Lipid compounds or pharmaceutically acceptable salts thereof and their preparation methods, lipid nanoparticles and applications

Abstract

This invention provides a lipid compound or a pharmaceutically acceptable salt thereof, a method for its preparation, lipid nanoparticles, and applications, belonging to the field of biomedical technology. The lipid compound or a pharmaceutically acceptable salt thereof has the structural formula shown in formula (I), as illustrated in the specification. R represents substituted or unsubstituted C1~C1. 10 Alkyl, substituted or unsubstituted C2~C 10 Ether; X is C1~C 20 Straight-chain or branched alkyl; Y is C1~C 20 Straight-chain alkyl, or C2~C 20 Straight-chain or branched unsaturated aliphatic hydrocarbon group; A is a C1~C5 alkylene ester group; Z is a C 11 ~C 18 Branched alkyl; where a substituent is present on the substituted or unsubstituted group described above, the substituent is selected from hydroxyl groups.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to a lipid compound or a pharmaceutically acceptable salt thereof, a method for its preparation, lipid nanoparticles, and their applications. Background Technology

[0002] In nucleic acid therapy, the efficiency and safety of delivery systems are among the key technological bottlenecks. To address issues such as the easy degradation of nucleic acid molecules in vivo, poor targeting, and insufficient cell penetration, various delivery systems have been extensively developed. Among these, lipid-based nucleic acid delivery systems, especially lipid nanoparticles, offer advantages such as simple lipid component synthesis, scalable preparation processes, and a wide range of packaging applications. Lipid compounds are important components of lipid nanoparticles and can influence the delivery and expression of nucleic acid drugs.

[0003] Therefore, more lipid compounds capable of efficiently delivering nucleic acid molecules need to be studied. Summary of the Invention

[0004] In view of this, in order to at least partially solve the aforementioned technical problems, the present invention provides a lipid compound or a pharmaceutically acceptable salt thereof, a method for preparing the same, lipid nanoparticles, and applications.

[0005] According to one aspect of the present invention, a lipid compound or a pharmaceutically acceptable salt thereof is provided, having the structural formula shown in formula (I):

[0006] Formula (I);

[0007] Where R represents substituted or unsubstituted C1~C 10 Alkyl, substituted or unsubstituted C2~C 10 Ether; X is C1~C 20 Straight-chain or branched alkyl; Y is C1~C 20 Straight-chain alkyl, or C2~C 20 Straight-chain or branched unsaturated aliphatic hydrocarbon group; A is a C1~C5 alkylene ester group; Z is a C 11 ~C 18 Branched alkyl; where a substituent is present on the substituted or unsubstituted group described above, the substituent is selected from hydroxyl groups.

[0008] According to another aspect of the present invention, a method for preparing the lipid compound or a pharmaceutically acceptable salt thereof as described above is provided, comprising: under alkaline conditions, reacting the compound of formula (A) with... The compound shown in formula (B) A nucleophilic substitution reaction is carried out in a first organic solvent under the catalysis of a first catalyst to obtain a lipid compound as shown in formula (I) or a pharmaceutically acceptable salt thereof.

[0009] According to another aspect of the present invention, a lipid nanoparticle is provided, comprising a carrier comprising a lipid compound as described above or a pharmaceutically acceptable salt thereof.

[0010] According to another aspect of the present invention, the use of a lipid compound as described above or a pharmaceutically acceptable salt thereof, or lipid nanoparticles as described above, in nucleic acid drug delivery is provided.

[0011] The lipid compounds or pharmaceutically acceptable salts thereof according to embodiments of the present invention have triple ester bonds (which can be understood as ester bonds at three different sites). Ester bonds are biodegradable chemical bonds, and their hydrolysis products have good biocompatibility, avoiding inflammatory responses or tissue toxicity caused by long-term accumulation of lipid compounds in vivo. Simultaneously, the hydrolysis rate of ester bonds can be regulated by chain length, substituent steric hindrance, etc., to achieve precise matching of the release rate of bioactive substances. Furthermore, the ester bonds synergistically act with other hydrophilic and hydrophobic functional units to precisely regulate the overall hydrophilic / hydrophobic balance of the lipid compound molecule, taking into account both the stability of the constructed delivery system and the release rate of bioactive substances. Attached Figure Description

[0012] The above and other objects, features and advantages of the present invention will become clearer from the following description of embodiments of the invention with reference to the accompanying drawings.

[0013] Figure 1 The 1H NMR spectrum of compound 1 in Example 1;

[0014] Figure 2 The 1H NMR spectrum of compound 2 in Example 2;

[0015] Figure 3 The 1H NMR spectrum of compound 3 in Example 3;

[0016] Figure 4 The 1H NMR spectrum of compound 4 in Example 4;

[0017] Figure 5 The 1H NMR spectrum of compound 5 in Example 5;

[0018] Figure 6 The 1H NMR spectrum of compound 6 in Example 6;

[0019] Figure 7 The structural diagrams are of compounds 1 to 6 in Examples 1 to 6 and commercially available ALC-0315;

[0020] Figure 8Bioluminescence images of various organs in mice after intravenous injection of lipid nanoparticles constructed from compounds 1-3 in Examples 1-3 and commercially available ALC-0315. Detailed Implementation

[0021] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the invention. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the invention for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0022] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0023] In this invention, the term "pharmaceutically acceptable salt" refers to a relatively non-toxic organic or inorganic acid addition salt of a compound of formula (I). Inorganic acids include, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, etc., while organic acids include, formic acid, acetic acid, acetoacetic acid, pyruvic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, cinnamic acid, picric acid, trifluoromethanesulfonic acid, dodecyl sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid, itaconic acid, naphthalenedisulfonic acid, malic acid, adipic acid, alginic acid, maleic acid, D-gluconic acid, aspartic acid, etc.

[0024] In this invention, C a ~C b The expression indicates that the group has a to b number of carbon atoms. Unless otherwise specified, this number of carbon atoms generally does not include the number of carbon atoms of the substituents.

[0025] In this invention, the term "alkyl" may include a branched or straight-chain saturated aliphatic monovalent hydrocarbon group having a specified number of carbon atoms. For example, as C1~C 10 Examples of alkyl groups include: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.

[0026] In the process of realizing this invention, it was discovered that triple ester bonds, as connecting bonds, can work synergistically with other functional units to improve the biodegradability, good stability and targeting of lipid compound molecules, and the lipid nanoparticles constructed therefrom have high in vivo transfection efficiency.

[0027] Specifically, according to one aspect of the present invention, a lipid compound or a pharmaceutically acceptable salt thereof is provided, having the structural formula shown in formula (I):

[0028] Formula (I);

[0029] Where R represents substituted or unsubstituted C1~C 10 Alkyl, substituted or unsubstituted C2~C 10 Ether; X is C1~C 20 Straight-chain or branched alkyl; Y is C1~C 20 Straight-chain alkyl, or C2~C 20 Straight-chain or branched unsaturated aliphatic hydrocarbon group; A is a C1~C5 alkylene ester group; Z is a C 11 ~C 18 Branched alkyl; where a substituent is present on the substituted or unsubstituted group described above, the substituent is selected from hydroxyl groups.

[0030] According to embodiments of the present invention, the synergistic effect of various parts of the molecular structure of the lipid compounds or their pharmaceutically acceptable salts facilitates the construction of a delivery system with high loading efficiency, good in vivo stability, excellent biocompatibility, and precise delivery performance. Specifically, the triple ester bond, as a degradable linker, can undergo rapid hydrolysis in the intracellular environment after the lipid compound is successfully delivered, degrading the lipid compound molecule into non-toxic small molecule fragments with good biocompatibility. These fragments can be cleared by the body through normal metabolic pathways, reducing the risk of lipid compound accumulation in vivo and improving the biosafety of the delivery process. By precisely controlling the chemical structure of each structural unit (such as functional group type, chain length, and substituents), the loading performance, stability, release behavior, and biocompatibility of the lipid compound can be synergistically optimized, ultimately constructing a highly structure-function-adaptive delivery system.

[0031] It is understandable that the unsaturated aliphatic hydrocarbon groups here include unsaturated olefin groups, unsaturated alkyne groups, or combinations of the aforementioned groups.

[0032] In some embodiments, the lipid compound or a pharmaceutically acceptable salt thereof has any one of the structural formulas shown in formula (I-1) or formula (I-2):

[0033] Formula (I-1);

[0034] Formula (I-2).

[0035] The aforementioned structure further enhances the specific recognition and binding ability of liver tissue, thereby determining that the delivery system constructed based on this lipid compound has liver-targeting function and can improve the enrichment concentration and selective expression level of bioactive substances (such as nucleic acids and small molecule drugs) in liver tissue.

[0036] In some implementations, X is selected from any of the following structures:

[0037] ,

[0038] In this diagram, the dashed lines represent connection sites. After preparing a basic lipid nanoparticle delivery system using ionizable lipid compound molecules with the aforementioned X-group, the hydrophobicity and spatial arrangement of its branches can balance the membrane fluidity and structural stability of the lipid nanoparticles. This ensures that they do not easily disintegrate prematurely in blood circulation and can respond rapidly and trigger escape in the acidic environment of lysosomes, effectively preventing the degradation of nucleic acid drugs by lysosomal nucleases and improving the lysosomal escape efficiency of nucleic acid drugs.

[0039] In some implementations, Y is selected from any of the following structures:

[0040] ;

[0041] R is selected from any of the following structures:

[0042] ; where the dashed lines represent connection points.

[0043] According to embodiments of the present invention, in ionizable lipid compounds, by screening R, specific recognition and efficient loading of bioactive substances (such as nucleic acids, small molecule drugs, and proteins) can be achieved through charge-mediated binding, hydrogen bonding, or hydrophilic matching. Simultaneously, this imparts good dispersibility to the lipid nanoparticles, preventing particle aggregation. By screening Y, hydrophobic interactions and van der Waals forces drive the lipid compound molecules to provide a stable encapsulation space for bioactive substances. Furthermore, its chain length and spatial conformation can regulate the membrane fluidity and hydrophobicity of the lipid nanoparticles, optimizing their interaction with the cell membrane and improving the intracellular delivery efficiency of bioactive substances. Further, the introduction of the aforementioned unsaturated bonds and the regulation of the degree of unsaturation optimize the compatibility and interaction matching between the lipid compound and the cell membrane, reducing interfacial repulsion effects and accelerating endocytosis-mediated intracellular transport.

[0044] In some embodiments, the lipid compound or a pharmaceutically acceptable salt thereof has any one of the structures shown in formulas (I1) to (I6):

[0045] Equation (I1);

[0046] Equation (I2);

[0047] Equation (I3);

[0048] Equation (I4);

[0049] Equation (I5);

[0050] Equation (I6).

[0051] According to embodiments of the present invention, the above-mentioned lipid compounds have superior stability and biodegradability, which are more conducive to nucleic acid drug delivery and gene expression.

[0052] According to another aspect of the present invention, a method for preparing the lipid compound or a pharmaceutically acceptable salt thereof as described above is provided, comprising: under alkaline conditions, reacting the compound of formula (A) with... The compound shown in formula (B) A nucleophilic substitution reaction is carried out in a first organic solvent under the catalysis of a first catalyst to obtain a lipid compound as shown in formula (I) or a pharmaceutically acceptable salt thereof.

[0053] Specifically, the preparation method of this lipid compound or its pharmaceutically acceptable salt can be as follows:

[0054] .

[0055] It is understood that the preparation method of the present invention is not limited to the above-described method, and other methods can be used as needed.

[0056] In some embodiments, the molar ratio of the compound shown in formula (A) to the compound shown in formula (B) is 1:(1 to 1.2), for example, it can be 1:1, 1:1.1 or 1:1.2, preferably 1:1.1; the temperature of the nucleophilic substitution reaction is 80 to 90°C, for example, it can be 80°C, 82°C, 85°C, 87°C or 90°C, preferably 85°C; the reaction time is 3 to 5 h, for example, it can be 3 h, 4 h or 5 h, preferably 4 h; or a range consisting of any two of the above values.

[0057] To increase the rate of the nucleophilic substitution reaction, the first catalyst may include potassium iodide. To improve the solubility of the reactants, the first organic solvent may include acetonitrile.

[0058] In some embodiments, the compound represented by formula (A) can be prepared by reacting the compound represented by formula (C) under alkaline conditions. The compound shown in formula (D) The aminolysis reaction was carried out in the first organic solvent to give the compound shown in formula (A).

[0059] Specifically, the compound shown in formula (A) can be prepared as follows:

[0060] .

[0061] In some implementations, the temperature of the aminolysis reaction is 50-70°C, and the reaction time is 1-3 hours.

[0062] In some embodiments, the compound shown in formula (D) can be prepared by: taking the compound shown in formula (E) The compound shown in formula (F) The first condensation reaction was carried out in a second organic solvent under the action of a second catalyst to obtain the compound shown in formula (D).

[0063] Specifically, the compound shown in formula (D) can be prepared as follows:

[0064] .

[0065] Of course, it can also be prepared using other reactants, and the present invention does not impose any particular limitation on this.

[0066] In some embodiments, the temperature of the first condensation reaction is 10-25°C and the reaction time is 11-13 h. In the first condensation reaction, the second catalyst may be a combination of N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine or a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 4-dimethylaminopyridine. The second organic solvent includes dichloromethane.

[0067] In some embodiments, the compound shown in formula (B) can be prepared by: taking the compound shown in formula (G) The compound shown in formula (H) A second condensation reaction is carried out in a second organic solvent under the action of a second catalyst to obtain the compound shown in formula (B).

[0068] Specifically, the compound shown in formula (B) can be prepared as follows:

[0069] .

[0070] In some embodiments, the temperature of the second condensation reaction is 10~25°C and the reaction time is 11~13h. In the second condensation reaction, the second catalyst and the second organic solvent are as described above and will not be repeated.

[0071] In some embodiments, the compound represented by formula (G) can be prepared by: taking the compound represented by formula (I) The oxidation reaction is carried out in a third organic solvent under the action of an oxidant and a third catalyst to obtain the compound shown in formula (G).

[0072] Specifically, the compound represented by formula (G) can be prepared as follows:

[0073] .

[0074] In some embodiments, the oxidation reaction is carried out at a temperature of 10-25°C for 1-3 hours, the oxidant includes sodium periodate, the third catalyst includes ruthenium chloride, and the third organic solvent may include a mixed solution of dichloromethane and acetonitrile.

[0075] Of course, the compounds shown in formula (A) and / or formula (B) can be prepared by conventional experimental methods in the art, and the present invention does not impose any particular limitation on them.

[0076] According to another aspect of the present invention, a lipid nanoparticle is provided, comprising a carrier comprising a lipid compound as described above or a pharmaceutically acceptable salt thereof.

[0077] According to embodiments of the present invention, the above-mentioned lipid nanoparticles isolate bioactive substances from the external environment through a carrier, thereby improving the stability and controllability of delivery and thus enhancing the targeted recognition and expression of bioactive substances.

[0078] In some implementations, the carrier also includes structural lipids. The structural lipids effectively stabilize the structure of the carrier.

[0079] Structural lipids include, but are not limited to, one or more of cholesterol, campesterol, stigmasterol, brassosterol, sitosterol, ergosterol, nonsteroidal, corticosteroids, ursolic acid, tomatine, and α-tocopherol.

[0080] The molar ratio of the lipid compound of the present invention to the above-mentioned structural lipid is 1:1 to 10:1, preferably 1:1 to 5:1, more preferably 1:1 to 4:1, and even more preferably 1:1 to 2:1.

[0081] In some embodiments, the carrier also includes neutral lipids. Neutral lipids are disclosed or undisclosed lipid molecules existing in a neutral zwitterionic form within a selected pH range.

[0082] Neutral lipids include one or more of ceramides, sphingomyelins, phosphatidylcholine, phosphatidylethanolamine, and their derivatives. Further, neutral lipids include, but are not limited to, one or more of 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 2-(((2,3-bis(oleoyloxy)propyl))dimethylammonium phosphate)ethylhydrogen (DOCP), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), sphingomyelin (SM), ceramides, sterols, and their derivatives.

[0083] The molar ratio of the lipid compound of the present invention to the above-mentioned neutral lipid is 1:2 to 20:1, preferably 1:1 to 10:1, and more preferably 3:1 to 6:1.

[0084] In some embodiments, the carrier also includes polymer-conjugated lipids. Polymer-conjugated lipids can improve the stability of lipid nanoparticles and reduce the protein uptake of lipids.

[0085] Polymer-conjugated lipids primarily include polyethylene glycol (PEG)-modified lipid compounds. Further, PEG-modified lipid compounds include one or more of PEG-modified phosphatidylethanolamine, PEG-modified ceramide, PEG-modified diacylglycerol, PEG-modified phosphatidic acid, PEG-modified dialkylamine, and PEG-modified dialkylglycerol. Specifically, PEG-modified lipid compounds include, but are not limited to, one or more of PEG-modified bis(tetradecylacetamide) (mPEG-DTDAM), PEG-modified phosphatidylethanolamine (PEG-DMG), PEG-modified dimyristoyl phosphatidylethanolamine (PEG-DMPE), PEG-modified dipalmitoyl phosphatidylcholine (PEG-DPPC), PEG-modified dilauroyl phosphatidylethanolamine (PEG-DLPE), PEG-modified distearate phosphatidylethanolamine (PEG-DSPE), PEG-modified cholesterol (Chol-PEG), and PEG-modified ceramide (Ceramide-PEG).

[0086] Preferably, the PEG-modified lipid compound is mPEG-DTDAM or PEG-DMG, and more preferably, the relative molecular mass of PEG is 2000.

[0087] The molar ratio of the lipid compound of the present invention to the above-mentioned polymer conjugated lipid is (10~200):1. Preferably, it is (10~100):1, more preferably (10~50):1, and even more preferably (25~35):1.

[0088] In some embodiments, when the above-mentioned structural lipids, neutral lipids, and polymer conjugated lipids are present simultaneously, the molar ratio of the lipid compound of the present invention to the above-mentioned structural lipids, neutral lipids, and polymer conjugated lipids is (15~60):(15~45):(1~20):(0.5-2), preferably (20~35):(20~35):(1~10):(0.5-1.5).

[0089] In some implementations, the carrier may also include one or more other charged lipid compounds.

[0090] Charged lipid compounds include, but are not limited to, 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioloxy-N,N-dimethylaminopropane (DODMA), and N-[1-(2,3-dioleenyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). The following are one or more of the following: N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dimyristoleoyl-sn-glycero-3-ethylcholine phosphate (MOEPC), (R)-5-(dimethylamino)pentane-1,2-diyldioleoyl ester hydrochloride (DODAPen-Cl), (R)-5-guanidinopentane-1,2-diyldioleoyl ester hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentane-1-ammonium chloride (DOTAPen).

[0091] In some embodiments, the lipid nanoparticles also include preventative or therapeutic agents. These preventative or therapeutic agents may include, for example, nucleic acids, peptides, proteins, etc. The preventative or therapeutic agents are encapsulated or attached to a carrier.

[0092] In some embodiments, the mass ratio of the carrier to the preventive or therapeutic agent is (1~100):1. Preferably, it is (5~60):1, more preferably (8~40):1, even more preferably (10~30):1, or a range consisting of any two of the above values.

[0093] In some embodiments, the average particle size of the lipid nanoparticles is 80 nm to 190 nm, preferably 90 nm to 150 nm, and more preferably 90 nm to 125 nm. The polydispersity index of the lipid nanoparticles is ≤0.3.

[0094] In some further preferred embodiments, the lipid nanoparticles have an average particle size of 100–125 nm and a polydispersity index ≤0.2. This configuration helps to further ensure in vivo circulation stability and improve target cell recognition specificity, thereby facilitating the delivery and release of nucleic acid drugs.

[0095] Optionally, the average particle size of the lipid nanoparticles can be, for example, 100 nm, 110 nm, 120 nm or 125 nm, preferably around 110 nm.

[0096] In some embodiments, the preventive or therapeutic agent may be a nucleic acid, including but not limited to single-stranded DNA, double-stranded DNA, siRNA, shRNA, miRNA, mRNA, dsRNA, tRNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), and other forms of RNA molecules known in the art, preferably RNA.

[0097] Preferably, the nucleic acid may contain at least one type of mRNA.

[0098] According to another aspect of the present invention, the present invention also provides a lipid nanoparticle composition comprising the aforementioned lipid nanoparticles, and further comprising one or more of pharmaceutically commonly used excipients or diluents.

[0099] According to another aspect of the present invention, the use of a lipid compound as described above or a pharmaceutically acceptable salt thereof, or lipid nanoparticles as described above, in nucleic acid drug delivery is provided.

[0100] According to embodiments of the present invention, the ionizable lipid compounds of the present invention can be used to construct lipid nanoparticles, and their structural advantages enable precise targeting of target cells (e.g., hepatocytes). Lipid nanoparticles can efficiently deliver loaded nucleic acid drugs (e.g., mRNA) into target cells and release them specifically, mediating the desired gene expression regulation of nucleic acid drugs in target cells, which is of great significance for the prevention or treatment of diseases.

[0101] The technical solution of the present invention will be further illustrated below through specific embodiments. It should be noted that the specific embodiments described below are merely illustrative examples, and the scope of protection of the present invention is not limited thereto.

[0102] Example 1: Synthesis of compound 1 (i.e., formula (I1))

[0103] Compound 1 was synthesized according to the following route:

[0104] .

[0105] 1. Synthesis of Compound 1-1

[0106] At 25 °C, N,N'-dicyclohexylcarboimide (DCC) (7.50 g, 36.40 mmol) was added to 120 mL of dichloromethane solution containing 2-hexyldecanoic acid (7.70 g, 30.00 mmol), and the resulting solution was stirred at room temperature for 10 min. Subsequently, 6-bromohexanol (4.46 g, 24.80 mmol) and 4-dimethylaminopyridine (DMAP) (170 mg) were added sequentially. The reaction mixture was stirred at room temperature for 12 h, and the reaction was monitored by thin-layer chromatography (TLC). After the reaction was complete, solid impurities were filtered off, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:20) to give a colorless oily product, compound 1-1 (9.80 g, yield 79.60%).

[0107] Compound 1-1 was analyzed by 1H NMR spectroscopy. 1 HNMR (400MHz, CDCl3) δ4.07 (t, J=6.6Hz, 2H), 3.41 (t, J=6.8Hz, 2H), 2.31 (ddd, J= 14.3, 9.1, 5.4Hz, 1H), 1.97–1.81 (m, 2H), 1.69–1.20 (m, 30H), 0.97–0.78 (m, 6H).

[0108] 2. Synthesis of compounds 1-2

[0109] Compound 1-1 (2.00 g, 2.18 mmol), 4-aminobutanol (7.20 g, 82.00 mmol), and potassium carbonate (K₂CO₃) (894.00 mg, 6.48 mmol) were added to a reaction flask containing 10 mL of acetonitrile. The mixture was stirred at 60 °C for 2 hours under nitrogen protection, and the reaction progress was monitored by thin-layer chromatography. After the reaction was complete, the mixture was concentrated under reduced pressure. The resulting residue was redissolved in 50 mL of dichloromethane and washed with saturated brine (3 × 40 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the yellow oily product, compound 1-2 (1.60 g, yield 80.10%).

[0110] The existence of this structure was verified by performing 1H NMR spectroscopy on compounds 1-2.

[0111] 3. Synthesis of compounds 1-3

[0112] At 25 °C, 6-bromohexanoic acid (6.84 g, 35.00 mmol), 1-undecen-5-ol (5.24 g, 30.80 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (6.20 g, 40.00 mmol), and 4-dimethylaminopyridine (DMAP) (0.94 g, 7.70 mmol) were sequentially added to a round-bottom flask containing 120 mL of dichloromethane, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the reaction solution was washed three times with saturated brine, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:20) to give a colorless oily product, compound 1-3 (6.47 g, yield 57.90%).

[0113] The existence of this structure was verified by performing 1H NMR spectroscopy on compounds 1-3.

[0114] 4. Synthesis of compounds 1-4

[0115] At 25 °C, compounds 1-3 (4.00 g, 11.70 mmol) were dissolved in a mixed solvent of 40 mL dichloromethane and 40 mL acetonitrile, and ruthenium trichloride hydrate (RuCl3·H2O) (0.18 g, 0.90 mmol) was added. Sodium periodate (NaIO4) (16.00 g, 75.50 mmol) was dissolved in 80 mL water and added dropwise to the above reaction system under ice bath cooling, followed by stirring at room temperature for 2 hours. After the reaction was completed, the organic solvent was removed by rotary evaporation, and the aqueous phase was extracted three times with 100 mL dichloromethane. The combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, and concentrated to directly give the oily product 1-4 (3.60 g, yield 90.00%).

[0116] The existence of this structure was verified by performing 1H NMR spectroscopy on compounds 1-4.

[0117] 5. Synthesis of compounds 1-5

[0118] At 25 °C, N,N'-dicyclohexylcarboimide (DCC) (2.50 g, 12.10 mmol) was added to 50 mL of dichloromethane solution containing compounds 1-4 (3.60 g, 10.00 mmol), and the resulting reaction solution was stirred at room temperature for 10 min. Subsequently, (2E,6E)-non-2,6-dien-1-ol (1.70 g, 9.00 mmol) and 4-dimethylaminopyridine (DMAP) (100 mg) were added sequentially. The reaction system was stirred at room temperature for 12 h, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, solid impurities were filtered off, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:20) to give the colorless oily product compounds 1-5 (4.30 g, yield 81.10%).

[0119] The existence of this structure was verified by performing 1H NMR spectroscopy on compounds 1-5.

[0120] 6. Synthesis of Compound 1

[0121] Compounds 1-2 (0.097 g, 0.20 mmol), 1-5 (0.093 g, 0.22 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.20 mmol) were added sequentially to a 50 mL round-bottom flask. 10 mL of acetonitrile was added as a solvent, and the mixture was stirred and refluxed at 85 °C for 4 hours. After the reaction was complete, the acetonitrile was removed by rotary evaporation. The residue was dissolved in dichloromethane and washed three times with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated to remove the solvent. Finally, the mixture was purified by silica gel column chromatography (eluent: ethyl acetate / methanol = 50:1) to give compound 1 (0.082 g, 0.07 mmol, yield 43.20%), a pale yellow oil.

[0122] Compound 1 was analyzed by ESI-MS [M+H] + 834.72 and 1 HNMR confirmed. Figure 1 This is the 1H NMR spectrum of compound 1 from Example 1. Figure 1 As shown, the specific structure of compound 1 is confirmed. 1HNMR (400MHz, CDCl3) δ5.78 (d, J=15.3Hz, 1H), 5.65–5.54 (m, 1H), 5.36 (ddd, J=28.5, 10.8, 5.9Hz, 2H), 4.87 (d, J=12.5Hz, 1H), 4.53 ( t, J=10.1Hz, 2H), 4.06 (t, J=6.7Hz, 2H), 3.55 (s, 2H), 2.53–2.21 (m, 10H), 2.17–1.97 (m, 6H), 1.95–1.03 (m, 55H), 1.01–0.79 (m, 12H).

[0123] Example 2: Synthesis of compound 2 (i.e., formula (I2))

[0124] Compound 2 was synthesized according to the following route:

[0125] .

[0126] 1. Synthesis of Compound 2-1

[0127] At 25 °C, N,N'-dicyclohexylcarboimide (DCC) (2.50 g, 12.10 mmol) was added to 50 mL of dichloromethane solution containing compound 1-4 (3.60 g, 10.00 mmol), and the resulting reaction solution was stirred at room temperature for 10 min. Subsequently, 1-octyne-2-ol (1.20 g, 9.00 mmol) and 4-dimethylaminopyridine (DMAP) (100 mg) were added sequentially. The reaction system was stirred at room temperature for 12 h, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, solid impurities were filtered off, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:20) to give the colorless oily product compound 2-1 (3.20 g, yield 76.20%).

[0128] The existence of this structure was verified by performing 1H NMR spectroscopy on compound 2-1.

[0129] 2. Synthesis of Compound 2

[0130] Compound 2-1 (0.095 g, 0.20 mmol), compound 1-2 (0.093 g, 0.22 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and potassium iodide (KI) (0.03 g, 0.20 mmol) were added sequentially to a 50 mL round-bottom flask. 10 mL of acetonitrile was added as a solvent, and the mixture was stirred and refluxed at 85 °C for 4 hours. After the reaction was complete, the acetonitrile was removed by rotary evaporation. The residue was dissolved in dichloromethane and washed three times with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate, concentrated to remove the solvent, and finally purified by silica gel column chromatography (eluent: ethyl acetate / methanol = 50:1) to give compound 2 (0.073 g, 0.07 mmol, yield 38.10%), a pale yellow oil.

[0131] Compound 2 was analyzed by ESI-MS [M+H]. + 820.71 and 1 HNMR confirmed. Figure 2 This is the 1H NMR spectrum of compound 2 in Example 2. Figure 2 As shown, the specific structure of compound 2 is confirmed. 1 HNMR (400MHz, CDCl3) δ4.89 (t, J=12.5Hz, 1H), 4.72–4.62 (m, 2H), 4.11–4.01 (m, 2H), 3.66–3 .57 (m, 2H), 2.68–2.21 (m, 12H), 2.02–1.80 (m, 3H), 1.74–1.21 (m, 58H), 0.93–0.82 (m, 12H).

[0132] Example 3: Synthesis of compound 3 (i.e., formula (I3))

[0133] Compound 3 was synthesized according to the following route:

[0134] .

[0135] 1. Synthesis of compound 3-1

[0136] At 25°C, N,N'-dicyclohexylcarboimide (DCC) (2.50 g, 12.1 mmol) was added to 50 mL of dichloromethane solution containing compound 1-4 (3.60 g, 10.00 mmol), and the resulting reaction solution was stirred at room temperature for 10 min. Subsequently, 1-octanol (1.20 g, 9.00 mmol) and 4-dimethylaminopyridine (DMAP) (100 mg) were added sequentially. The reaction system was stirred at room temperature for 12 h, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, solid impurities were filtered off, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:20) to give the colorless oily product compound 3-1 (3.30 g, yield 76.80%).

[0137] The existence of this structure was verified by performing 1H NMR spectroscopy on compound 3-1.

[0138] 2. Synthesis of Compound 3

[0139] Compound 3-1 (0.095 g, 0.20 mmol), compound 1-2 (0.093 g, 0.22 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.20 mmol) were added sequentially to a 50 mL round-bottom flask. 10 mL of acetonitrile was added as a solvent, and the mixture was stirred and refluxed at 85 °C for 4 hours. After the reaction was complete, the acetonitrile was removed by rotary evaporation. The residue was dissolved in dichloromethane and washed three times with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate, concentrated to remove the solvent, and finally purified by silica gel column chromatography (eluent: ethyl acetate / methanol = 50:1) to give a pale yellow oily compound 3 (0.063 g, 0.07 mmol, yield 33.1%).

[0140] Compound 3 was analyzed by ESI-MS [M+H]. + 824.74 and 1 HNMR confirmed. Figure 3 The image shows the 1H NMR spectrum of compound 3 in Example 3. Figure 3 As shown, the specific structure of compound 3 is confirmed. 1 HNMR (400MHz, CDCl3) δ4.89 (t, J=12.5Hz, 1H), 4.09 (dt, J=13.5, 7.0Hz, 4H), 3.55 (s, 2H), 2.42 (d, J=30.9Hz, 6H), 2 .33–2.26 (m, 4H), 2.02–1.78 (m, 5H), 1.61 (ddd, J=23.3, 14.3, 7.2Hz, 14H), 1.40–1.21 (m, 48H), 0.91–0.83 (m, 12H).

[0141] Example 4: Synthesis of compound 4 (i.e., formula (I4))

[0142] Compound 4 was synthesized according to the following route:

[0143] .

[0144] 1. Synthesis of compound 4-1

[0145] At 25 °C, 6-bromohexanoic acid (6.84 g, 35.00 mmol), 6-hepten-3-ol (3.51 g, 30.80 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (6.20 g, 40.00 mmol), and 4-dimethylaminopyridine (DMAP) (0.94 g, 7.70 mmol) were sequentially added to a round-bottom flask containing 120 mL of dichloromethane, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the reaction solution was washed three times with saturated brine, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:20) to give a colorless oily product, compound 4-1 (5.88 g, yield 61.50%).

[0146] The existence of this structure was verified by performing 1H NMR spectroscopy on compound 4-1.

[0147] 2. Synthesis of compound 4-2

[0148] At 25 °C, compound 4-1 (3.40 g, 11.70 mmol) was dissolved in a mixed solvent of 40 mL dichloromethane and 40 mL acetonitrile, and RuCl3·H2O (0.18 g, 0.90 mmol) was added. NaIO4 (16.00 g, 75.50 mmol) was dissolved in 80 mL water and added dropwise to the above reaction system under ice bath cooling, followed by stirring at room temperature for 2 hours. After the reaction was completed, the organic solvent was removed by rotary evaporation, and the aqueous phase was extracted three times with 100 mL dichloromethane. The combined organic phases were washed twice with saturated brine, dried over anhydrous sodium sulfate, and concentrated to give the oily product 4-2 (3.00 g, yield 88.20%).

[0149] The existence of this structure was verified by performing 1H NMR spectroscopy on compound 4-2.

[0150] 3. Synthesis of compound 4-3

[0151] At 25°C, N,N'-dicyclohexylcarboimide (DCC) (2.50 g, 12.10 mmol) was added to 50 mL of dichloromethane solution containing compound 4-2 (3.10 g, 10.00 mmol), and the resulting reaction solution was stirred at room temperature for 10 min. Subsequently, (2E,6E)-non-2,6-dien-1-ol (1.70 g, 9.00 mmol) and 4-dimethylaminopyridine (DMAP) (100 mg) were added sequentially. The reaction system was stirred at room temperature for 12 h, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, solid impurities were filtered off, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:20) to give the colorless oily product compound 4-3 (3.90 g, yield 72.20%).

[0152] The existence of this structure was verified by performing 1H NMR spectroscopy on compound 4-3.

[0153] 4. Synthesis of compound 4-4

[0154] 8-Bromooctanoic acid (5.20 g, 23.00 mmol), heptadecano-9-ol (5.20 g, 20.00 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (5.55 g, 29.00 mmol), and DMAP (0.48 g, 4.00 mmol) were dissolved in 120 mL of dichloromethane. The mixture was stirred at room temperature for 12 h, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, the reaction solution was washed three times with saturated brine, the organic phase was separated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was further purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:20) to give a colorless oily product, compound 4-4 (7.20 g, yield 74.20%).

[0155] The existence of this structure was verified by performing 1H NMR spectroscopy on compound 4-4.

[0156] 5. Synthesis of compounds 4-5

[0157] Compound 4-4 (2.00 g, 2.16 mmol), 2-aminoethanol (5.00 g, 82.00 mmol), and K₂CO₃ (894.00 mg, 6.48 mmol) were added to a reaction flask containing 10 mL of acetonitrile. The mixture was stirred at 60 °C for 2 h under nitrogen protection, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was complete, the reaction solution was evaporated to dryness under reduced pressure. The residue was redissolved in 50 mL of dichloromethane and washed with saturated brine (3 × 40 mL). The organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the yellow oily product compound 4-5 (1.60 g, yield 76.20%).

[0158] The existence of this structure was verified by performing 1H NMR spectroscopy on compounds 4-5.

[0159] 6. Synthesis of Compound 4

[0160] Compounds 4-5 (0.088 g, 0.20 mmol), 5-3 (0.094 g, 0.22 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.20 mmol) were added sequentially to a 50 mL round-bottom flask. 10 mL of acetonitrile was added as a solvent, and the mixture was stirred and refluxed at 85 °C for 4 hours. After the reaction was complete, the acetonitrile was removed by rotary evaporation. The residue was dissolved in dichloromethane and washed three times with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated to remove the solvent. Finally, the mixture was purified by silica gel column chromatography (eluent: ethyl acetate / methanol = 50:1) to give compound 4 (0.065 g, yield 36.50%), a pale yellow oil.

[0161] Compound 4 was analyzed by ESI-MS [M+H]. + 792.67 and 1 HNMR confirmed. Figure 4 This is the 1H NMR spectrum of compound 4 in Example 4. Figure 4 As shown, the specific structure of compound 4 is confirmed. 1 HNMR (400MHz, CDCl3) δ5.78 (d, J=15.4Hz, 1H), 5.64–5.52 (m, 1H), 5.46–5.26 (m, 2H), 4.92–4.78 (m, 2H), 4.52 (d, J=6.2Hz, 2H), 3.54 (t, J=5. 3Hz, 2H), 2.59 (t, J=5.2Hz, 2H), 2.46 (dd, J=14.1, 6.1Hz, 4H), 2.38–2.23 (m, 5H), 2.17–2.00 (m, 5H), 1.77–1.22 (m, 50H), 1.10–0.69 (m, 12H).

[0162] Example 5: Synthesis of compound 5 (i.e., formula (I5))

[0163] Compound 5 was synthesized according to the following route:

[0164] .

[0165] 1. Synthesis of Compound 5-1

[0166] At 25°C, N,N'-dicyclohexylcarboimide (DCC) (2.50 g, 12.10 mmol) was added to 50 mL of dichloromethane solution containing compound 4-2 (3.10 g, 10.00 mmol), and the resulting reaction solution was stirred at room temperature for 10 min. Subsequently, 1-octyne-2-ol (1.20 g, 9.00 mmol) and 4-dimethylaminopyridine (DMAP) (100 mg) were added sequentially. The reaction system was stirred at room temperature for 12 h, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, solid impurities were filtered off, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:20) to give the colorless oily product compound 5-1 (3.67 g, yield 70.30%).

[0167] The existence of this structure was verified by performing 1H NMR spectroscopy on compound 5-1.

[0168] 2. Synthesis of Compound 5

[0169] Compounds 4-5 (0.088 g, 0.20 mmol), 5-1 (0.092 g, 0.22 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.20 mmol) were added sequentially to a 50 mL round-bottom flask. 10 mL of acetonitrile was added as a solvent, and the mixture was stirred and refluxed at 85 °C for 4 hours. After the reaction was complete, the acetonitrile was removed by rotary evaporation. The residue was dissolved in dichloromethane and washed three times with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated to remove the solvent. Finally, the mixture was purified by silica gel column chromatography (eluent: ethyl acetate / methanol = 50:1) to give compound 5 (0.062 g, yield 36.30%), a pale yellow oil.

[0170] Compound 5 was analyzed by ESI-MS [M+H]. + 778.67 and 1 HNMR confirmed. Figure 5 This is the 1H NMR spectrum of compound 5 in Example 5. Figure 5 As shown, the specific structure of compound 5 is confirmed. 1HNMR (400MHz, CDCl3) δ4.91–4.80 (m, 2H), 4.66 (dt, J=15.4, 7.7Hz, 2H), 3.60 (s, 2H ), 2.76–2.15 (m, 14H), 1.98–1.81 (m, 2H), 1.73–1.14 (m, 52H), 1.02–0.73 (m, 12H).

[0171] Example 6: Synthesis of compound 6 (i.e., formula (I6))

[0172] Compound 6 was synthesized according to the following route:

[0173] .

[0174] 1. Synthesis of Compound 6-1

[0175] At 25°C, N,N'-dicyclohexylcarboimide (DCC) (2.50 g, 12.10 mmol) was added to 50 mL of dichloromethane solution containing compound 4-2 (3.10 g, 10.00 mmol), and the resulting reaction solution was stirred at room temperature for 10 min. Subsequently, 1-octanol (1.17 g, 9.00 mmol) and 4-dimethylaminopyridine (DMAP) (100 mg) were added sequentially. The reaction system was stirred at room temperature for 12 h, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, solid impurities were filtered off, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:20) to give the colorless oily product compound 6-1 (3.46 g, yield 69.20%).

[0176] The existence of this structure was verified by performing 1H NMR spectroscopy on compound 6-1.

[0177] 2. Synthesis of Compound 6

[0178] Compounds 4-5 (0.088 g, 0.20 mmol), 6-1 (0.092 g, 0.22 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.20 mmol) were added sequentially to a 50 mL round-bottom flask. 10 mL of acetonitrile was added as a solvent, and the mixture was stirred and refluxed at 85 °C for 4 hours. After the reaction was complete, the acetonitrile was removed by rotary evaporation. The residue was dissolved in dichloromethane and washed three times with saturated brine. The combined organic phases were dried over anhydrous sodium sulfate and concentrated to remove the solvent. Finally, the mixture was purified by silica gel column chromatography (eluent: ethyl acetate / methanol = 50:1) to give compound 6 (0.058 g, yield 35.82%), a pale yellow oil.

[0179] Compound 6 was analyzed by ESI-MS [M+H]. + 782.66 and 1 HNMR confirmed. Figure 6 This is the 1H NMR spectrum of compound 6 in Example 6. Figure 6 As shown, the specific structure of compound 6 is confirmed. 1 HNMR (400MHz, CDCl3) δ4.87 (dd, J=12.5, 6.2Hz, 2H), 4.06 (t, J=6.8Hz, 2H), 3.54 (t, J=5.2Hz, 2H), 2.62–2.44 (m, 6 H), 2.31 (dt, J=15.5, 5.7Hz, 6H), 1.59 (ddd, J=27.2, 17.8, 10.2Hz, 22H), 1.44–1.25 (m, 38H), 0.98–0.79 (m, 12H).

[0180] Example 7: Preparation and Physicochemical Characterization of Lipid Nanoparticles

[0181] 1. Preparation of lipid nanoparticles

[0182] Compounds 1-6 prepared in Examples 1-6 and a commercially available ionizable lipid compound (ALC-0315) were dissolved in 300 μL of ethanol with DSPC, cholesterol, and mPEG-DTDAM (ALC-0159) at a molar ratio of 46.3:9.4:42.7:1.6 to prepare ethanol lipid solutions. Separately, 200 μL of a 0.5 μg / μL solution of messenger RNA (Fluc mRNA) encoding firefly luciferase was dissolved in 700 μL of 20 mM citrate buffer at pH 4 to prepare the aqueous phase. The aqueous and organic phases were mixed using a microfluidic mixer to prepare lipid nanoparticles (LNPs) encapsulating Fluc mRNA. The prepared lipid nanoparticles were rapidly added to an ultrafiltration tube containing 10 volumes of PBS (pH 7.4) standard solution and centrifuged at 5000 rpm for 30 min to remove ethanol and citrate buffer. The desired concentration of lipid nanoparticle solution was obtained using an Amicon Ultra centrifuge filter. The lipid nanoparticles prepared from compounds 1-6 and ALC-0135 were named LNP-1, LNP-2, LNP-3, LNP-4, LNP-5, LNP-6, and LNP-0315, respectively. The molecular structures of the lipid compounds are shown below. Figure 7 As shown.

[0183] 2. Determination of lipid nanoparticle size, polydispersity index and encapsulation efficiency

[0184] The particle size and polydispersity index (PDI) of the lipid nanoparticles LNP-1~LNP-6 and LNP-0315 obtained above were determined using a Malvern particle size analyzer (Malvern UK). The results are shown in Table 1.

[0185] The mRNA encapsulation efficiency (EE%) was detected using RiboGreen dye (Thermo Fisher Scientific, Cat No. 5, R11491). First, to determine the free mRNA in the lipid nanoparticles, 10 μL of each of the LNP-1~LNP-6 and LNP-0315 lipid nanoparticle solutions were placed in centrifuge tubes and diluted with 990 μL of 1:1 Tris-EDTA buffer (Nanjing Novizan Biotechnology Co., Ltd.). 100 μL of the diluted solution was then added to a 96-well plate. Next, 100 μL of RiboGreen dye was added to each well, and the plate was incubated for 5 min. The concentration of free mRNA was measured using a spectrophotometer (Thermo Fisher Scientific, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. To determine the total mRNA content in lipid nanoparticles, 10 μL of each of the LNP-1~LNP-6 and LNP-0315 lipid nanoparticle solutions were placed in centrifuge tubes and diluted with 990 μL of 2% TE-Triton buffer (Nanjing Novizan Biotechnology Co., Ltd.). 100 μL of the diluted solution was then added to a 96-well plate. Next, 100 μL of RiboGreen dye was added to each well, and the plate was incubated for 5 min. The total mRNA concentration was measured using a spectrophotometer (Thermo Fisher Scientific, USA) at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The formula for calculating EE (%) is as follows.

[0186] EE (%) = (Total mRNA concentration - Free mRNA concentration) / Total mRNA concentration × 100%.

[0187] The results are shown in Table 1.

[0188] Table 1 Physicochemical properties of LNP-1~LNP-6 and LNP-0315 lipid nanoparticles

[0189]

[0190] As shown in Table 1, the LNP-1~LNP-6 lipid nanoparticles prepared in this invention and the LNP-0315 lipid nanoparticles prepared from commercially available ALC-0315 have particle sizes ranging from 100 to 125 nm, and their PDI values ​​are all less than 0.2. This indicates that the above seven lipid nanoparticles all possess excellent physicochemical properties. Furthermore, the LNP-1~LNP-6 lipid nanoparticles prepared in this invention and the LNP-0315 lipid nanoparticles prepared from commercially available ALC-0315 all exhibit high mRNA encapsulation efficiency.

[0191] Example 8: Cytotoxicity Experiment

[0192] HEK293 cells or DC2.4 cells were seeded in 96-well plates (2000 cells / well) and cultured for 12 h. After cell attachment, the prepared LNP-1~LNP-6 and LNP-0315 lipid nanoparticle solutions were added to the 96-well plates, and the mRNA concentration was controlled at 2 μg / ml. After 24 hours of incubation, cytotoxicity was assessed using the Cell Counting Kit-8 (CCK-8, APExBIO, USA). The OD value at 450 nm was measured using a microplate reader. Cell viability was calculated based on the OD values, and the results are shown in Table 2.

[0193] Table 2. In vitro toxicity data of LNP-1~LNP-6 and LNP-0315 lipid nanoparticles

[0194]

[0195] As shown in Table 2, the above seven lipid nanoparticles exhibit low cytotoxicity and have promising prospects for in vivo application.

[0196] Example 9: Bioluminescence detection in vivo

[0197] To investigate the in vivo mRNA delivery of compounds 1-6 and commercially available ALC-0315, 21 female BALB / c mice weighing 16-18g were randomly divided into 7 groups of 3 mice each. Each group of mice was intramuscularly injected with 100μL of a solution containing LNP-1-LNP-6 and LNP-0315 lipid nanoparticles prepared in Example 7, and 2μg of Fluc mRNA per mouse. Six hours after injection, 100μL of luciferase substrate (30mg / mL) was injected intraperitoneally, and the reaction was allowed to proceed for 5 minutes. Bioluminescence signal images were obtained using a mouse imaging system (Perkin Elmer). For example, comparing LNP-1-LNP-3 and LNP-0315 prepared from commercially available ALC-0315, the major organs of the mice ( Figure 8 The organs in the image, from left to right, are heart, liver, spleen, lung, and kidney. The imaging results are as follows: Figure 8As shown, the liver bioluminescence effect of LNP-1 to LNP-3 is significantly higher than that of LNP-0315. Furthermore, the average bioluminescence intensity of LNP-1 to LNP-6 and LNP-0315 in the liver is shown in Table 3.

[0198] Table 3. In vivo bioluminescence data of LNP-1~LNP-6 and LNP-0315 lipid nanoparticles.

[0199]

[0200] From Table 3 and Figure 8 It is evident that LNP-1~LNP-6 and LNP-0315 lipid nanoparticles can effectively deliver mRNA in vivo and complete expression. Furthermore, the in vivo delivery effect of lipid nanoparticles prepared by compounds 1~6 in the embodiments of the present invention is better than that of lipid nanoparticles prepared by commercially available ALC-0315 in the control group. In particular, the nucleic acid delivery efficiency of the lipid compounds in Examples 1 and 2 in the liver is 3.1 times and 2.3 times higher than that of commercially available ALC-0315 in the control group, respectively, showing good application prospects.

[0201] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A lipid compound or a pharmaceutically acceptable salt thereof, characterized in that, It has the structural formula shown in equation (I): Equation (I); Where R is a hydroxyl-substituted C1~C 10 alkyl; X is selected from any of the following structures: ； Y is selected from any of the following structures: In this context, the dashed lines represent connection points; A is a C1~C5 alkylene ester group; Z is C 11 ~C 18 Branched alkyl groups.

2. The lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The lipid compound or its pharmaceutically acceptable salt has any one of the structural formulas shown in formula (I-1) or formula (I-2): Equation (I-1); Equation (I-2).

3. The lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, R is selected from any of the following structures: ; where the dashed lines represent connection points.

4. The lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The lipid compound or its pharmaceutically acceptable salt has any one of the structures shown in formulas (I1) to (I6): Equation (I1); Equation (I2); Equation (I3); Equation (I4); Equation (I5); Equation (I6).

5. A method for preparing a lipid compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 4, characterized in that, include: Under alkaline conditions, the compound shown in formula (A) The compound shown in formula (B) A nucleophilic substitution reaction is carried out in a first organic solvent under the catalysis of a first catalyst to obtain a lipid compound as shown in formula (I) or a pharmaceutically acceptable salt thereof.

6. A lipid nanoparticle, characterized in that, Includes a carrier, said carrier comprising a lipid compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 4.

7. The lipid nanoparticles according to claim 6, characterized in that, The lipid nanoparticles may also include preventative or therapeutic agents; The preventive or therapeutic agent is encapsulated or attached to the carrier.

8. The lipid nanoparticles according to claim 7, characterized in that, The lipid nanoparticles have an average particle size of 100~125nm and a polydispersity index of ≤0.

2. The preventive or therapeutic agent is a nucleic acid.

9. The use of a lipid compound as described in any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, or a lipid nanoparticle as described in any one of claims 6 to 8, in nucleic acid drug delivery.

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