Ionizable heterocyclic lipid molecules and their application in the preparation of lipid nanoparticles

Abstract

The present invention relates to the field of nucleic acid drug delivery technology, and more specifically to ionizable heterocyclic lipid molecules and their use in the preparation of lipid nanoparticles. Compared to currently marketed lipid nanoparticles using MC3 lipids, the lipid nanoparticles obtained from the ionizable heterocyclic lipid molecules of the present invention produce more uniform and stable mRNA LNP particles, exhibit more significant cell transfection advantages, and enable efficient and low-frequency drug delivery, thereby reducing potential toxicity risks.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid drug delivery, and in particular to ionizable heterocyclic lipid molecules and applications thereof in preparing lipid nanoparticles. Background Art

[0002] RNA-based therapies are currently gaining widespread attention in the treatment of various diseases, including those associated with "undruggable" targets. RNA drugs mainly include antisense oligonucleotides (ASOs), small interfering RNA (siRNA), micro RNA (miRNA) and mRNA. Over the past decade, a series of RNA-based therapeutics have been approved for the treatment of different types of diseases, such as macular degeneration, spinal muscular atrophy, hypercholesterolemia and amyloidosis. In 2020, two encapsulated mRNA drugs, BNT162b2 and mRNA-1273, have received emergency use authorization (EUA) from the FDA and EMA as SARS-CoV-2 vaccines for the prevention of coronavirus disease 2019 (COVID-19).

[0003] Although RNA drugs have great potential in the treatment of various diseases, due to the fact that RNA drugs are easily degraded by enzymes and the extremely low cellular uptake rate of free RNA drugs, it is necessary to introduce some carriers to package and deliver RNA in order to expand the application of RNA.

[0004] Among these delivery vectors, lipid nanoparticles (LNP) are the most important non-viral delivery vectors used for nucleic acid drugs. The mRNA vaccines developed by BioNTech / Pfizer and Moderna are both delivered using lipid nanoparticles, and the lipid complexes in their lipid nanoparticles are composed of cationic lipids, auxiliary neutral phospholipids, cholesterol and PEG lipids.

[0005] Cationic lipids can form ionic interactions with negatively charged RNA through their positive charge to form complexes. Cationic lipids are further divided into lipids with permanent positive charge and ionizable lipids. Cationic lipids with permanent positive charge include DOTAP and DOTMA. In the early stages of nucleic acid drug delivery, only cationic lipids with permanent positive charge were used to encapsulate nucleic acids. Although they showed a high in vitro transfection rate in vitro, the permanent positive charge and non-degradable properties of such cationic lipids limited their in vivo efficacy and may also cause cytotoxicity problems. Lipofectin is a mixture of DOTMA and DOPE. By adding the auxiliary lipid DOPE to reduce cytotoxicity, it can load mRNA for in vivo expression. Another type of cationic lipid, ionizable lipid, refers to a class of lipids that have almost no charge under normal neutral physiological pH conditions but are positively charged at acidic pH, including DODMA and siRNA lipid nanoparticle drugs. Dlin-MC3-DMA, as well as ALC0315 and SM102 used in the mRNA vaccines currently being developed by BioNTech / Pfizer and Moderna, etc. The pH-sensitive properties of ionizable lipids prevent them from being metabolized by the reticuloendothelial system (RES), thereby extending their half-life. They are also less likely to cause immune activation or interact with serum proteins, thereby improving their safety.

[0006] In addition to cationic lipids, other lipids included in the lipid nanoparticle formulation also have their own functions. Auxiliary neutral lipids, such as DSPC, are used to support the lipid bilayer structure; cholesterol acts as a stabilizer to enhance the stability of the preparation; and PEG-lipids can prolong the half-life of the preparation, reduce nonspecific interactions with plasma proteins, improve the stability of the preparation and prevent aggregation.

[0007] Although current RNA delivery systems are primarily based on lipid nanoparticles, lipid nanoparticles themselves also have some shortcomings. The key lipids in currently marketed nucleic acid lipid nanoparticle formulations are ionizable cationic lipids. After being taken up by cells, they can be protonated in acidic endosomes and interact with anionic endosomal phospholipids to form conical ion pairs that are incompatible with the bilayer. These cation-anionic lipid pairs drive the transition from the bilayer structure to the inverted hexagonal HII phase, which facilitates membrane fusion / disruption, endosomal escape, and drug release into the cytoplasm. At the same time, these ionizable cationic lipids also affect the stability of RNA formulations, including expression stability. In order to maintain good stability and efficacy, the current ionizable cationic lipid content in the lipid nanoparticle formulation must be kept above 50%, as disclosed in Chinese Patent CN102119217B. Therefore, whether the ionizable cationic lipids in currently marketed lipid nanoparticles are the optimal choice for RNA delivery remains a question.

[0008] In order to overcome the above problems, the present invention:

[0009] 1. By improving the structure of ionizable amino lipid molecules, introducing a saturated heterocyclic piperazine group, a hydroxyl hydrophilic head, a diol connecting functional group and / or an insignificant long tail chain, a new type of ionizable amino lipid was obtained;

[0010] 2. Lipid nanoparticles are obtained by combining the novel ionizable amino lipids with neutral phospholipids, cholesterol, and PEG lipids. The obtained lipid nanoparticles can well encapsulate mRNA and increase the translation expression level of mRNA in cells; and

[0011] 3. Compared with the currently marketed lipid nanoparticles using MC3 lipids, the mRNA LNP particles formed by the obtained lipid nanoparticles are more uniform and stable, indicating that this new type of ionizable lipid has better stability and adaptability, and is less affected by the formulation content factors compared with MC3. At the same time, this new type of ionizable lipid exhibits more significant cell transfection advantages than MC3 lipids in low-content formulations, which can achieve efficient and low-frequency drug delivery, thereby reducing potential toxicity risks. Summary of the Invention

[0012] Based on the problems existing in the prior art, the present invention provides ionizable heterocyclic lipid molecules and applications thereof in preparing lipid nanoparticles.

[0013] The present invention provides a lipid compound represented by formula (I) or a pharmaceutically acceptable salt thereof:

[0014]

[0015] in,

[0016] R1 and R2 are each independently C6-C 20 Alkyl, C6-C 20 Alkenyl or C6-C 20 Alkynyl;

[0017] X is independently -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=S)O-, -OC(=S)-, -OC(=S)O-, -C(=O)S-, -SC(=O)-, -OC(=O)S-, -O-, -S-, -C1-C6 alkylene-O-, -O-C1-C6 alkylene-, -C1-C6 alkylene-S-, or -S-C1-C6 alkylene-;

[0018] R3 is independently a 5-6 membered saturated heterocyclyl containing 1 or 2 ring heteroatoms independently selected from N, O and S, which is optionally substituted by R4;

[0019] R4 is independently C1-C6 alkyl, which is optionally substituted with -OH;

[0020] m and n are each independently selected from integers of 1-10.

[0021] In one embodiment of the present invention, the lipid compound represented by formula (I) has a structure represented by the following formula (II):

[0022]

[0023] In one embodiment of the present invention, in the lipid compound represented by the above formula (I) or formula (II), R1 is C6-C 10 Straight chain alkyl, and R2 is C 10 -C 20 Branched alkyl groups.

[0024] In one embodiment of the present invention, in the lipid compound represented by the above formula (I) or formula (II), R1 is a C9 straight chain alkyl group, and R2 is

[0025] In one embodiment of the present invention, in the lipid compound represented by the above formula (I) or formula (II), X is independently -C(=O)O-, -OC(=O)-, -C1-C6 alkylene-O- or -O-C1-C6 alkylene-.

[0026] In one embodiment of the present invention, in the lipid compound represented by the above formula (I) or formula (II), X is independently -OC(=O)- or -O-CH2-.

[0027] In one embodiment of the present invention, in the lipid compound represented by the above formula (I) or formula (II), R4 is independently -CH3, -CH2CH2OH, -CH2CH2CH2OH or -CH2CH2CH2CH2OH.

[0028] In one embodiment of the present invention, in the lipid compound represented by the above formula (I) or formula (II), m and n are each independently 6.

[0029] In one embodiment of the present invention, the lipid compound represented by formula (I) has a structure represented by the following formula (III):

[0030]

[0031] In one embodiment of the present invention, in the lipid compound represented by the above formula (III), X is independently -OC(=O)- or -O-CH2-, and R4 is independently -CH3, -CH2CH2OH, -CH2CH2CH2OH or -CH2CH2CH2CH2OH.

[0032] In one embodiment of the present invention, in the lipid compound represented by the above formula (I), formula (II) or formula (III), the compound is selected from:

[0033]

[0034]

[0035] The present invention also provides a lipid nanoparticle composition, which comprises the lipid compound represented by the above formula (I), formula (II) or formula (III) described in the present application, or a pharmaceutically acceptable salt thereof.

[0036] In one embodiment of the present invention, the above lipid nanoparticle composition further comprises neutral lipids, cholesterol and PEG lipids.

[0037] In one embodiment of the present invention, in the above lipid nanoparticle composition, the neutral lipid is selected from DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, SOPE and 1,2-dipentanoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE), and the PEG lipid is selected from PEG-DMG, PEG-dipalmitoylglycerol, PEG-DSPE, PEG-dilaurylglyceramide, PEG-dimyristylglyceramide, PEG-dipalmitoylglyceramide and PEG-distearoylglyceramide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[β]-oxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB, PEG2k-DMG, PEG2k-DSPE, PEG2k-DSG, PEG2k-DMA and PEG2k-DSA.

[0038] In one embodiment of the present invention, in the above lipid nanoparticle composition, the neutral lipid is DSPC, and the PEG lipid is PEG2k-DMG.

[0039] In one embodiment of the present invention, in the above lipid nanoparticle composition, the lipid compound described herein accounts for 30-50% by mole of the total lipid components of the composition.

[0040] In one embodiment of the present invention, in the above lipid nanoparticle composition, the lipid compound described herein accounts for 40-50% by mole of the total lipid components of the composition.

[0041] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 30-50% of the lipid compound, 8-12% of the neutral lipid, 35-60% of the cholesterol, and 1-3% of the PEG lipid.

[0042] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 31.5% of the lipid compound, 10% of the neutral lipid, 56% of the cholesterol, and 2.5% of the PEG lipid.

[0043] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 43.3% of the lipid compound, 8.7% of the neutral lipid, 46.5% of the cholesterol, and 1.5% of the PEG lipid.

[0044] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 46.3% of the lipid compound, 9.4% of the neutral lipid, 42.7% of the cholesterol, and 1.6% of the PEG lipid.

[0045] In one embodiment of the present invention, in the above lipid nanoparticle composition, the molar percentages of the lipid components described in the present application in the total lipid components of the composition are: 50% of the lipid compound, 10% of the neutral lipid, 38.5% of the cholesterol, and 1.5% of the PEG lipid.

[0046] In one embodiment of the present invention, the above lipid nanoparticle composition further comprises a nucleic acid molecule selected from the group consisting of mRNA, siRNA, antisense oligonucleotide (ASO), saRNA and miRNA.

[0047] The present invention also provides a method for delivering a nucleic acid into a cell, comprising delivering the lipid nanoparticles described above into the cell.

[0048] In one embodiment of the present invention, the cell is a mammalian cell, preferably the cell is a human cell.

[0049] In one embodiment of the present invention, the cell is a mammalian precancerous lesion cell, preferably a human precancerous lesion cell.

[0050] In one embodiment of the present invention, the cell is a tumor cell, preferably the cell is a human tumor cell.

[0051] The present invention also provides a use of the lipid nanoparticles described herein in preparing a drug for treating a disease.

[0052] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as indefinite or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0053] The term "pharmaceutically acceptable" as used in this application refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0054] The term "pharmaceutically acceptable salt" as used herein refers to a salt of a compound of the invention, prepared from a compound having a specific substituent discovered by the present invention and a relatively nontoxic acid or base. When a compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such compound with a sufficient amount of a base in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, organic acid salts, salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid (see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds of the present invention contain basic and acidic functional groups and can be converted into either a base or acid addition salt.

[0055] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, the salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent or a mixture of the two.

[0056] The term "alkyl" or "alkylene" as used herein is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, "C1-C10 Alkyl (or alkylene) is intended to include C1, C2, C3, C4, C5, C6, C7, C8, C9 and C 10 Alkyl (or alkylene). The alkyl group may be unsubstituted or substituted, wherein at least one hydrogen is replaced by another chemical group. Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl), etc.

[0057] As used herein, the term "alkenyl" is intended to include hydrocarbon chains having a linear or branched configuration and having one or more carbon-carbon double bonds that may be present at any stable point along the chain. For example, "C2-C6 alkenyl" is intended to include C2, C3, C4, C5, and C6 alkenyl groups; for example, ethenyl, propenyl, butenyl, pentenyl, and hexenyl.

[0058] As used herein, the term "alkynyl" is intended to include hydrocarbon chains having a straight or branched configuration and having one or more carbon-carbon triple bonds that may exist at any stable point along the chain. For example, "C2-C6 alkynyl" is intended to include C2, C3, C4, C5, and C6 alkynyl groups; for example, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0059] As used herein, the term "heterocycle" or "heterocyclyl" refers to a stable monocyclic, bicyclic, or tricyclic ring containing a heteroatom or heteroatom group, which may be saturated, partially unsaturated, or unsaturated (aromatic), and which comprises carbon atoms and 1, 2, 3, or 4 ring heteroatoms independently selected from N, O, and S. The nitrogen atom may be substituted or unsubstituted. Exemplary monocyclic heterocyclic groups include azetidinyl, pyrrolidinyl, oxetanyl, imidazolinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, 1-pyridonyl, 4-piperidonyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolane and tetrahydro-1,1-dioxothiphenyl. Exemplary bicyclic heterocyclic groups include quinuclidine. The "heterocycle" employed in the present invention is preferably a 5-6 membered saturated heterocyclic group containing 1 or 2 ring heteroatoms independently selected from N, O and S, more preferably a piperazinyl.

[0060] The term "neutral lipid" as used herein refers to any of a variety of lipids that exist in the form of uncharged or neutral zwitterions at physiological pH. Representative neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside. In addition, the neutral lipids of the present invention can also be selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and oleoylphosphatidylethanolamine 4-(N-maleimidomethyl)- Cyclohexane-1 carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE) and 1,2-dipentanoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE), preferably, the neutral lipid is selected from DSPC.

[0061] As used herein, the term "PEG lipid" may be selected from the group consisting of PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilaurylglyceramide, PEG-dimyristylglyceramide, PEG-dipalmitoylglyceramide and PEG-distearoylglyceramide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[β]-oxy)formamido-3',6'-dioxaoctyl]amino Formyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB (3,4-di-tetradecylbenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (Cat. No. 880120C, obtained from Avanti Polar Lipids, Alabaster, Alabama, USA), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA) and 1,2-distearoyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA), preferably, the PEG lipid is selected from PEG2k-DMG.

[0062] The terms "nucleic acid" or "nucleic acid molecule" as used herein will be recognized and understood by those of ordinary skill in the art, for example, to mean a molecule comprising, preferably consisting of, a nucleic acid component. The term nucleic acid molecule preferably refers to a DNA or RNA molecule. It is preferably used synonymously with the term polynucleotide. Preferably, a nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers that are covalently linked to each other via phosphodiester bonds of a sugar / phosphate backbone. Preferably, the nucleic acid molecule is selected from mRNA, siRNA, antisense oligonucleotide (ASO), saRNA or miRNA.

[0063] The ionizable heterocyclic lipid molecules and lipid nanoparticles prepared therefrom provided by the present invention have the following beneficial effects:

[0064] 1. By improving the structure of ionizable amino lipid molecules, introducing a saturated heterocyclic piperazine group, a hydroxyl hydrophilic head, a diol connecting functional group and / or an insignificant long tail chain, a new type of ionizable amino lipid was obtained;

[0065] 2. Lipid nanoparticles are obtained by combining the novel ionizable amino lipids with neutral phospholipids, cholesterol, and PEG lipids. The obtained lipid nanoparticles can well encapsulate mRNA and increase the translation expression level of mRNA in cells; and

[0066] 3. Compared with the currently marketed lipid nanoparticles using MC3 lipids, the mRNA LNP particles formed by the obtained lipid nanoparticles are more uniform and stable, indicating that this new type of ionizable lipid has better stability and adaptability, and is less affected by the formulation content factors compared with MC3. At the same time, this new type of ionizable lipid exhibits more significant cell transfection advantages than MC3 lipids in low-content formulations, which can achieve efficient and low-frequency drug delivery, thereby reducing potential toxicity risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 : Flow chart of the preparation of lipid nanoparticles encapsulating nucleic acids;

[0068] Figure 2 Results of in vitro transfection experiments using different mRNA-encapsulated lipid nanoparticles. Luciferase mRNA was encapsulated in lipid nanoparticles prepared with different ionizable amino lipids and then transfected into HeLa cells in vitro. Transfection efficiency was measured 24 hours after transfection. The lipid nanoparticle formulation was based on Dlin-MC3-DMA.

[0069] Figure 3 :The in vitro transfection experimental results of different lipid nanoparticles encapsulating mRNA, wherein after adopting MC3-LNP of different prescriptions and lipid 8-LNP of the present invention to encapsulate luciferase mRNA, Hela cells are carried out in vitro transfection, and the transfection efficiency is detected 24h after transfection.The prescription of lipid nanoparticles is based on the prescription of Dlin-MC3-DMA.

[0070] Figure 4 :The in vitro transfection experimental results of different lipid nanoparticles encapsulating mRNA, wherein after adopting MC3-LNP of different prescriptions and lipid II-LNP of the present invention to encapsulate luciferase mRNA, Hela cells are carried out in vitro transfection, and the transfection efficiency is detected 24h after transfection.The prescription of lipid nanoparticles is based on the prescription of Dlin-MC3-DMA. DETAILED DESCRIPTION

[0071] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are merely illustrative and illustrative of the present invention and should not be construed as limiting the scope of protection of the present invention. Any technical solutions implemented based on the content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0072] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0073] Example Synthesis of Different Ionizable Lipids

[0074] Objective: To synthesize a series of ionizable amino lipid compounds and conduct corresponding evaluation and screening of lipids.

[0075] Example 1: Preparation of lipid 1

[0076]

[0077] 80g (1mol, 1.0eq) diethyl malonate and 300g (1.75mol, 2.5eq) p-toluenesulfonic acid monohydrate are dissolved in 150ml acetonitrile, stirred to clarify, then 9g (0.2mol, 1.08eq) N-bromosuccinimide is added, temperature rising reflux reaction 12 hours. After reaction, desolventizing is concentrated under reduced pressure, appropriate amount of water and dichloromethane are added, stirred and stratified, and collected organic layer is dried over anhydrous sodium sulfate. Suction filtration, filter cake is washed with a small amount of dichloromethane, desolventizing is carried out to obtain 130g crude product, and crude product is further purified by silica gel column, eluent is from petroleum ether to petroleum ether: ethyl acetate=1:10, obtains 65.2g oily matter (0.27mol, compound 1), yield 34.6%.

[0078] 65.2 g (0.27 mol, 1.0 eq) of compound 1, 2.2 g (0.27 mol, 1.0 eq) of N-methylpiperazine, and 156.4 g (0.41 mol, 10.05 eq) of potassium carbonate were dissolved in 80 ml of acetonitrile and reacted at room temperature for 48 hours. After completion of the reaction, the mixture was concentrated under reduced pressure to remove the solvent. Appropriate amounts of water and ethyl acetate were added, and the mixture was stirred and allowed to stand for stratification. The organic layer was collected and dried over anhydrous sodium sulfate. The mixture was filtered, and the filter cake was washed with a small amount of ethyl acetate. The solvent was removed under reduced pressure to obtain 100 g of the crude product. The crude product was further purified on a silica gel column with the eluent varying from petroleum ether to dichloromethane:alcohol = 1:20 to obtain 39 g of an oil (0.15 mol, compound 2), with a yield of 55.5%. 1H NMR (400MHz, CDCl3) δ4.28 (q, J = 7.2Hz, 4H), 4.07 (s, 1H), 2.84 (t, J = 4.7Hz, 4H), 2.64–2.36 (m, 4H), 2.34 (s, 3H), 1.33 (t, J = 7.1Hz, 6H).

[0079] 19.5 g (0.25 mol, 12.5 eq) of LAH was dissolved in 40 ml of THF, and then 66 g (0.3 mol, 3.0 eq) of compound 2 (dissolved in 10 ml of THF) was gradually added dropwise under low temperature conditions and allowed to react for 12 hours under the same conditions. After the reaction, 200 ml of NaOH was slowly added dropwise to the reaction solution, resulting in a vigorous exotherm and gas evolution. The temperature was kept constant, and a large amount of solid precipitated. 100 ml of dichloromethane was then added to dilute the solution, filtered with suction, and the filtrate was concentrated and washed with petroleum ether / ethyl acetate to obtain 3.5 g of an oil (0.02 mol, compound 3), with a yield of 20%. 1H NMR (400MHz, DMSO) δ4.26 (brs, 2H), 3.71–3.22 (m, 6H), 2.62 (t, J = 4.8Hz, 4H), 2.46 (t, J = 6.1Hz, 1H), 2.28 (s, 2H), 2.14 (s, 3H).

[0080] 0.87 g (0.005 mol, 1.0 eq) of compound 3, 2.8 g (0.01 mol, 2.0 eq) of linoleic acid, 10.93 g (0.15 mol, 3.0 eq) of DIPEA, 4.49 g (0.04 mol, 0.8 eq) of DMAP, and 12.39 g (0.125 mol, 2.5 eq) of EDCI were dissolved in 30 ml of DCM and reacted at room temperature for 48 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure, and the crude product was further purified on a silica gel column with the eluent changing from petroleum ether to petroleum ether:dichloromethane = 1:100 to obtain 170 mg of an oil (0.00024 mol, lipid 1), with a yield of 4.86%. 1H NMR (400MHz, CDCl3) δ5.39(td,J=11.9,5.0Hz,8H),4.32(dd,J=11.7,6.2Hz,2H),4.13(dd,J=11.8,5.5Hz,2H),2.78(dt,J=22. 3,5.7Hz,8H),2.54–2.21(m,9H),2.09(q,J=6.9Hz,8H),1.65(t,J=7.3Hz,4H),1.36(d,J=13.6Hz,32H),0.93(t,J=6.5Hz,6H).

[0081] Example 2: Preparation of lipid 2

[0082]

[0083] 15.12 g (0.02 mol, 1.0 eq) of 9-heptadecanol, 2.4 g (0.02 mol, 1.0 eq) of suberic acid, 13.9 g (0.03 mol, 1.5 eq) of DIPEA, 11 g (0.008 mol, 0.4 eq) of DMAP, and 4.16 g (0.024 mol, 1.2 eq) of EDCI were dissolved in 150 ml of DCM and reacted at room temperature for 48 hours. After completion of the reaction, the solvent was concentrated under reduced pressure and the crude product was further purified on a silica gel column with the eluent increasing from dichloromethane to dichloromethane:methanol = 1:100 to obtain 1.1 g of an oil (0.0027 mol, compound 4), with a yield of 13.75%. 1H NMR (400MHz, CDCl3) δ4.92 (q, J=6.3Hz, 1H), 2.40 (dd, J=26.3, 6.3Hz, 4H), 1.7 5(p,J=3.5Hz,4H),1.57(q,J=6.2Hz,4H),1.32(s,26H),0.94(t,J=6.7Hz,6H).

[0084] 0.98g (0.002, 2.0eq) of compound 4, 0.12g (0.001, 1.0eq) of compound 3, 1.39g (0.003mol, 3.0eq) of DIPEA, 1.1g (0.0008mol, 0.8eq) of DMAP, and 1.5g (0.0025mol, 2.5eq) of EDCI were reacted at room temperature for 48 hours. After completion of the reaction, the mixture was concentrated under reduced pressure and desolvated. The crude product was further purified on a silica gel column with the eluent changed from dichloromethane to dichloromethane:methanol = 1:100 to obtain 100mg of an oil (0.0001mol, lipid 2), with a yield of 10%. 1H NMR (400MHz, CDCl3) δ4.90(p,J=6.3Hz,2H),4.31(dd,J=11.6,6.3Hz,2H),4.12(dd,J=11.6,5.6Hz,2H),2.75(t,J=4.8Hz,4H),2.45( s, 3H), 2.38–2.23 (m, 13H), 1.65 (h, J = 7.4Hz, 10H), 1.53 (t, J = 6.2Hz, 9H), 1.43–1.31 (m, 24H), 1.29 (s, 31H), 0.91 (t, J = 6.7Hz, 15H).

[0085] Example 3: Preparation of lipid 3

[0086]

[0087] 12.5 g (0.02 mol, 2.0 eq) of compound 3, 21.8 g (0.02 mol, 3.0 eq) of linoleic acid, 3.19 g (0.03 mol, 1.5 eq) of DIPEA, 11 g (0.008 mol, 0.4 eq) of DMAP, and 41.6 g (0.024 mol, 1.2 eq) of EDCI were dissolved in 350 ml of DCM and reacted at room temperature for 48 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The crude product was further purified on a silica gel column using an eluent ratio of dichloromethane to dichloromethane:methanol = 1:100 to obtain 2.2 g of an oil (0.005 mol, compound 5), with a yield of 25%. NMR(400MHz, CDCl3) δ5.39(p,J=6.6Hz,4H),4.27(dd,J=11.7,6.6Hz,1H),4.07(dd,J=11.7,5.6Hz ,1H),3.57(dd,J=10.8,5.1Hz,1H),3.45(t,J=10.2Hz,1H),3.00(dq,J=10.6,5.7Hz,1H),2.90(d,J =9.5Hz,2H),2.80(q,J=8.4Hz,3H),2.65(t,J=8.5Hz,2H),2.51(s,4H),2.33(d,J=10.0Hz,5H),2. 09(q,J=6.9Hz,4H),1.76–1.53(m,1H),1.38(d,J=7.6Hz,4H),1.34(s,11H),0.93(t,J=6.6Hz,3H).

[0088] 1.94 g (0.00229 mol, 1.0 eq) of compound 4, 11 g (0.00229 mol, 2.0 eq) of compound 5, 10.44 g (0.00343 mol, 1.15 eq) of DIPEA, 10.11 g (0.00009 mol, 1.4 eq) of DMAP, and 0.53 g (0.00275 mol, 1.2 eq) of EDCI were dissolved in 100 ml of DCM and reacted at room temperature for 48 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The crude product was further purified on a silica gel column using dichloromethane:ethanol = 1:100 as the eluent to obtain 200 mg of an oil (0.00024 mol, lipid 3), with a yield of 10.5%. NMR (400MHz, CDCl3) δ5.38 (tt, J=9.8, 5.9Hz, 4H), 4.32 (dd, J=11.6, 6.3Hz, 2H), 4. 13(dd,J=11.7,5.5Hz,2H),3.70(ddt,J=36.7,13.8,4.5Hz,3H),2.78(dt,J=22.5, 5.7Hz, 6H), 2.45 (s, 2H), 2.33 (d, J=10.9Hz, 10H), 2.09 (q, J=6.9Hz, 5H), 1.64 (dt, J=15.0,7.5Hz,8H),1.36(d,J=13.9Hz,25H),1.30(s,21H),0.93(q,J=6.1Hz,11H).

[0089] Example 4: Preparation of lipid 4

[0090]

[0091] 12 g (0.0115 mol, 3.0 eq) of compound 3, 31.27 g (0.0115 mol, 4.0 eq) of stearic acid, 12.22 g (0.0172 mol, 1.0 eq) of DIPEA, 0.516 g (0.0046 mol, 0.14 eq) of DMAP, and 2.614 g (0.0138 mol, 11.2 eq) of EDCI were dissolved in 330 ml of DCM and reacted at room temperature for 48 hours. After completion of the reaction, the solvent was concentrated under reduced pressure and the crude product was further purified on a silica gel column with a dichloromethane:ethanol ratio of 1:100 as the eluent to obtain 0.5 g of an oil (0.0012 mol, compound 6) in a yield of 10.4%.

[0092] 0.46 g (0.0011 mol, 1.0 eq) of compound 4, 10.5 g (0.0011 mol, 11.0 eq) of compound 6, 20.22 g (0.0017 mol, 1.25 eq) of DIPEA, 6.05 g (0.00004 mol, 0.8 mol) of DMAP, and 10.26 g (0.0014 mol, 9.2 eq) of EDCI were dissolved in 1000 ml of DCM and reacted at 40° C. for 48 hours. After completion of the reaction, the solvent was concentrated under reduced pressure and the crude product was further purified on a silica gel column with a dichloromethane:ethanol ratio of 1:100 as the eluent to obtain 120 mg of an oily product (0.00014 mol, lipid 4) in a yield of 13.06%. 1H NMR (400MHz, CDCl3) δ4.36–4.23(m,2H),4.13(dd,J=11.8,5.5Hz,1H),3.81–3.59(m,4H),3.50(t,J=6.6Hz,1H),2.80(s,1H),2.36(d ,J=8.4Hz,4H),2.34–2.28(m,3H),1.69–1.50(m,11H),1.48–1.35(m,7H),1.33(s,14H),1.30(s,36H),0.94(dt,J=13.6,7.1Hz,11H).

[0093] Example 5: Preparation of lipid 5

[0094]

[0095] 10.8 g (0.002 mol, 1.0 eq) of compound 4, 0.14 g (0.002 mol, 11.0 eq) of compound 3, 20.39 g (0.003 mol, 3.02 eq) of DIPEA, 10.1 g (0.0008 mol, 3.8 eq) of DMAP, and 7.5 g (0.0025 mol, 21.5 eq) of EDCI were dissolved in 1000 ml of DCM and reacted at room temperature for 48 hours. After completion of the reaction, the mixture was concentrated under reduced pressure to remove the solvent. The crude product was further purified on a silica gel column with the eluent varying from petroleum ether to dichloromethane:ethyl acetate = 1:50 to obtain 0.6 g of an oil (0.0011 mol, compound 7) in a yield of 54.54%.

[0096] 15 g (0.0287 mol, 1.0 eq) of suberic acid, 112.12 g (0.043 mol, 32.0 eq) of DIPEA, 12.4 g (0.0115 mol, 10.4 eq) of DMAP, and 36.6 g (0.0344 mol, 1.2 eq) of EDCI were dissolved in 1500 ml of DCM. The reaction mixture was stirred for 24 hours, and then 24.14 g (0.0287 mol, 1.0 eq) of n-nonanol was added and allowed to react at room temperature for 48 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure, and the crude product was further purified on a silica gel column with the eluent varying from petroleum ether to dichloromethane:ethyl acetate = 1:80 to obtain 1 g of an oil (0.003 mol, compound 8) in a yield of 10.45%.

[0097] 0.93 g (0.001 mol, 1.0 eq) of compound 8, 10.55 g (0.001 mol, 1.0 eq) of compound 7, 2.26 g (0.0015 mol, 2.0 eq) of DIPEA, 7.05 g (0.0004 mol, 0.49 eq) of DMAP, and 10.23 g (0.0012 mol, 1.2 eq) of EDCI were dissolved in 1000 ml of DCM and reacted at room temperature for 48 hours. After completion of the reaction, the solvent was concentrated under reduced pressure and the crude product was further purified on a silica gel column with the eluent changing from petroleum ether to dichloromethane:ethyl acetate = 1:90 to obtain 200 mg of an oil (0.00024 mol, lipid 5) with a yield of 23.5%. 1H NMR (400MHz, CDCl3) δ4.90(p,J=6.4Hz,1H),4.32(dd,J=11.7,6.7Hz,2H),4.10(q,J=5.8Hz,4H),3.03(s,5H),2.63(s,3H ), 2.34(dt,J=20.9,7.2Hz,8H),1.67(q,J=7.1Hz,10H),1.54(d,J=6.9Hz,2H),1.45–1.19(m,48H),0.92(t,J=6.6Hz,9H).

[0098] Example 6: Preparation of lipid 6

[0099]

[0100] 40 g (0.1 mol, 1.0 eq) of NaH was dissolved in 40 ml of THF, and 4.4 g (0.1 mol, 1.0 eq) of n-nonanol (dissolved in 500 ml of THF) was added dropwise. The mixture was stirred for 48 hours, followed by the dropwise addition of 93.3 g (0.1 mol, 1.0 eq) of 8-bromooctanoic acid. The mixture was allowed to react at room temperature for 48 hours. After completion of the reaction, the pH of the reaction solution was adjusted to 6. The organic layer was concentrated and purified on a silica gel column using a 1:50 ratio of dichloromethane to methanol as the eluent to afford 20 g of an oil (0.067 mol, compound 9) in a 66.67% yield.

[0101] 10.3 g (0.001 mol, 1.0 eq) of compound 9, 10.55 g (0.001 mol, 1.0 eq) of compound 7, 0.216 g (0.0015 mol, 1.5 eq) of DIPEA, 0.095 g (0.0004 mol, 0.4 eq) of DMAP, and 0.93 g (0.0012 mol, 1.2 eq) of EDCI were dissolved in 10 ml of DCM and reacted at room temperature for 48 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure. The crude product was further purified on a silica gel column with a dichloromethane:ethanol ratio of 1:100 as the eluent to obtain 200 mg of an oil (0.00024 mol, lipid 6), with a yield of 23.89%. NMR (400MHz, CDCl3) δ4.32 (dd, J=11.6, 6.3Hz, 1H), 4.12 (dd, J=11.8, 5.0Hz, 1H), 3.77(d,J=11.3Hz,1H),3.68(t,J=5.1Hz,1H),3.55(dt,J=15.9,6.0Hz,2H),3.43( t,J=6.7Hz,3H),2.80(t,J=4.8Hz,2H),2.55(s,5H),2.49–2.33(m,7H),2.32(dd, J=11.9,5.6Hz,3H),1.73–1.45(m,1H),1.42–1.21(m,33H),0.92(t,J=6.6Hz,6H).

[0102] Example 7: Preparation of lipid 7

[0103]

[0104] 55.85 g (0.15 mol, 1.0 eq.) of compound 1 was dissolved in 40 mL of acetonitrile. 61.85 g (0.225 mol, 1.5 eq.) of N-BOC-piperazine and 91.05 g (0.225 mol, 1.5 eq.) of solid potassium carbonate were added at room temperature. After addition, the mixture was heated at 40°C and allowed to react for 48 hours. The reaction mixture was sampled and tested by TLC to confirm the reaction completion. The reaction mixture was concentrated to remove the acetonitrile and dissolved in 30 mL of ethyl acetate. The mixture was then washed with saturated citric acid until acidic. The organic phase was separated and washed once with saturated sodium chloride solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to yield 47.0 g (0.1366 mol, compound 10) as a colorless oily liquid, with a crude yield of 90.7%. The crude product was directly used in the next reaction.

[0105] 47.0 g (0.14 mol, 1.0 eq.) of compound 10 was dissolved in ethanol. 121 g (0.56 mol, 4.0 eq.) of sodium borohydride was added portionwise at room temperature. After addition, the mixture was heated to 40°C and allowed to react for 48 hours. The reaction solution was sampled and TLC was performed to confirm the completion of the reaction. The reaction solution was concentrated to remove ethanol, dissolved in ethyl acetate, and filtered. The filtrate was mixed with silica gel and purified on a silica gel column to obtain 20.0 g of a light yellow oily liquid (0.077 mol, compound 11), with a yield of 56.3%. 1H NMR (400MHz, CDCl3) δ3.86 (s, 2H), 3.74 (d, J = 4.9Hz, 4H), 3.50 (s, 4H), 2.82 (d, J = 7.3Hz, 1H), 2.78 (s, 4H), 1.49 (s, 9H). ESI-MS: m / z 261.0(M+1)+; m / z 283.0(M+Na)+.

[0106] 22.6 g (0.01 mol, 1.0 eq) of compound 11, 15.6 g (0.02 mol, 2.0 eq) of linoleic acid, 33.9 g (0.03 mol, 3.0 eq) of DIEA, 10 g (0.008 mol, 0.8 eq) of DMAP, and 34.8 g (0.025 mol, 2.5 eq) of EDCI were dissolved in 10 ml of DCM and reacted at room temperature for 48 hours. After completion of the reaction, the solvent was concentrated under reduced pressure and the crude product was further purified on a silica gel column with a dichloromethane:ethanol ratio of 1:50 as the eluent to afford 5.4 g of an oil (0.007 mol, compound 12), in a 70% yield.

[0107] 25.4 g (0.007 mol, 1.0 eq) of compound 12 and 50 ml of TFA were dissolved in 10 ml of DCM and reacted at room temperature for 48 hours. After the reaction, the solvent was removed by concentration under reduced pressure. The crude compound 13 was directly used for the next reaction.

[0108] 4.7 g (0.007 mol, 1.0 eq) of compound 13, 10.1 g (0.008 mol, 1.2 eq) of potassium carbonate, and 18 g (0.008 mol, 1.2 eq) of 2-bromoethanol were dissolved in 10 ml of THF and reacted at room temperature for 48 hours. After completion of the reaction, the mixture was concentrated under reduced pressure to remove the solvent. The crude product was further purified on a silica gel column using an eluent ranging from petroleum ether to dichloromethane:ethanol (1:80) to obtain 700 mg of an oil (0.00096 mol, lipid 7), with a yield of 13.72%. NMR (400MHz, CDCl3) δ5.40 (tt, J=11.3, 4.7Hz, 8H), 4.33 (dd, J=11.5, 6.2Hz, 2H), 4. 14(dd,J=11.5,5.6Hz,2H),3.65(t,J=5.3Hz,2H),3.04(q,J=6.0Hz,1H),2.78(dt,J= 25.0,5.6Hz,8H),2.56(dd,J=12.1,6.3Hz,6H),2.35(t,J=7.6Hz,5H),2.08(p,J=7.8 Hz, 9H), 1.66 (p, J = 7.0 Hz, 5H), 1.37 (dd, J = 16.5, 5.6 Hz, 34H), 0.93 (t, J = 6.6 Hz, 6H).

[0109] Example 8: Preparation of lipid 8

[0110]

[0111] 121.38 g (0.03 mol, 1.0 eq.) of compound 4 was dissolved in dichloromethane. 18.9 g (0.0315 mol, 1.05 eq.) of compound 11, 19.3 g (0.09 mol, 3.0 eq.) of DIPEA, 10.8 g (0.015 mol, 0.5 eq.) of DMAP, and 28.3 g (0.045 mol, 1.5 eq.) of EDCI were added sequentially at room temperature. The reaction mixture was allowed to react for 48 hours at room temperature after the addition was complete. The reaction mixture was sampled and TLC was performed to confirm the reaction completion. 10 mL of 90% aqueous citric acid solution was added to the reaction mixture, stirred, and then separated. The organic phase was washed once with water and once with saturated aqueous sodium chloride solution. The organic phase was separated and dried over anhydrous sodium sulfate, filtered, and the filtrate was mixed with silica gel. The sample was separated and purified on a silica gel column to obtain 7.66 g of a light yellow oily liquid (0.0117 mol, compound 14) in a yield of 39.8%.

[0112] ESI-MS: m / z 655.2 (M+1) + .

[0113] 13.67 g (0.0056 mol, 1.0 eq.) of compound 14 was dissolved in dichloromethane. 21.52 g (0.0084 mol, 1.5 eq.) of compound 8, 11.81 g (0.014 mol, 2.5 eq.) of DIPEA, 21.34 g (0.0028 mol, 0.5 eq.) of DMAP, and 12.15 g (0.0112, 2.0 eq.) of EDCI were added sequentially at room temperature. The reaction mixture was allowed to react at room temperature for 48 hours after the addition was complete. The reaction mixture was sampled and TLC was performed to confirm the reaction completion. 300 mL of citric acid aqueous solution was added to the reaction mixture, stirred, and then separated. The organic phase was washed once with water and once with saturated sodium chloride aqueous solution. The organic phase was separated and dried over anhydrous sodium sulfate, filtered, and the filtrate was mixed with silica gel. The sample was separated and purified on a silica gel column to obtain 5.10 g of a light yellow oily liquid (0.005 mol, compound 15) in a yield of 89.2%.

[0114] ESI-MS: m / z 938.2 (M+1)+.

[0115] 55.10 g (0.005 mol, 1.0 eq.) of compound 15 was dissolved in dichloromethane, and TFA was added at room temperature, followed by reaction at room temperature for 48 hours. The reaction solution was sampled and analyzed by TLC to confirm the completion of the reaction. The reaction solution was directly concentrated to dryness, clarified by addition of EA and water, and the pH was adjusted to 6 with solid sodium carbonate. The organic phase was separated and washed once with aqueous solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain 4.8 g of a yellow oily liquid (0.005 mol, compound 16). The crude yield was 100%.

[0116] 24.8 g (0.005 mol, 1.0 eq.) of compound 16 was dissolved in acetonitrile. 20.94 g (0.0075 mol, 1.5 eq.) of 2-bromoethanol, 12.07 g (0.015 mol, 3.0 eq.) of potassium carbonate, and 10.36 g (0.0025 mol, 0.5 eq.) of sodium iodide were added sequentially at room temperature. After addition, the mixture was allowed to react at room temperature for 48 hours. The reaction mixture was sampled and TLC was performed to determine if the reaction of the raw materials had completed. Water was added directly to the reaction mixture and the product was extracted with ethyl acetate. The organic phase was washed once with aqueous solution, separated, and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was mixed with silica gel and purified by silica gel column separation to obtain 1.10 g of a light yellow oily liquid (lipid 8, 0.00125 mol) in a yield of 42.6%. 1H NMR (400MHz, CDCl3) δ4.98–4.83(m,1H),4.33(dd,J=11.5,6.3Hz,2H),4.18–4.05(m,4H),3.70(t,J=5.0Hz,2H),3.08–3.00(m,1H),2 .90(s,4H),2.79(s,4H),2.63(d,J=4.6Hz,6H),2.40–2.29(m,10H),1.67(s,12H),1.39(s,12H),1.30(s,32H),0.92(t,J=6.3Hz,9H).

[0117] ESI-MS: m / z 881.3 (M+1)+.

[0118] Example 9: Preparation of lipid 9

[0119]

[0120] 22.5 g (0.003 mol, 1.0 eq.) of compound 16 was dissolved in acetonitrile. 10.63 g (0.0045 mol, 1.5 eq.) of 3-bromopropanol, 13.24 g (0.009 mol, 3.0 eq.) of potassium carbonate, and 10.23 g (0.0015 mol, 0.5 eq.) of sodium iodide were added sequentially at room temperature. The reaction mixture was allowed to react for 48 hours at room temperature after the addition was complete. The reaction mixture was sampled and TLC was performed to determine the complete reaction of the raw materials. Water was then added to the reaction mixture and the product was extracted with ethyl acetate. The organic phase was washed once with aqueous solution, separated, and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was mixed with silica gel and purified on a silica gel column to obtain 1.20 g of a light yellow oily liquid (lipid 9, 0.00134 mol) in a yield of 45.8%. 1H NMR (400MHz, CDCl3) δ4.95–4.84(m,1H),4.31(dd,J=11.4,6.2Hz,2H),4.09(dd,J=13.2,6.2Hz,4H),3.60(s,2H),3.05–2.97(m,1H),2.77(s ,4H),2.55(s,4H),2.43(s,2H),2.37–2.28(m,9H),1.67(d,J=18.3Hz,16H),1.37(s,12H),1.29(s,34H),0.91(t,J=6.2Hz,9H).ESI-MS:m / z 895.3(M+1)+.

[0121] Example 10: Preparation of lipid 10

[0122]

[0123] 10.5 g (0.0003 mol, 1.0 eq.) of compound 16 was dissolved in 3 mL of acetonitrile. 10.07 g (0.00045 mol, 1.5 eq.) of 4-bromobutanol, 20.12 g (0.0009 mol, 3.0 eq.) of potassium carbonate, and 40.02 g (0.00015 mol, 0.5 eq.) of sodium iodide were added sequentially at room temperature. The reaction mixture was allowed to react for 48 hours at room temperature after the addition was complete. The reaction mixture was sampled and TLC was performed to determine if the reaction was complete. Water was added directly to the reaction mixture and the product was extracted with ethyl acetate. The organic phase was washed once with aqueous sodium chloride solution, separated, and dried over anhydrous sodium sulfate. The organic phase was filtered, and the filtrate was mixed with silica gel. The sample was separated and purified on a silica gel column to obtain 915 mg of an orange-yellow oily liquid (lipid 10, 0.00005 mol), with a yield of 17.1%. 1H NMR (400MHz, CDCl3) δ4.95–4.83(m,1H),4.29(dd,J=11.5,6.3Hz,2H),4.09(d d,J=14.1,6.7Hz,4H),3.82(t,J=4.9Hz,2H),3.02(dd,J=11.5,5.7Hz,2H),2.7 3(s,4H),2.62(dd,J=22.3,17.0Hz,6H),2.37–2.28(m,10H),1.79–1.71(m,2H) ,1.65(s,12H),1.37(s,12H),1.29(s,34H),0.91(t,J=6.2Hz,9H).ESI-MS:m / z 909.3(M+1)+.

[0124] Example 11: Preparation of lipid 11

[0125]

[0126] 3.67 g (0.0056 mol, 1.0 eq.) of compound 14 was dissolved in dichloromethane. 2.40 g (0.0084 mol, 1.5 eq.) of compound 9, 2.17 g (0.0168 mol, 3.0 eq.) of DIPEA, 0.34 g (0.0028 mol, 0.5 eq.) of DMAP, and 2.15 g (0.0112 mol, 2.0 eq.) of EDCI were added sequentially at room temperature. After addition, the mixture was allowed to react at room temperature for 8 hours. The reaction solution was sampled and TLC was performed to determine the complete reaction of the raw materials. The reaction solution was directly added with aqueous citric acid, stirred, and then separated. The organic phase was washed once with the aqueous solution, separated, and the organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was mixed with silica gel and purified on a silica gel column to obtain 3.62 g of a light yellow oily liquid (compound 17, 0.0039 mol) in a yield of 70.1%. ESI-MS: m / z 923.3 (M+1)+.

[0127] 295 mg (0.00032 mol, 1.0 eq.) of compound 17 (1.0 eq.) was dissolved in dichloromethane, and TFA was added at room temperature, followed by reaction at room temperature for 4 hours. The reaction solution was sampled and TLC was performed to confirm the completion of the reaction. The reaction solution was directly concentrated to dryness, clarified by addition of EA and water, and the pH was adjusted to 8-9 with solid sodium hydroxide. The organic phase was separated and washed once with aqueous solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain 260 mg of a yellow oily liquid (compound 18, 0.0003 mol), with a crude yield of 97.3%.

[0128] 260 mg (0.0003 mol, 1.0 eq.) of compound 18 was dissolved in acetonitrile, and 56 mg (0.00045 mol, 1.5 eq.) of 2-bromoethanol, 124 mg (0.0009 mol, 3.0 eq.) of potassium carbonate, and 23 mg (0.00015 mol, 0.5 eq.) of sodium iodide were added sequentially at room temperature. After the addition was complete, the reaction mixture was allowed to react at room temperature for 12 hours. The reaction mixture was sampled and TLC was performed to determine if the reaction of the raw materials had completed. 10 mL of water was added directly to the reaction mixture, and the product was extracted with ethyl acetate. The organic phase was washed once with aqueous solution, separated, and dried over anhydrous sodium sulfate. The organic phase was filtered, and the filtrate was mixed with silica gel. The sample was separated and purified on a silica gel column to obtain 115.0 mg of a light yellow oily liquid (lipid 11, 0.00013 mol) in a yield of 42.9%. 1H NMR (400MHz, CDCl3) δ4.93–4.85(m,1H),4.38–4.24(m,2H),4.12(dd,J=11.0,4.9Hz,2H),3.65(s,2H),3.42(t,J=6.4Hz,4H),3.07–2.99(m,1 H),2.75(s,4H),2.57(d,J=5.5Hz,6H),2.39–2.29(m,7H),1.63–1.48(m,10H),1.36(s,8H),1.29(s,40H),0.91(d,J=6.0Hz,9H).ESI-MS:m / z 867.7(M+1)+.

[0129] Example 12: Preparation of lipid 12

[0130]

[0131] 260 mg (0.0003 mol, 1.0 eq.) of compound 18 was dissolved in acetonitrile, and 63 mg (0.00045 mol, 1.5 eq.) of 3-bromopropanol, 124 mg (0.0009 mol, 3.0 eq.) of potassium carbonate, and 23 mg (0.00015 mol, 0.5 eq.) of sodium iodide were added sequentially at room temperature. After the addition was complete, the reaction mixture was allowed to react at room temperature for 12 hours. The reaction mixture was sampled and TLC was performed to determine if the reaction of the raw materials had completed. 10 mL of water was added directly to the reaction mixture, and the product was extracted with ethyl acetate. The organic phase was washed once with aqueous solution, separated, and dried over anhydrous sodium sulfate. The organic phase was filtered, and the filtrate was mixed with silica gel. The sample was separated and purified on a silica gel column to obtain 118.0 mg of a light yellow oily liquid (lipid 12, 0.00013 mol), with a yield of 44.5%.

[0132] Example 13: Preparation of lipid 13

[0133]

[0134] 260 mg (0.0003 mol, 1.0 eq.) of compound 18 was dissolved in acetonitrile, and 69 mg (0.00045 mol, 1.5 eq.) of 4-bromobutanol, 124 mg (0.0009 mol, 3.0 eq.) of potassium carbonate, and 23 mg (0.00015 mol, 0.5 eq.) of sodium iodide were added sequentially at room temperature. The reaction mixture was allowed to react for 12 hours at room temperature after the addition was complete. The reaction mixture was sampled and TLC was performed to determine if the reaction of the raw materials had completed. 10 mL of water was added directly to the reaction mixture, and the product was extracted with ethyl acetate. The organic phase was washed once with aqueous solution, separated, and dried over anhydrous sodium sulfate. The organic phase was filtered, and the filtrate was mixed with silica gel. The sample was separated and purified on a silica gel column to obtain 60.0 mg of a light yellow oily liquid (lipid 13, 0.00007 mol) in a yield of 22.3%.

[0135] Test example

[0136] Experimental Example 1: Physicochemical Properties and In Vitro Transfection Efficiency of mRNA LNPs Prepared Using Different Ionizable Amino Lipid Compounds

[0137] 1.1 Experimental Objectives

[0138] The physicochemical properties and in vitro transfection efficiency of mRNA LNPs prepared with different ionizable amino lipid compounds were investigated.

[0139] 1.2 Test materials and instruments

[0140] Luc mRNA was provided by Nanjing Jimai Biotechnology Co., Ltd. The lipid materials used in the experiments were as follows: lipids 1–10 were homemade, Dlin-MC3-DMA (Shanghai Avanto Pharmaceutical Technology Co., Ltd.), DSPC (Xi'an Ruixi Biotechnology Co., Ltd.), cholesterol (Sigma-Aldrich Shanghai Trading Co., Ltd.), PEG2k-DMG (Avanti Polar Lipids, Inc.), and Tf-PEG2k-DMG (Xi'an Ruixi Biotechnology Co., Ltd.).

[0141] The equipment used for LNP preparation is Maiana's small-scale microfluidic device, model: INano L;

[0142] The particle size was measured using a dynamic light scattering laser particle size analyzer (Zetasizer Ultra, Malvern Panalytical Ltd).

[0143] Encapsulation efficiency Fluorescence detection was performed using the ELISA kit (Thermo Fisher Scientific Inc).

[0144] 1.3 Test methods

[0145] 1.3.1 Preparation of LNPs using different ionizable lipid compounds

[0146] Press Attach Figure 1 Ionizable amino lipid mRNA LNPs were prepared as shown. The following solutions were prepared: A. Lipid ethanol solution, with the molar ratio of each lipid being: Dlin-MC3-DMA or lipid 1-10:DSPC:cholesterol:PEG2k-DMG = 50:10:38.5:1.5; B. Malate buffer containing mRNA, pH = 4.0; Solution A and Solution B were mixed in a 1:3 volume ratio in a microfluidic chip, and the LNP intermediate was then transferred to a dialysis cassette (Slide-A-Lyzer TM , MWCO = 20k), dialyzed in pH = 7.4 PBS for 16 h, and ethanol was removed to obtain the LNP product.

[0147] 1.3.2 Physical and chemical properties test

[0148] Product particle size, particle size distribution (PDI), and zeta potential were measured using a dynamic light scattering laser particle size analyzer (Zetasizer Ultra, Malvern). Samples were diluted 50-fold with 5 mM NaCl solution and transferred to a DTS1070 cuvette. The measurement mode was 173° backscattered light. Each sample was equilibrated in the instrument for 120 seconds and allowed to reach 25°C before measurement began.

[0149] The product encapsulation efficiency was detected by a Ribogreen-based nucleic acid fluorescence method. The LNP sample was diluted 100-fold with 1X PBS to obtain a test solution for determining the free and total mRNA in the sample. A series of standard solutions with a concentration gradient were prepared by diluting the free mRNA solution with 1X PBS, and the LNP sample test solution and mRNA standard solution were added to a 96-well blackboard. TritonX100 and Ribogreen working solutions were prepared according to the instructions, added to a 96-well blackboard, mixed with the LNP sample test solution and mRNA standard solution, and then slowly shaken at room temperature in the dark for 10 minutes. Sample fluorescence was measured using a microplate reader at an excitation light of 480 nm and an emission light of 520 nm, and the sample encapsulation efficiency was calculated based on the sample fluorescence value.

[0150] 1.3.3 Cell assay

[0151] The transfection efficiency of each lipid was evaluated in vitro on Hela cells. Hela cells were cultured routinely with DMEM + 10% FBS medium to ensure that the cells were in the logarithmic growth phase; one day before transfection, the cells were seeded into 96-well culture plates at an appropriate cell density and grown overnight. During transfection, the cells were required to reach 70–90% confluency; we prepared different concentrations of LNP-mRNA in serum-free medium and added 10ul to the 96-cell culture plate to make the concentrations in each well reach 100, 50, and 25ng, respectively. Firefly luciferase mRNA of the corresponding concentration was used as a positive reference. mRNA transfection was performed using a MessengerMax (Thermo). After incubation for 24 h in a 37°C, 5% CO2 incubator, an equal volume of ONE GLO (Promega) detection reagent was added to the wells, pipetted evenly, and then transferred to a 96-well white plate. Light intensity was measured using a microplate reader in Lumi mode.

[0152] 1.4 Test results

[0153] The particle size, particle size distribution (PDI), Zeta potential, encapsulation efficiency and transfection efficiency relative to MC3 of the product are shown in Table 1:

[0154] Table 1. Particle size, PDI, Zeta potential, encapsulation efficiency, and transfection efficiency of mRNA LNPs prepared using different ionizable amino lipids

[0155]

[0156] The results of the cell experiment on Hela cells were used to evaluate the transfection efficiency of each lipid in vitro. Figure 2 shown.

[0157] 1.5 Test Conclusion

[0158] This study evaluated the in vitro transfection efficiency of mRNA LNPs prepared using different novel ionizable amino lipids. MC3 is a commercially available ionizable amino lipid for RNA delivery. Lipid 1 is the lipid disclosed in patent WO2010030739A1. Lipid 7 is a simple structural modification of lipid 1 with the addition of a hydroxyethyl functional group. Lipids 6, 8, 9, and 10 are novel ionizable amino lipids prepared based on the lipid structure we designed and protected. The results showed that LNPs prepared using lipids 6, 8, 9, and 10 all had particle sizes around 100 nm or less, similar to those of MC3 LNPs. Their PDIs were <0.1, smaller than those of MC3, lipid 1, and lipid 7 LNPs, and the particles were uniform. Their encapsulation efficiencies were >90%, higher than those of lipid 1 and lipid 7 LNPs. Their transfection efficiencies were ≥ that of MC3 LNPs, with lipid 6 and 8 LNPs exhibiting expression levels exceeding five times that of MC3 LNPs.

[0159] Experimental Example 2: In vitro transfection efficiency of mRNA LNPs prepared under different formulation conditions using ionizable amino lipid compounds

[0160] 1.1 Experimental Objectives

[0161] The differences in in vitro transfection efficiency between our designed lipids (such as lipid 8) and MC3 lipids under different formulation conditions were investigated.

[0162] 1.2 Test materials and instruments

[0163] The materials and instruments used in this test are the same as those in Test Example 1 above.

[0164] 1.3 Test methods

[0165] 1.3.1 Preparation of LNPs of Ionizable Lipid Compounds

[0166] Press Attach Figure 1 Prepare each ionizable amino lipid mRNA LNP as shown. Prepare the following solutions respectively:

[0167] A. Ethanol solutions of lipids, wherein the molar ratios of the lipids in different samples were prepared as shown in Table 2 below.

[0168] Table 2. Molar ratio of each lipid in the sample

[0169] Sample number The molar ratio of each lipid in the sample 1 Dlin-MC3-DMA / DSPC / cholesterol / PEG2k-DMG=31.5 / 10 / 56 / 2.5 2 Dlin-MC3-DMA / DSPC / cholesterol / PEG2k-DMG=43.3 / 8.7 / 46.5 / 1.5 3 Dlin-MC3-DMA / DSPC / cholesterol / PEG2k-DMG=46.3 / 9.4 / 42.7 / 1.6 4 Dlin-MC3-DMA / DSPC / cholesterol / PEG2k-DMG=50 / 10 / 38.5 / 1.5 5 Lipid 8 / DSPC / cholesterol / PEG2k-DMG=31.5 / 10 / 56 / 2.5 6 Lipid 8 / DSPC / cholesterol / PEG2k-DMG=43.3 / 8.7 / 46.5 / 1.5 7 Lipid 8 / DSPC / cholesterol / PEG2k-DMG=46.3 / 9.4 / 42.7 / 1.6 8 Lipid 8 / DSPC / cholesterol / PEG2k-DMG=50 / 10 / 38.5 / 1.5

[0170] B. Malate buffer containing mRNA, pH = 4.0.

[0171] Solution A and solution B were mixed in a microfluidic chip at a volume ratio of 1:3, and then the LNP intermediate was transferred to a dialysis cassette (Slide-A-LyzerTM , MWCO = 20k), dialyzed in pH = 7.4 PBS for 16 h, and ethanol was removed to obtain the LNP product.

[0172] 1.3.2 Physicochemical property tests and cell experiments

[0173] Product particle size, particle size distribution (PDI), and zeta potential were measured using a dynamic light scattering laser particle size analyzer (Zetasizer Ultra, Malvern). Product encapsulation efficiency was determined using a Ribogreen-based nucleic acid fluorescence assay. The transfection efficiency of each lipid was evaluated in vitro on HeLa cells. The specific experimental procedures were the same as those in Experiment 1 above.

[0174] 1.4 Test results

[0175] The particle size, particle size distribution (PDI), Zeta potential, encapsulation efficiency and transfection efficiency relative to MC3 of the product are shown in Table 3:

[0176] Table 3. Particle size, PDI, Zeta potential, encapsulation efficiency, and transfection efficiency of mRNA LNPs prepared using MC3 and lipid 8 under different formulation conditions

[0177]

[0178]

[0179] The results of the cell experiment on Hela cells were used to evaluate the transfection efficiency of each lipid in vitro. Figure 3 shown.

[0180] 1.5 Test Conclusion

[0181] The results showed that the particle size of LNPs prepared with different formulations using lipid 8 was <100 nm, consistent with that of MC3 LNPs. Their PDIs were all <0.1. However, as the proportion of MC3 lipids decreased, the PDI of MC3 LNPs gradually increased, reaching above 0.2. This indicates that MC3 LNPs become increasingly heterogeneous and unstable with increasing ionizable amino lipid content, whereas lipid 8 LNPs do not suffer from this problem and are more stable than MC3 LNPs. Furthermore, cell transfection experiments revealed that when the ionizable amino lipid content in the LNPs decreases, LNPs formed from lipid 8 exhibit a significant transfection advantage over MC3 LNPs. In particular, when the ionizable amino lipid content is reduced to 43.3%, at a dose of 50 ng / well, the LNPs exhibited an 18.05-fold increase in cell transfection compared to MC3 LNPs. This advantage was even more pronounced at lower doses, reaching a 35.02-fold increase. This shows that the mRNALNP particles formed by the lipids we designed (such as lipid 8) are more uniform and stable than the lipid nanoparticles using MC3 lipids currently on the market, indicating that this type of new ionizable lipid has better stability and adaptability, and is less affected by the formulation content factors compared to MC3; at the same time, this type of new ionizable lipid exhibits more significant cell transfection advantages than MC3 lipids in low-content formulations, which can achieve efficient and low-frequency drug delivery, thereby reducing potential toxicity risks.

[0182] Experimental Example 3: In vitro transfection efficiency of mRNA LNPs prepared under different formulation conditions using ionizable amino lipid compounds

[0183] 1.1 Experimental Objectives

[0184] The differences in in vitro transfection efficiency between our designed lipids (such as lipid 11) and MC3 lipids under different formulation conditions were investigated.

[0185] 1.2 Test materials and instruments

[0186] The materials and instruments used in this test are the same as those in Test Example 1 above.

[0187] 1.3 Test methods

[0188] 1.3.1 Preparation of LNPs of Ionizable Lipid Compounds

[0189] Press Attach Figure 1 Prepare each ionizable amino lipid mRNA LNP as shown. Prepare the following solutions respectively:

[0190] A. Ethanol solutions of lipids, wherein the molar ratios of the lipids in different samples are prepared as shown in Table 4 below.

[0191] Table 4. Molar ratio of each lipid in the sample

[0192]

[0193]

[0194] B. Malate buffer containing mRNA, pH = 4.0.

[0195] Solution A and solution B were mixed in a microfluidic chip at a volume ratio of 1:3, and then the LNP intermediate was transferred to a dialysis cassette (Slide-A-Lyzer TM , MWCO = 20k), dialyzed in pH = 7.4 PBS for 16 h, and ethanol was removed to obtain the LNP product.

[0196] 1.3.2 Physicochemical property tests and cell experiments

[0197] Product particle size, particle size distribution (PDI), and zeta potential were measured using a dynamic light scattering laser particle size analyzer (Zetasizer Ultra, Malvern). Product encapsulation efficiency was determined using a Ribogreen-based nucleic acid fluorescence assay. The transfection efficiency of each lipid was evaluated in vitro on HeLa cells. The specific experimental procedures were the same as those in Experiment 1 above.

[0198] 1.4 Test results

[0199] The particle size, particle size distribution (PDI), Zeta potential, encapsulation efficiency and transfection efficiency relative to MC3 of the product are shown in Table 5:

[0200] Table 5. Particle size, PDI, Zeta potential, encapsulation efficiency, and transfection efficiency of mRNA LNPs prepared using MC3 and lipid 11 under different formulation conditions

[0201]

[0202] The results of the cell experiment on Hela cells were used to evaluate the transfection efficiency of each lipid in vitro. Figure 4 shown.

[0203] 1.5 Test Conclusion

[0204] The results showed that the particle size of LNPs prepared with different lipid 11 formulations was <100 nm, consistent with that of MC3 LNPs. Their PDIs were all <0.13. However, as the proportion of MC3 lipids decreased, the PDI of MC3 LNPs gradually increased, reaching above 0.2. This suggests that MC3 LNPs become increasingly heterogeneous and unstable with increasing ionizable amino lipid content, whereas lipid 11 LNPs exhibited no such problem and were more stable than MC3 LNPs. Furthermore, cell transfection experiments revealed that when the ionizable amino lipid content in the LNPs decreased, LNPs formed with lipid 11 exhibited a significant transfection advantage over MC3 LNPs. In particular, when the ionizable amino lipid content was reduced to 43.3%, at a dose of 50 ng / well, LNPs exhibited an 85.29-fold increase in cell transfection compared to MC3 LNPs. This advantage was even more pronounced at lower doses, reaching a 124.76-fold increase. This shows that the mRNA LNP particles formed by the lipids we designed (such as lipid 11) are more uniform and stable than the lipid nanoparticles using MC3 lipids currently on the market, indicating that this type of new ionizable lipid has better stability and adaptability, and is less affected by the formulation content factors compared to MC3; at the same time, this type of new ionizable lipid exhibits more significant cell transfection advantages than MC3 lipids in low-content formulations, which can achieve efficient and low-frequency drug delivery, thereby reducing potential toxicity risks.

Claims

1. A lipid compound represented by formula (II) or a pharmaceutically acceptable salt thereof: in, R1 is C6-C 10 Straight chain alkyl, R2 is R4 is a C1-C6 alkyl group which is optionally substituted with -OH, X is -OC(=O)-, -C(=O)-O- or -O-CH2-, and m and n are each independently selected from an integer of 1-10.

2. The lipid compound according to claim 1, wherein R1 is a C9 straight chain alkyl group.

3. The lipid compound according to any one of claims 1 to 2, wherein R4 is -CH3, -CH2CH2OH, -CH2CH2CH2OH or -CH2CH2CH2CH2OH.

4. The lipid compound according to any one of claims 1 to 2, wherein m and n are each independently 6.

5. The lipid compound according to any one of claims 1 to 2, wherein the lipid compound has a structure represented by the following formula (III):

6. The lipid compound according to claim 5, wherein X is -OC(=O)- or -O-CH2-.

7. The lipid compound according to claim 5, wherein R4 is -CH3, -CH2CH2OH, -CH2CH2CH2OH or -CH2CH2CH2CH2OH.

8. The lipid compound according to any one of claims 1 to 2, wherein the compound is selected from the group consisting of:

9. A lipid nanoparticle composition comprising the lipid compound of claim 1 or a pharmaceutically acceptable salt thereof.

10. The lipid nanoparticle composition according to claim 9, further comprising a neutral lipid, cholesterol and a PEG lipid.

11. The lipid nanoparticle composition according to claim 10, wherein the neutral lipid is selected from the group consisting of DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, SOPE and 1,2-dipentanoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE), and the PEG lipid is selected from the group consisting of PEG-DMG, PEG-dipalmitoylglycerol, PEG-DSPE, PEG-dilaurylglyceramide, PEG-dimyristylglyceramide, PEG-dipalmitoylglyceramide and PEG-distearoylglyceramide, PEG-cholesterol (1-[8'-(cholest-5-en-3[β]-oxy)formamido-3',6'-dioxaoctyl]carbamoyl-[ω]-methyl-poly(ethylene glycol), PEG-DMB, PEG2k-DMG, PEG2k-DSPE, PEG2k-DSG, PEG2k-DMA and PEG2k-DSA.

12. The lipid nanoparticle composition according to claim 10 or 11, wherein the neutral lipid is DSPC and the PEG lipid is PEG2k-DMG.

13. The lipid nanoparticle composition according to any one of claims 9 to 11, wherein the lipid compound accounts for 30-50% by mole of the total lipid content of the composition.

14. The lipid nanoparticle composition according to claim 13, wherein the lipid compound accounts for 40-50% by mole of the total lipid content of the composition.

15. The lipid nanoparticle composition according to any one of claims 9 to 11, further comprising a nucleic acid molecule selected from the group consisting of mRNA, siRNA, antisense oligonucleotide (ASO), saRNA and miRNA.

16. Use of the lipid nanoparticle composition according to any one of claims 9 to 11 in the preparation of a medicament for treating a disease.

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