 5:1, R...) f=0.6) indicates that the raw material has been consumed and a new spot has formed. The reaction solution was poured into 100 mL of saturated ammonium chloride aqueous solution on ice, and DCM (60 mL * 3) was added for extraction. The organic phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness, dissolved, mixed, and column chromatography was performed with EtOAc (8%) as the mobile phase. Product 4 (2.7 g, 0.0076 mol) was obtained as a light yellow oil at room temperature.

[0368] Step 7:

[0369] 9A (200 mg, 0.0015 mol) was added to a 100 mL single-necked flask, dissolved in 15 mL of MeOH, followed by 0.1 mL of TEA. While stirring, 4 (510 mg, 0.0014 mol) was added, along with 50 mg of glacial acetic acid. Sodium cyanoborohydride (483.38 mg, 0.0077 mol) was added to the reaction solution. The mixture was stirred at room temperature for one hour. LC-MS analysis showed no residual starting material, and the main peak was the product. The reaction solution was evaporated to dryness, and 30 mL of water was added. A saturated sodium bicarbonate solution was added to adjust the pH to approximately 8. The mixture was extracted three times with 30 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain 9 (225 mg, 0.0035 mol, crude product, unpurified).

[0370] Step 8:

[0371] Add 9 (225 mg, 0.0035 mol) to a 100 mL single-necked flask, dissolve in MeOH (15 mL), add TEA (0.1 mL), and add 8 (531 mg, 0.0015 mol) while stirring. Add 50 mg of glacial acetic acid, and add sodium cyanoborohydride (483.38 mg, 0.0077 mol) to the above reaction solution. Stir at room temperature for one hour. LCMS analysis showed no residue of the starting material, and the main peak was the product. The reaction solution was evaporated to dryness, and then 30 mL of water was added. A saturated sodium bicarbonate solution was added to adjust the pH to approximately 8, and the solution was extracted three times with ethyl acetate (30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness. The crude product was dissolved and mixed. The mobile phase for column chromatography was 12% MeOH:88% EtOAC. Evaporation to dryness yielded AL08-009 (222 mg, 0.00027 mol), a light yellow oily substance.

[0372] 1.2.8: Synthesis of AL08-010 / 014 / 015 molecules

[0373] Since the synthesis steps of molecules such as AL08-010 / 014 / 015 are similar to those of molecules such as AL08-009, they will not be described in detail.

[0374] 1.3: Characterization Data

[0375] 1.3.1: AL08-001

[0376] The proton spectrum of AL08-001 is shown in Figure 1, and the mass spectrometry and NMR data are shown below:

[0377] LC-MS:(ES,m / z):780.0[M+H] + ;

[0378] 1 H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.7Hz,4H),3.75(t,J=5.2Hz,2H),3.36–3.12(m,6H),3.11–2.73(m,6H),2.60–2.48(m ,3H),2.38–2.30(m,2H),2.11–2.03(m,1H),1.71–1.55(m,9H),1.50–1.42(m,7H),1.40–1.18(m,48H),0.89(t,J=7.0Hz,13H).

[0379] 1.3.2: AL08-003

[0380] The proton spectrum of AL08-003 is shown in Figure 2, and the mass spectrometry and NMR data are shown below:

[0381] LC-MS:(ES,m / z):836.2[M+H] + ;

[0382] 1 H NMR(600MHz,Chloroform-d)δ4.07(t,J=6.7Hz,1H),3.68–3.38(m,1H),2.95–2.72(m,0H),2.48(q,J=9.2,7.4Hz,1H),2.32(d t,J=8.9,4.0Hz,0H),2.12–1.71(m,1H),1.76–1.51(m,3H),1.53–1.33(m,3H),1.29(d,J=22.4Hz,12H),0.89(t,J=6.9Hz,3H).

[0383] 1.3.3: AL08-004

[0384] The proton spectrum of AL08-004 is shown in Figure 3, and the mass spectrometry and NMR data are shown below:

[0385] LC-MS:(ES,m / z):808.1[M+H] + ;

[0386] 1 H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.6Hz,3H),3.73(t,J=5.2Hz,2H),3.55–3.46(m,1H),3.07–2.71(m,4H ),2.61–2.46(m,2H),2.33(tt,J=9.0,5.3Hz,1H),1.67–1.56(m,6H),1.54–1.13(m,45H),0.89(t,J=7.0Hz,9H).

[0387] 1.3.4: AL08-005

[0388] The proton spectrum of AL08-005 is shown in Figure 4, and the mass spectrometry and NMR data are shown below:

[0389] LC-MS:(ES,m / z):864.1[M+H] + ;

[0390] 1 H NMR(400MHz,Chloroform-d)δ4.08(t,J=6.7Hz,1H),3.68(t,J=5.3Hz,0H),3.50–3.38(m,0H),3.02–2.38(m,3H),2.32(t, J=5.3Hz,0H),2.01(s,0H),1.82(dd,J=13.0,7.3Hz,0H),1.63(d,J=6.7Hz,1H),1.52–1.15(m,15H),0.89(t,J=6.7Hz,3H).

[0391] 1.3.5: AL08-006

[0392] The proton spectrum of AL08-006 is shown in Figure 5, and the mass spectrometry and NMR data are shown below:

[0393] LC-MS:(ES,m / z):920.0[M+H] + ;

[0394] 1 H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.7Hz,0H),3.73(t,J=5.2Hz,0H),3.07–2.44(m,0H) ,2.35–2.26(m,0H),2.04(s,0H),1.69–1.53(m,0H),1.50–1.21(m,1H),0.90(t,J=7.0Hz,0H).

[0395] 1.3.6: AL08-007

[0396] The proton spectrum of AL08-007 is shown in Figure 6, and the mass spectrometry and NMR data are shown below:

[0397] LC-MS:(ES,m / z):892.2[M+H] + ;

[0398] 1 H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.6Hz,3H),3.73(t,J=5.2Hz,2H),3.55–3.46(m,1H),3.07–2.71(m,4H ),2.61–2.46(m,2H),2.33(tt,J=9.0,5.3Hz,1H),1.67–1.56(m,6H),1.54–1.13(m,45H),0.89(t,J=7.0Hz,9H).

[0399] 1.3.7: AL08-008

[0400] The proton spectrum of AL08-008 is shown in Figure 7, and the mass spectrometry and NMR data are shown below:

[0401] LC-MS:(ES,m / z):864.1[M+H] + ;

[0402] 1 H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.7Hz,4H),3.70(t,J=5.3Hz,2H),3.53(p,J=7.3Hz,1H),3.09(s,2H),2.88–2.54 (m,10H),2.33(tt,J=8.9,5.3Hz,2H),1.91–1.83(m,1H),1.72–1.51(m,13H),1.47–1.20(m,69H),0.90(t,J=7.0Hz,14H).

[0403] 1.3.8: AL08-009

[0404] The proton spectrum of AL08-009 is shown in Figure 8, and the mass spectrometry and NMR data are shown below:

[0405] LC-MS:(ES,m / z):822.1[M+H] + ;

[0406] 1H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.6Hz,2H),3.73(t,J=5.2Hz,1H),3.49(p,J=7.5Hz,1H),3.05–2.70(m,3H),2.5 2(p,J=7.2,5.6Hz,2H),2.38–2.29(m,1H),2.10–2.02(m,1H),1.92–1.85(m,0H),1.68–1.20(m,35H),0.94–0.82(m,6H).

[0407] 1.3.9: AL08-010

[0408] The proton spectrum of AL08-010 is shown in Figure 9, and the mass spectrometry and NMR data are shown below:

[0409] LC-MS:(ES,m / z):808.1[M+H] + ;

[0410] 1 H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.7Hz,0H),3.73(t,J=5.2Hz,0H),3.47(q,J=7.7Hz,0H),3.04–2.97(m,0H),2.97–2.65(m,0H),2.56–2 .39(m,0H),2.32(dq,J=9.3,5.3,4.6Hz,0H),2.08–2.00(m,0H),1.91–1 .80(m,0H),1.71–1.53(m,0H),1.52–1.16(m,1H),0.89(t,J=7.0Hz,0H).

[0411] 1.3.10: AL08-011

[0412] The proton spectrum of AL08-011 is shown in Figure 10, and the mass spectrometry and NMR data are shown below:

[0413] LC-MS:(ES,m / z):976.2[M+H]+;

[0414] 1H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.7Hz,5H),3.72(t,J=5.2Hz,2H),3.56–3. 46(m,1H),3.32–3.10(m,4H),2.98–2.91(m,1H),2.88–2.80(m,2H),2.75–2.68(m,1 H),2.63–2.49(m,3H),2.36–2.29(m,2H),2.06(q,J=6.7Hz,1H),1.88–1.83(m,1H), 1.71–1.53(m,9H),1.54–1.38(m,12H),1.34–1.21(m,75H),0.90(t,J=7.0Hz,13H).

[0415] 1.3.11: AL08-012

[0416] The proton spectrum of AL08-012 is shown in Figure 11, and the mass spectrometry and NMR data are shown below:

[0417] LC-MS:(ES,m / z):836.1[M+H] + ;

[0418] 1 H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.7Hz,4H),3.69(t,J=5.3Hz,2H),3.47–3.40(m,1H),2.92–2.87(m,1H),2.85–2.56(m,11H),2.53–2 .43(m,4H),2.37–2.26(m,2H),2.02(s,2H),1.85–1.77(m,1H),1.70–1 .56(m,9H),1.51–1.41(m,9H),1.39–1.20(m,58H),0.93–0.83(m,13H).

[0419] 1.3.12: AL08-014

[0420] The proton spectrum of AL08-014 is shown in Figure 12, and the mass spectrometry and NMR data are shown below:

[0421] LC-MS:(ES,m / z):807.0[M+H] + ;

[0422] 1H NMR(600MHz,Chloroform-d)δ4.08(td,J=6.7,1.8Hz,1H),3.70(t,J=5.3Hz,0H),3.50–3.35(m,0H),3.01–2.57(m,1H),2.57–2.43(m,1H),2.31 (t,J=7.6Hz,1H),2.08–1.81(m,0H),1.83–1.75(m,0H),1.70–1.58(m,1 H),1.47–1.40(m,1H),1.40–1.20(m,12H),0.90(td,J=7.1,2.1Hz,2H).

[0423] 1.3.13: AL08-015

[0424] The proton spectrum of AL08-015 is shown in Figure 13, and the mass spectrometry and NMR data are shown below:

[0425] LC-MS:(ES,m / z):780.0[M+H] + ;

[0426] 1 H NMR(600MHz,Chloroform-d)δ4.14–4.01(m,1H),3.78(t,J=5.1Hz,0H),3.56(t,J=7.6Hz,0H),3.20–2.73(m,1H),2.61–2.50(m, 1H),2.31(t,J=7.6Hz,1H),2.14–2.05(m,0H),2.00–1.87(m,0H),1.64(d,J=7.2Hz,1H),1.54–1.16(m,14H),1.10–0.80(m,3H).

[0427] 1.3.14: AL08-018

[0428] The proton spectrum of AL08-018 is shown in Figure 14, and the mass spectrometry and NMR data are shown below:

[0429] LC-MS:(ES,m / z):836.1[M+H] + ;

[0430] 1H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.7Hz,4H),3.77(t,J=5.2Hz,2H),3.16–2.78(m,10H),2.67–2.53(m,3H ),2.39–2.28(m,2H),2.19–2.08(m,1H),1.96(s,1H),1.71–1.58(m,7H),1.57–1.18(m,63H),0.98–0.84(m,12H).

[0431] 1.3.15: AL08-019

[0432] The proton spectrum of AL08-019 is shown in Figure 15, and the mass spectrometry and NMR data are shown below:

[0433] LC-MS:(ES,m / z):780.0[M+H] + ;

[0434] 1 H NMR(600MHz,Chloroform-d)δ4.09(t,J=6.7Hz,1H),3.75(q,J=6.7,5.9Hz,1H), 3.08–2.68(m,2H),1.73–1.50(m,3H),1.50–1.20(m,16H),0.90(t,J=7.1Hz,3H).

[0435] 1.3.16: AL08-020

[0436] The proton spectrum of AL08-020 is shown in Figure 16, and the mass spectrometry and NMR data are shown below:

[0437] LC-MS:(ES,m / z):780.0[M+H] + ;

[0438] 1 H NMR(600MHz,Chloroform-d)δ4.19–4.07(m,3H),3.78(t,J=5.2Hz,1H),3.08–3.01(m,1H),2.99–2.81(m,2H),2.77(q,J=7.0Hz,1H),2.73–2.65(m ,1H),2.37–2.29(m,3H),1.69(p,J=6.9Hz,2H),1.60(ddd,J=21.4,11.1, 7.1Hz, 4H), 1.50–1.34 (m, 5H), 1.34–1.22 (m, 22H), 0.90 (t, J = 7.0Hz, 6H).

[0439] 1.3.17: AL08-021

[0440] The proton spectrum of AL08-021 is shown in Figure 17, and the mass spectrometry and NMR data are shown below:

[0441] LC-MS:(ES,m / z):864.1[M+H] + ;

[0442] 1 H NMR(600MHz,Chloroform-d)δ4.14(q,J=7.1Hz,1H),4.09(t,J=6.7Hz,2H),3.75(q,J=6.7,5.9Hz,1H),3.08–3.01(m,1H),2.98–2.91(m,1H),2. 89–2.77(m,2H),2.37–2.29(m,1H),2.07(s,2H),1.65–1.56(m,4H),1.4 9–1.40(m,2H),1.36(s,7H),1.34–1.23(m,23H),0.90(t,J=7.1Hz,6H).

[0443] 1.3.18: AL08-022

[0444] The proton spectrum of AL08-022 is shown in Figure 18, and the mass spectrometry and NMR data are shown below:

[0445] LC-MS:(ES,m / z):808.0[M+H] + ;

[0446] 1 H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.7Hz,4H),3.67(t,J=5.3Hz,2H),3.00(s,2H),2.78–2.72(m,3H),2.69–2.61(m,2H), 2.52(dq,J=13.6,6.8,5.7Hz,4H),2.33(tt,J=9.0,5.3Hz,2H),1.68–1.56(m,8H),1.52–1.19(m,61H),0.89(t,J=7.0Hz,13H).

[0447] 1.3.19: AL08-023

[0448] The proton spectrum of AL08-023 is shown in Figure 19, and the mass spectrometry and NMR data are shown below:

[0449] LC-MS:(ES,m / z):864.1[M+H] + ;

[0450] 1 H NMR(600MHz,Chloroform-d)δ4.09(t,J=6.7Hz,2H),3.78(t,J=5.2Hz,1H),3.09–2.98(m,1H),2.93–2.41(m,8H),2.33 (tt,J=8.8,5.3Hz,1H),2.17(s,1H),1.73–1.53(m,7H),1.51–1.34(m,7H),1.33–1.19(m,29H),0.90(t,J=7.0Hz,7H).

[0451] 1.3.20: AL08-024

[0452] The proton spectrum of AL08-024 is shown in Figure 20, and the mass spectrometry and NMR data are shown below:

[0453] LC-MS:(ES,m / z):780.0[M+H] + ;

[0454] 1 H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.7Hz,1H),3.63(t,J=5.4Hz,1H),3.40 (t,J=7.6Hz,0H),3.27(q,J=6.7Hz,1H),2.80–2.49(m,1H),2.48–2.37(m,1H),2 .35–2.27(m,0H),1.97(d,J=4.8Hz,0H),1.83–1.71(m,0H),1.74–1.58(m,2H),1 .52(t,J=7.4Hz,1H),1.47–1.36(m,2H),1.37–1.20(m,22H),0.99–0.78(m,6H).

[0455] 1.3.21: AL08-025

[0456] The proton spectrum of AL08-025 is shown in Figure 21, and the mass spectrometry and NMR data are shown below:

[0457] LC-MS:(ES,m / z):807.1[M+H] + ;

[0458] 1H NMR(600MHz,Chloroform-d)δ5.66(t,J=6.0Hz,1H),4.09(t,J=6.7Hz,2H),3.73(t, J=5.2Hz,2H),3.63(q,J=7.7,7.2Hz,1H),3.28(q,J=6.4Hz,2H),3.01–2.60(m,13H) ,2.33(tt,J=8.9,5.3Hz,1H),2.17–2.07(m,1H),2.01(dd,J=9.3,5.0Hz,1H),1.93( dt,J=13.9,7.4Hz,1H),1.71–1.51(m,12H),1.50–1.19(m,56H),0.96–0.84(m,12H).

[0459] 1.3.22: AL08-026

[0460] The proton spectrum of AL08-026 is shown in Figure 22, and the mass spectrometry and NMR data are shown below:

[0461] LC-MS:(ES,m / z):835.0[M+H] + ;

[0462] 1 H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.7Hz,1H),3.71(t,J=5.3Hz,1H),3.48(t,J=7.6Hz,0H),3.27(q,J=6.7Hz ,1H),3.00–2.61(m,2H),2.50(q,J=8.0Hz,2H),2.23–1.79(m,1H),1.71–1.12(m,29H),0.89(td,J=7.1,4.4Hz,6H).

[0463] 1.3.23: AL08-027

[0464] The proton spectrum of AL08-027 is shown in Figure 23, and the mass spectrometry and NMR data are shown below:

[0465] LC-MS:(ES,m / z):835.0[M+H] + ;

[0466] 1H NMR(600MHz,Chloroform-d)δ4.08(t,J=6.7Hz,1H),3.69(t,J=5.3Hz,1H),3.45(d,J=7.6Hz,0H),3.27(q,J=6.7Hz,1H),2.88(t,J=8.7Hz,0H),2.81–2.59( m,2H),2.49(d,J=8.8Hz,2H),2.33(dt,J=9.0,4.0Hz,0H),1.96(dt,J=9.3,4. 5Hz, 0H), 1.82 (dd, J=13.2, 7.2Hz, 0H), 1.73–0.98 (m, 30H), 0.99–0.71 (m, 6H).

[0467] Example 2: Preparation of LNP-mRNA using ionizable lipid-encapsulated mRNA

[0468] This invention uses the ionizable lipids prepared in Example 1 and the control ionizable lipid ALP801 (see compound AXT-8 in patent CN116514696B) to encapsulate tool mRNA (mCherry) and assemble it into LNP-mRNA. The steps include:

[0469] 1) Ionizable lipids (AL08-001, AL08-003 to AL08-012, AL08-014, AL08-015, AL08-018 to AL08-027 prepared in Example 1), DSPC (distearylphosphatidylcholine, 1,2-distearyl-sn-glycerol-3-phosphocholine), cholesterol, and DMG-PEG2000 were mixed thoroughly in an ethanol phase in different proportions to form an organic phase;

[0470] 2) The mRNA was dissolved in sodium acetate buffer (pH=4.0) solution and thoroughly mixed to form the aqueous phase;

[0471] 3) Transfer the aqueous phase and organic phase separately into suitable syringes, removing as many air bubbles as possible;

[0472] 4) Using a PNI nanoparticle preparation instrument, the mixture was prepared at a volume ratio of 3:1 between the aqueous phase and the organic phase and a flow rate of 12 mL / min. The mixture was then purified, concentrated, and filtered for sterilization to obtain a product containing lipid nanoparticles (LNP-mRNA) encapsulated with mRNA.

[0473] 5) Perform physicochemical quality control on the final product LNP-mRNA.

[0474] Physicochemical quality control methods and results of LNP-mRNA:

[0475] 1) Particle size and polydispersity index (PDI): The particle size and distribution of LNP were detected using a nanoparticle size analyzer. The results are detailed in Table 3. The particle size is basically around 100 nm; the PDI is mostly around 0.1.

[0476] 2) Encapsulation Efficiency (EE%): After staining total mRNA and free mRNA with RiboGreen, the encapsulation efficiency was measured by microplate reader and calculated. The results are detailed in Table 3. The encapsulation efficiency was mostly maintained at 95%.

[0477] The above results indicate that this type of ionizable lipid of the present invention has superior properties for forming LNPs.

[0478] Table 3: Physicochemical properties and cell experiment data of different N / P formulations Note: a The ratio of ALP08-XY / AXT-8 represents the ratio of corresponding parameters between the ALP08-XY formulation and the AXT-8 formulation (where X represents a positive integer from 1 to 27, indicating the compound number, and Y represents a positive integer from 1 to 5, indicating the formulation number). The formulation of ALP801 LNP is: AXT-8:DSPC:Chol:DMG-PEG2000 = 50:10:38.5:1.5, with N / P of 3:1.

[0479] " / " indicates that the physicochemical properties are not up to standard and no cell experiments were performed.

[0480] Y corresponds to different formulations. Y = 1, 2, and 3 represent formulations of 50:10:38.5:1.5 (ionizable lipids:DSPC:cholesterol:DMG-PEG2000, molar ratio, with N / P ratios of 3:1, 4:1, and 5:1 respectively, where AL08-024-3 and AL08-025-3 have an N / P ratio of 6:1); Y = 4 corresponds to a formulation ratio of 40:20:38.5:1.5, with an N / P ratio of 3:1; Y = 5 corresponds to a formulation ratio of 60:10:28.5:1.5, with an N / P ratio of 3:1.

[0481] Example 3: Detection of LNP-mRNA expression in human primary T cells

[0482] To elucidate the ability of the above LNP-mRNA to transfect human T cell lines in vitro, an experiment was designed to transfect human primary T cells with LNP-mCherry, and the expression level of mCherry in the cells was detected. The steps are as follows:

[0483] 1) Isolation of primary T cells from human PBMCs: magnetic bead labeling (hereinafter referred to as 10) 7 Taking cells as an example, increase the corresponding reagents proportionally according to the number of cells. (Every 10) 7 Resuspend the total cells in 80 μL buffer, add 20 μL CD3 MicroBeads, incubate in a refrigerator for 15 minutes (2-8℃), wash the cells with buffer, and resuspend in 500 μL buffer for up to 10 cells. 8 Cells. Place the MS column on a magnetic rack and sort T cells using the MS column.

[0484] 2) LNP-mRNA transfection of primary T cells: Adjust the T cell count to 1*10 6 / mL, 24-well plate, 200uL of cells seeded into each well, i.e., 2*10 5 Add 6 μg / mL of LNP-mRNA (quantified by mRNA) to each well and replenish X-vivo medium to 1 mL. Add IL-2 to each well to a final concentration of 100 IU / mL. Incubate at 37°C for 24 h and then perform flow cytometry analysis.

[0485] 3) Flow cytometry analysis: Collect cells into 1.5 mL EP tubes, centrifuge at 400 g for 5 min, and discard the supernatant. Wash cells once with 1 mL of 2% FBS (prepared with PBS), centrifuge at 400 g for 5 min, and discard the supernatant. Add the appropriate antibody to the cell suspension and stain for 20 min. Wash cells once with 1 mL of 2% FBS (prepared with PBS), centrifuge at 400 g for 5 min, and discard the supernatant. Resuspend cells in 50 μL of 2% FBS (prepared with PBS) and analyze by flow cytometry. Virulence is determined by cell viability (see Table 3 for details).

[0486] As shown in Table 3, after transfection of primary T cells with different formulations (at a dose of 6 μg / mL), the cell viability of most formulations was higher than 90%, demonstrating their high safety.

[0487] The flow cytometry data for some LNP-mRNAs (formulas ALP801, ALP08-005-1, ALP08-019-1, ALP08-012-1, ALP08-022-1, and ALP08-025-2) are shown in Figure 24 and Table 4. Figure 24 shows that all listed formulations achieved transfection efficiencies of over 70%.

[0488] Table 4: Transfection efficiency and expression levels of primary T cells transfected with different formulations

[0489] The results in Table 4 were used as a baseline to evaluate the transfection efficiency of other LNP-mRNAs. The formulation of ALP801 was: AXT08:DSPC:Chol:DMG-PEG2000 = 50:10:38.5:1.5, with an N / P ratio of 4:1.

[0490] Table 4 shows the ratio of CD3+ positivity rates after transfection of human primary T cells with different formulations to the positive control ALP801 (mCherry). The ratio results show that the transfection efficiency of T cells with formulations such as ALP08-0019-1, ALP08-012-1, ALP08-005-1, ALP08-022-1, ALP08-025-2, and ALP08-017-2 is higher than or equal to that of ALP801.

[0491] Table 4 shows the MFI (mean fluorescence intensity) ratios of different formulations transfected human primary T cells with the positive control ALP801 (mCherry) transfected cells. As shown in Figure 24, the mean fluorescence intensity of T cells transfected with ALP08-005-1, ALP08-019-1, ALP08-012-1, ALP08-022-1, ALP08-016-1, and ALP08-017-2 was higher than or equal to that of ALP801.

[0492] Example 4: Preparation of Ab-LNP

[0493] Preparation of LNP-mal:

[0494] Ionizable lipids: DSPC:Chol:DSPE-PEG2k:DSPE-PEG5k-Mal: (50:10:38:X:Y, molar ratio; AL08-005 is used as an example of ionizable lipids here, X+Y=2, Y can be 0.25 to 2), lipids are dissolved in ethanol, mCherry is dissolved in sodium acetate aqueous solution at pH 4.0, and LNP-mal is prepared by microfluidic control.

[0495] Antibody activation:

[0496] Measure a certain volume of LNP-mal solution and calculate the molar amount of DSPE-PEG5k-mal. Calculate the molar amount of Ab antibody using the molar ratio of Ab antibody to mal derivative of 1:2. Based on the molar ratio of TCEP-activated Ab antibody (hCD8 antibody used here) of 20:1, measure the corresponding volume of TCEP solution and mix it evenly with the above Ab antibody. Incubate at room temperature for 0.5 hours and remove excess TCEP using a G25 desalting column.

[0497] A certain amount of LNP-mal solution was added to the above TCEP activated antibody solution and incubated for 1 hour to form Ab-LNP linked to the antibody. The antibody concentration was detected using a BCA protein detection kit.

[0498] The physicochemical properties characterization results of Ab-LNP are shown in Table 5.

[0499] Table 5: Physicochemical Properties of Ab-LNP

[0500] The physicochemical properties of Ab-LNP show that, compared with LNP without antibody linkage, its particle size is increased by approximately 15 nm, PDI is less than 0.2, encapsulation efficiency is as high as 91.15%, and antibody concentration is 1.678 mg / mL, demonstrating that it has superior nanoparticle formation characteristics.

[0501] Example 5: Ab-LNP transfection of hPBMC

[0502] Primary PBMCs (unactivated, e.g., not activated with anti-CD3 / anti-CD28 antibodies) were rapidly dissolved in a 37°C water bath, added to culture medium restored to 37°C, centrifuged at 300g for 10 min, resuspended in X-vivo 15 medium, and AO / PI cell counted. The cell volume was adjusted to 2.5*10⁻⁶ cells / mL. 6 / mL, 24-well plate, 200uL of cells seeded into each well, i.e., 5*10 5 / well. Add the appropriate dose of Ab-LNP (containing CD8 antibody) prepared in Example 4 (6 μg / mL) and supplement with X-vivo medium to 1 mL. After incubation at 37°C for 24 h, collect the cells into 1.5 mL EP tubes, centrifuge at 400 g for 5 min, discard the supernatant, and wash the cells once with 1 mL of 2% FBS (prepared with PBS), repeating the washing twice. Add the flow cytometry antibody to the cell suspension, stain at room temperature for 20 min, then wash the cells once with 1 mL of 2% FBS (prepared with PBS), centrifuge at 400 g for 5 min, discard the supernatant, resuspend the cells again in 100 μL of 2% FBS (prepared with PBS), and perform flow cytometry analysis.

[0503] Figure 25 shows the results of Ab-LNP transfection of hPBMCs. The results of LNP and Ab-LNP transfection of hPBMCs demonstrate that the LNP of this invention has excellent immune cell transfection efficacy; compared to LNP without antibody modification, Ab-LNP exhibits superior and more precise targeting of CD8. + The ability of T cells is as high as 86%.

[0504] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An ionizable lipid, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein, The ionizable lipid is a compound of Formula (I): wherein, R1and R2are each independently selected from -(CH2) n - and n is selected from an integer from 1 to 14; L1 is selected from the group consisting of: -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; L2 is selected from the group consisting of: -O-, -(C=O)O-, -O(C=O)-, -(S-S)-, -O(S=O)-, -(C=O)S-, -S(C=O)-, -(C=S)O-, -NH(C=O)-, -(C=S)NH-, -NH(C=O)-, -NH(C=S)-, -(C=O)NH-, -CH(OH)-; R3, R4, R5, and R6are each independently selected from the group consisting of: H, C2-C 20 hydrocarbyl; R7 is a C1-C5 hydrocarbon group.

2. The ionizable lipid of claim 1, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein, n is selected from an integer from 5 to 8; Preferably, L1 is selected from the group consisting of: -(C=O)O-, -O(C=O)-, -(C=O)NH-, -NH(C=O)-; Preferably, L2 is selected from the group consisting of: -(C=O)O-, -O(C=0)-, -(C=O)NH-, -NH(C=O)-; Preferably R7 is a C2-C4 hydrocarbon group.

3. The ionizable lipid of claim 1, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein, The ionizable lipid is selected from the following structures:

4. A method of preparing an ionizable lipid, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, of any one of claims 1-3, wherein, The method comprises the following steps: (S1) reacting compound A1 and compound A2 under inert gas protection to obtain compound A3; (S2) removing tert-butyloxycarbonyl (Boc) from compound A3 under inert gas protection to obtain compound A4; (S3) reacting compound A4 with compound A5 or compound A6 under inert gas protection to obtain a compound shown in formula (I); wherein R1and R2are each independently -(CH2) n - wherein n is selected from an integer from 1 to 14, preferably from an integer from 5 to 8; G1 and G2 are each independently selected from the group consisting of: aldehyde group, halogen; L1 is selected from the group consisting of: -O-, -(C=O)O-, L2 is selected from the group consisting of: -O-, -(C=O)O-, R3, R4, R5, and R6are each independently selected from the group consisting of H, C2-C 20 hydrocarbyl; R7 is a C1-C5 hydrocarbon group, preferably selected from C2-C4 hydrocarbon group.

5. A lipid nanoparticle, wherein, The lipid nanoparticle comprises the ionizable lipid of any one of claims 1-3, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, or the ionizable lipid prepared by the method of claim 4, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof.

6. The lipid nanoparticle of claim 5, wherein, The lipid nanoparticle comprises a lipid component modified with a targeting agent selected from the group consisting of at least one of a ligand, a receptor, an antibody, an antigen-binding fragment of an antibody, an aptamer, a polypeptide.

7. A lipid nanoparticle pharmaceutical formulation, wherein, The lipid nanoparticle pharmaceutical formulation comprises: i) the lipid nanoparticle of claim 5; ii) a biologically active substance encapsulated in the lipid nanoparticle; and iii) a pharmaceutically acceptable carrier; The biologically active substance is selected from the group consisting of a polypeptide, a protein, an amino acid, a vitamin, a mannose, or a combination thereof. The method comprises:

8. A method of preparing the lipid nanoparticle pharmaceutical formulation of claim 7, wherein, (a) mixing the ionizable lipid of any one of claims 1-3, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, or the ionizable lipid prepared by the method of claim 4, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, and optionally a helper lipid, with an organic solvent, thereby obtaining a lipid organic phase; (b) mixing a biologically active substance with an aqueous solvent, thereby obtaining an aqueous phase containing the biologically active substance; (c) mixing the lipid organic phase of step (a) with the aqueous phase of step (b), thereby obtaining the lipid nanoparticle pharmaceutical.

9. Use of the ionizable lipid of any one of claims 1-3, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, or the ionizable lipid prepared by the method of claim 4, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, for the manufacture of a drug delivery system.

10. Use of the ionizable lipid of any one of claims 1-3, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, or the ionizable lipid prepared by the method of claim 4, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, for the manufacture of a lipid nanoparticle pharmaceutical formulation for the delivery of a biologically active substance to a cell.

11. Use of the ionizable lipid of any one of claims 1-3, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, or the ionizable lipid prepared by the method of claim 4, or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, for the manufacture of a medicament for prophylactic vaccines, treatment and / or prevention of tumors, autoimmune diseases, infectious diseases, and rare diseases. ​

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