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

Abstract

This invention provides a lipid compound or a pharmaceutically acceptable salt thereof, a method for preparing the same, 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 is C1~C1. 10 Hydroxyalkyl or substituted or unsubstituted C2~C 10 Ether; X is C1~C 20 Straight-chain or branched alkyl; Y is C1~C 20 Straight-chain or branched aliphatic hydrocarbon groups; with or without the above-mentioned substituted or unsubstituted C2~C 10 When the ether has a substituent, 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 recent years, the safe and efficient delivery of nucleic acid molecules to specific target cells and protection against intracellular degradation have been among the technological bottlenecks hindering the development of nucleic acid drugs. An ideal nucleic acid delivery vector must simultaneously meet the requirements of biosafety, in vivo circulation stability, and organ-targeting specificity, which is of great significance to the therapeutic efficacy of nucleic acid drugs.

[0003] Lipid nanoparticles, as an advanced nucleic acid delivery carrier, have shown application potential in the field of nucleic acid drug delivery. Lipid compounds, as key functional components of lipid nanoparticles, influence their stability and biocompatibility through their chemical structure and physicochemical properties. Specifically, lipid compounds self-assemble with nucleic acid molecules to form nanoparticles. These nanoparticles can mediate the penetration of nucleic acid molecules through the target cell membrane barrier, enabling the delivery and release of nucleic acid molecules into the target cell cytoplasm, thereby triggering targeted therapeutic effects or immunomodulatory functions.

[0004] Therefore, more lipid compounds capable of efficiently delivering nucleic acid molecules need to be studied. Summary of the Invention

[0005] 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.

[0006] 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):

[0007] Formula (I);

[0008] Where R is C1~C 10 Hydroxyalkyl or substituted or unsubstituted C2~C 10 Ether; X is C1~C 20 Straight-chain or branched alkyl; Y is C1~C 20 Straight-chain or branched aliphatic hydrocarbon groups; with or without the above-mentioned substituted or unsubstituted C2~C 10 When the ether has a substituent, the substituent is selected from hydroxyl groups.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] The lipid compounds or pharmaceutically acceptable salts thereof according to embodiments of the present invention have a molecular structure mainly comprising a hydrophilic head, a tetraester bond (i.e., ester bonds at four different sites), and a hydrophobic tail. Simultaneously, the two branches connected to the ester bond on the N atom are identical, exhibiting symmetry. The present invention optimizes the molecular structure of lipid compounds, enabling the two branches connected to the ester bond on the N atom to form a symmetrical structure. This symmetrical structure improves the interaction between the lipid compounds and nucleic acid drugs, thereby enhancing the delivery efficiency of the lipid compounds for nucleic acid drugs. Furthermore, the lipid compounds of the present invention possess excellent biodegradability, gradually degrading within target cells through enzymatic reactions, effectively reducing the toxicity of lipid compound accumulation within cells, improving biocompatibility, and exhibiting good chemical stability. They effectively protect nucleic acid drugs during in vivo circulation, preventing degradation due to external environmental factors, and allow for controlled release of nucleic acid drugs upon reaching target cells, ensuring drug efficacy. The lipid compounds of the present invention enrich the variety of lipid compounds, providing more options for nucleic acid drug delivery. The lipid nanoparticles formed exhibit high in vivo transfection efficiency, which is of great significance for disease prevention or treatment. Attached Figure Description

[0013] 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.

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

[0015] Figure 2 The 1H NMR spectrum of compound 2 in Example 2;

[0016] Figure 3 The 1H NMR spectrum of compound 3 in Example 3;

[0017] Figure 4 The 1H NMR spectrum of compound 4 in Example 4;

[0018] Figure 5 The 1H NMR spectrum of compound 5 in Example 5;

[0019] Figure 6 The 1H NMR spectrum of compound 6 in Example 6;

[0020] Figure 7 The structural diagrams are of compounds 1 to 10 in Examples 1 to 10 and commercially available ALC-0315;

[0021] Figure 8 Bioluminescence images of various organs in mice after intravenous injection of lipid nanoparticles constructed from compound 6 in Example 6 and commercially available ALC-0315, respectively. Detailed Implementation

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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. Examples of C1 to C5 alkyl groups include: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isopentyl, etc.

[0027] Lipid delivery systems offer advantages such as simple lipid component synthesis, scalable preparation processes, and wide applicability in packaging. Lipid nanoparticles have already found widespread use in drug delivery. Therefore, it is necessary to synthesize a series of structurally optimized lipid compounds for the delivery of nucleic acid drugs.

[0028] In the process of realizing this invention, it was discovered that the symmetrical structure on the N atom in the lipid compound helps to improve the delivery efficiency of nucleic acid drugs. Simultaneously, its synergistic effect with the hydrophilic head, tetraester bond, and hydrophobic tail in the lipid compound gives the lipid compound good biodegradability, chemical stability, and targeting, further ensuring the delivery efficiency of nucleic acid drugs.

[0029] 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):

[0030] Formula (I);

[0031] Where R is C1~C 10 Hydroxyalkyl or substituted or unsubstituted C2~C 10 Ether; X is C1~C 20 Straight-chain or branched alkyl; Y is C1~C 20 Straight-chain or branched aliphatic hydrocarbon groups; with or without the above-mentioned substituted or unsubstituted C2~C 10 When the ether has a substituent, the substituent is selected from hydroxyl groups.

[0032] According to embodiments of the present invention, the lipid compound or its pharmaceutically acceptable salt comprises a hydrophilic head, a tetraester bond, and a hydrophobic tail. Simultaneously, the two branches connected to the N atom by the ester bond have identical structures and exhibit symmetry. This highly symmetrical structure contributes to improving the delivery efficiency of the lipid compound for nucleic acid drugs. This symmetrical structure optimizes the molecular configuration and charge distribution of the lipid compound, promoting the formation of a functionally complementary overall structure with the hydrophilic head, tetraester bond, and hydrophobic tail, thereby collectively endowing the lipid compound with excellent comprehensive properties. The tetraester bond structure provides the structural basis for biodegradability, and combined with the optimization of molecular metabolic pathways by the symmetrical structure, enables the lipid compound to be efficiently degraded within cells through enzymatic hydrolysis, reducing cumulative toxicity. The stability regulation of the symmetrical structure by the hydrophilic head and hydrophobic tail endows the lipid compound with good chemical stability, ensuring that it maintains structural integrity during in vivo circulation and effectively protecting nucleic acid drugs from degradation. Furthermore, the synergistic effect of the above structures can also optimize the surface properties and particle size distribution of the constructed lipid nanoparticles, enhancing their targeted recognition and binding ability to target cells, further ensuring the targeted delivery efficiency of nucleic acid drugs.

[0033] It can be understood that the aliphatic hydrocarbon groups here include saturated alkane groups, unsaturated alkene groups, and unsaturated alkyne groups, or combinations of the aforementioned groups.

[0034] In some implementations, R is selected from any of the following structures:

[0035] In this diagram, the dashed lines represent connection sites. This arrangement helps to further regulate the surface charge and polarity of the lipid nanoparticles constructed from the lipid compounds of this invention, promoting their interaction with the cell membrane and facilitating the delivery and expression of nucleic acid drugs.

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

[0037] In this diagram, the dashed lines represent connection sites. Branching helps reduce the packing density of lipid tail chains, improves membrane fluidity, and makes it easier to disrupt cell membrane integrity when lysosomal acidification triggers lipid protonation, creating channels for nucleic acid escape and thus enhancing the lysosomal escape efficiency of nucleic acids.

[0038] In some implementations, Y is selected from any of the following structures:

[0039] In this diagram, the dashed lines represent connection sites. Screening for Y in ionizable lipid compounds helps optimize the stability of the lipid compounds and their encapsulation or loading efficiency for nucleic acid molecules, thereby forming uniform and stable lipid nanoparticles. Furthermore, unsaturated bonds help increase the interaction between lipid compounds and the cell membrane, thereby improving endocytosis.

[0040] In some embodiments, the lipid compound or a pharmaceutically acceptable salt thereof has the following formulas (I1) to (I2). 10 Any one of the structures shown:

[0041] Equation (I1);

[0042] Equation (I2);

[0043] Equation (I3);

[0044] Equation (I4);

[0045] Equation (I5);

[0046] Equation (I6);

[0047] Equation (I7);

[0048] Equation (I8);

[0049] Equation (I9);

[0050] Formula (I 10 ).

[0051] According to embodiments of the present invention, when the above-mentioned lipid compounds are used, they exhibit better stability and biodegradability during subsequent nucleic acid delivery, which is more conducive to gene expression in target cells.

[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:(2~2.5), for example, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4 or 1:2.5, preferably 1:2.2; the temperature of the nucleophilic substitution reaction is 80~90°C, for example, 80°C, 82°C, 85°C, 87°C or 90°C, preferably 85°C; the reaction time is 3~5 h, for example, 3 h, 4 h or 5 h, preferably 4 h. This configuration promotes the formation of the lipid compounds with the symmetrical structure described above and improves their reaction yield.

[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 shown in formula (B) can be prepared by: taking the compound shown in formula (C) and In the presence of a second catalyst, a condensation reaction is carried out in a second organic solvent to obtain the compound shown in formula (B).

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

[0060] .

[0061] In some embodiments, the condensation reaction is carried out at a temperature of 10-25°C for 11-13 hours. In the 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 may include dichloromethane.

[0062] In some embodiments, the compound shown in formula (C) can be prepared by: taking the compound shown in formula (D) 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 (C).

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

[0064] .

[0065] 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.

[0066] 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.

[0067] 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.

[0068] According to embodiments of the present invention, the above-mentioned lipid nanoparticles can efficiently deliver bioactive substances (such as nucleic acids) into cells, tissues or organs, thereby achieving efficient regulation of bioactive substances.

[0069] In some implementations, the carrier also includes structural lipids. The structural lipids effectively stabilize the structure of the carrier.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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).

[0077] Preferably, the PEG-modified lipid compound includes mPEG-DTDAM or PEG-DMG, and more preferably, the PEG has a relative molecular mass of 2000.

[0078] 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.

[0079] 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).

[0080] In some implementations, the carrier may also include one or more other charged lipid compounds.

[0081] 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).

[0082] 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.

[0083] In some embodiments, the mass ratio of the carrier to the preventive or therapeutic agent is (1~100):1, preferably 5:1~60:1, more preferably 8:1~40:1, more preferably 10:1~30:1, or a range consisting of any two of the above values.

[0084] In some embodiments, the average particle size of the lipid nanoparticles is 80 nm to 190 nm, preferably 90 nm to 150 nm. The polydispersity index of the lipid nanoparticles is ≤0.30. This configuration makes the lipid nanoparticles easier for cells to take up.

[0085] In some further preferred embodiments, the lipid nanoparticles have an average particle size of 100-130 nm and a polydispersity index ≤0.2. This configuration further facilitates the uptake of lipid nanoparticles by target cells, thereby enhancing the gene expression of nucleic acids in target cells.

[0086] In some embodiments, the preventive or therapeutic agent may be a nucleic acid, including but not limited to one or more of 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. RNA is preferred, and one or more of siRNA, shRNA, and mRNA are more preferred.

[0087] Preferably, the nucleic acid may contain at least one type of mRNA.

[0088] 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.

[0089] 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.

[0090] According to embodiments of the present invention, the biodegradable ionizable lipid compound of the present invention can be made into lipid nanoparticles with uniform particle size that are loaded with nucleic acid drugs. These nanoparticles can be precisely positioned at target cells (e.g., liver cells) to release nucleic acid drugs and achieve gene expression, thereby improving the efficiency of nucleic acid drug delivery. This is of great significance for designing nucleic acid drug delivery systems with better therapeutic effects.

[0091] 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.

[0092] Example 1: Synthesis of compound 1 (i.e., formula (I1))

[0093] Compound 1 was synthesized according to the following route:

[0094] .

[0095] 1. Synthesis of Compound 1-1

[0096] At 25°C, N,N'-dicyclohexylcarboimide (DCC) (10.00 g, 48.40 mmol) was added to 50 mL of dichloromethane solution containing 6-bromohexanoic acid (7.80 g, 40.00 mmol), and the resulting reaction solution was stirred at room temperature for 10 min. Subsequently, 4-penten-1-ol (6.80 g, 36.0 mmol) and 4-dimethylaminopyridine (DMAP) (200 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 a colorless oily product, compound 1-1 (10.20 g, yield 73.80%).

[0097] Compound 1-1 was analyzed by 1H NMR spectroscopy. 1 HNMR (400MHz, CDCl3) δ5.81 (ddt, J=16.9, 10.2, 6.6Hz, 1H), 5.15–4.95 (m, 2H), 4.09 (t, J=6.6Hz, 2H), 3.41 (t, J=6.8Hz, 2H ), 2.33 (t, J=7.4Hz, 2H), 2.13 (qd, J=6.4, 3.1Hz, 2H), 1.88 (dt, J=14.5, 6.8Hz, 2H), 1.78–1.59 (m, 4H), 1.54–1.40 (m, 2H).

[0098] 2. Synthesis of compounds 1-2

[0099] At 25 °C, compound 1-1 (3.10 g, 11.70 mmol) was 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 1-2 (2.90 g, yield 93.20%).

[0100] Compounds 1-2 were analyzed by 1H NMR spectroscopy. 1HNMR (400MHz, CDCl3) δ4.22–3.99 (m, 2H), 3.41 (t, J=6.7Hz, 2H), 2.63–2.40 (m, 2H), 2.33 (td, J=7.5, 2.0Hz, 2H), 2.02–1.81 (m, 4H), 1.74–1.39 (m, 4H).

[0101] 3. Synthesis of compounds 1-3

[0102] At 25 °C, compounds 1-2 (2.90 g, 10.30 mmol), 8-pentadecanol (2.20 g, 9.80 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (1.60 g, 10.00 mmol), and 4-dimethylaminopyridine (DMAP) (200 mg) were sequentially added to a round-bottom flask containing 40 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 (2.30 g, yield 52.30%).

[0103] Compounds 1-3 were analyzed by 1H NMR spectroscopy. 1 HNMR (400MHz, CDCl3) δ4.88 (p, J=6.3Hz, 1H), 4.11 (t, J=6.4Hz, 2H), 3.41 (t, J=6.8Hz, 2H), 2.35 (dt, J=2 0.1, 7.4Hz, 4H), 2.09–1.84 (m, 4H), 1.66 (dt, J=20.5, 7.4Hz, 2H), 1.58–1.14 (m, 26H), 1.00–0.75 (m, 6H).

[0104] 4. Synthesis of Compound 1

[0105] Compounds 1-3 (0.19 g, 0.44 mmol), ethanolamine (0.012 g, 0.20 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 41.20%), a pale yellow oil.

[0106] Compound 1 was analyzed by ESI-MS [M+H] + 882.74 and 1 HNMR confirmed. 1 HNMR (400MHz, CDCl3) δ4.91–4.84 (m, 2H), 4.10 (t, J=6.4Hz, 4H), 3.57 (s, 2H), 2.40 (ddd, J=52.9, 27.5, 26.5Hz, 14H), 1.97 (dd, J=14.3, 6.8Hz, 4H), 1.64 (dt, J=15.2, 7.6Hz, 10H), 1.52–1.24 (m, 50H), 0.88 (t, J=6.8Hz, 12H). 1 HNMR results show that Figure 1 middle.

[0107] Example 2: Synthesis of compound 2 (i.e., formula (I2))

[0108] Compound 2 was synthesized according to the following route:

[0109] .

[0110] 1. Synthesis of Compound 2-1

[0111] At 25°C, N,N'-dicyclohexylcarboimide (DCC) (2.50 g, 12.10 mmol) was added to 50 mL of dichloromethane solution containing compound 1-2 (2.80 g, 10.00 mmol), and the resulting reaction solution was stirred at room temperature for 10 min. Subsequently, 1-butyloctanol (1.7 g, 9.0 mmol) and 4-dimethylaminopyridine (DMAP) (50 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 71.20%).

[0112] Compound 2-1 was analyzed by 1H NMR spectroscopy. 1HNMR (400MHz, CDCl3) δ4.11 (t, J=6.4Hz, 2H), 3.98 (t, J=4.9Hz, 2H), 3.41 (t, J=6.7Hz, 2H), 2.36 (dt, J=2 9.6, 7.4Hz, 4H), 2.04–1.82 (m, 5H), 1.66 (dt, J=20.4, 7.4Hz, 2H), 1.55–1.18 (m, 18H), 1.00–0.83 (m, 6H).

[0113] 2. Synthesis of Compound 2

[0114] In a 50 mL round-bottom flask, compound 2-1 (0.24 g, 0.44 mmol), ethanolamine (0.012 g, 0.20 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.2 mmol) were added sequentially. 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 2 (0.076 g, yield 31.70%).

[0115] Compound 2 was analyzed by ESI-MS [M+H]. + 798.65 and 1 HNMR confirmed. 1 HNMR (400MHz, CDCl3) δ4.11 (t, J=6.4Hz, 4H), 3.98 (d, J=5.8Hz, 4H), 3.58 (s, 2H), 2.69–2.50 (m, 6H), 2.35 (dt, J=35. 0, 7.1Hz, 8H), 2.03–1.94 (m, 4H), 1.67–1.60 (m, 6H), 1.49 (d, J=7.0Hz, 4H), 1.37–1.22 (m, 36H), 0.94–0.85 (m, 12H). 1 HNMR results show that Figure 2 middle.

[0116] Example 3: Synthesis of compound 3 (i.e., formula (I3))

[0117] Compound 3 was synthesized according to the following route:

[0118] .

[0119] 1. Synthesis of compound 3-1

[0120] 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 3-1 (6.47 g, yield 57.90%).

[0121] Compound 3-1 was analyzed by 1H NMR spectroscopy. 1 HNMR (400MHz, CDCl3) δ5.78 (ddt, J=16.9, 10.2, 6.7Hz, 1H), 5.14–4.81 (m, 3H), 3.41 (t, J=6.8Hz, 2H), 2. 33 (dt, J=12.1, 7.5Hz, 2H), 2.10–1.98 (m, 2H), 1.94–1.81 (m, 2H), 1.74–1.15 (m, 16H), 0.95–0.83 (m, 3H).

[0122] 2. Synthesis of compound 3-2

[0123] At 25 °C, compound 3-1 (4.00 g, 11.7 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 directly give the oily product 3-2 (3.60 g, yield 90.00%).

[0124] Compound 3-2 was analyzed by 1H NMR spectroscopy. 1 HNMR (400MHz, CDCl3) δ4.90 (dd, J=12.0, 6.1Hz, 1H), 3.41 (t, J=6.7Hz, 2H), 2. 50–2.26 (m, 4H), 1.96–1.83 (m, 2H), 1.76–1.13 (m, 16H), 0.89 (t, J=6.9Hz, 3H).

[0125] 3. Synthesis of compound 3-3

[0126] At 25°C, N,N'-dicyclohexylcarboimide (DCC) (2.50 g, 12.10 mmol) was added to 50 mL of dichloromethane solution containing compound 3-2 (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) (50 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 3-3 (4.30 g, yield 81.10%).

[0127] Compound 3-3 was analyzed by 1H NMR spectroscopy. 1 HNMR (400MHz, CDCl3) δ5.77 (dt, J=6.3, 5.7Hz, 1H), 5.59 (dd, J=13.7, 7.3Hz, 1H), 5 .34 (ddq, J=10.5, 7.8, 6.8Hz, 2H), 4.90 (ddd, J=12.5, 5.8, 4.0Hz, 1H), 4.52 (d, J=6 .4Hz, 2H), 3.42 (ddd, J=8.7, 4.9, 1.5Hz, 2H), 2.37–2.28 (m, 4H), 2.16–1.77 (m, 10H ), 1.70–1.63 (m, 2H), 1.53–1.43 (m, 4H), 1.27 (d, J=3.1Hz, 8H), 1.00–0.88 (m, 6H).

[0128] 4. Synthesis of Compound 3

[0129] In a 50 mL round-bottom flask, compound 3-3 (0.19 g, 0.44 mmol), ethanolamine (0.012 g, 0.2 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.20 mmol) were added sequentially. 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 a pale yellow oily compound 3 (0.079 g, 0.07 mmol, yield 41.50%).

[0130] Compound 3 was analyzed by ESI-MS [M+H]. + 874.68 and 1 HNMR confirmed. 1 HNMR (400MHz, CDCl3) δ5.78 (dd, J=15.3, 5.4Hz, 2H), 5.62–5.52 (m, 2H), 5.36 (ddd, J=2 8.5, 10.8, 5.9Hz, 4H), 4.87 (d, J=12.1Hz, 2H), 4.53 (t, J=10.4Hz, 4H), 3.56 (s, 2H), 2.5 6 (d, J=49.0Hz, 6H), 2.40–2.21 (m, 8H), 2.09–1.99 (m, 5H), 1.95–1.84 (m, 5H), 1.64 (dd, J=15.3, 7.4Hz, 4H), 1.48 (d, J=6.0Hz, 4H), 1.29 (t, J=9.5Hz, 30H), 0.94–0.86 (m, 12H). 1 HNMR results show that Figure 3 middle.

[0131] Example 4: Synthesis of compound 4 (i.e., formula (I4))

[0132] Compound 4 was synthesized according to the following route:

[0133] .

[0134] 1. Synthesis of compound 4-1

[0135] At 25 °C, N,N'-dicyclohexylcarboimide (DCC) (2.50 g, 12.10 mmol) was added to 50 mL of dichloromethane solution containing compound 3-2 (3.60 g, 10.0 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) (50 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 4-1 (3.20 g, yield 76.20%).

[0136] Compound 4-1 was analyzed by 1H NMR spectroscopy. 1HNMR (400MHz, CDCl3) δ4.90 (ddd, J=12.5, 5.8, 4.0Hz, 1H), 4.63 (ddd, J=6.6Hz, 2H), 3.42 (ddd, J=8.7, 4.9, 1.5Hz, 2H), 2 .39–2.23 (m, 4H), 2.14–1.75 (m, 10H), 1.720–1.61 (m, 2H), 1.53–1.43 (m, 4H), 1.28–1.12 (m, 10H), 1.00–0.86 (m, 6H).

[0137] 2. Synthesis of Compound 4

[0138] In a 50 mL round-bottom flask, compound 4-1 (0.19 g, 0.44 mmol), ethanolamine (0.012 g, 0.2 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.20 mmol) were added sequentially. 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 4 (0.063 g, 0.07 mmol, yield 33.20%).

[0139] Compound 4 was analyzed by ESI-MS [M+H]. + 846.64 and 1 HNMR confirmed. 1 HNMR (400MHz, CDCl3) δ4.89 (t, J=12.4Hz, 2H), 4.77–4.58 (m, 4H), 3.56 (dd, J=12.4, 7.2Hz, 2H), 2.71–2.17 (m, 18H), 1.92–1.80 (m, 5H), 1.64 (dd, J=15.3, 7.6Hz, 4H), 1.51 (dt, J=14.3, 7.2Hz, 10H), 1.31 (ddd, J=22.7, 11.9, 6.5Hz, 29H), 0.94–0.86 (m, 12H). 1 HNMR results show that Figure 4 middle.

[0140] Example 5: Synthesis of compound 5 (i.e., formula (I5))

[0141] Compound 5 was synthesized according to the following route:

[0142] .

[0143] 1. Synthesis of Compound 5-1

[0144] At 25°C, N,N'-dicyclohexylcarboimide (DCC) (2.50 g, 12.10 mmol) was added to 50 mL of dichloromethane solution containing compound 3-2 (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) (50 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 (3.30 g, yield 76.80%).

[0145] Compound 5-1 was analyzed by 1H NMR spectroscopy. 1 HNMR (400MHz, CDCl3) δ4.90 (ddd, J=12.5, 5.8, 4.0Hz, 1H), 3.42 (ddd, J=8.7, 4.9, 1.5Hz, 2H), 2.35–2.21 ( m, 4H), 2.16–1.78 (m, 12H), 1.74–1.62 (m, 2H), 1.54–1.41 (m, 4H), 1.26–1.11 (m, 14H), 1.00–0.86 (m, 6H).

[0146] 2. Synthesis of Compound 5

[0147] In a 50 mL round-bottom flask, compound 5-1 (0.19 g, 0.44 mmol), ethanolamine (0.012 g, 0.20 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.20 mmol) were added sequentially. 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 5 (0.058 g, 0.07 mmol, yield 30.5%).

[0148] Compound 5 was analyzed by ESI-MS [M+H]. + 854.72 and 1 HNMR confirmed. 1HNMR (400MHz, CDCl3) δ4.89 (t, J=12.4Hz, 2H), 4.08 (dt, J=13.6, 6.8Hz, 4H), 3.57 (t, J=5.0Hz, 2H), 2.54 (dd, J=30.8, 23.7Hz, 6H), 2.40–2.26 (m, 8H), 1.88–1.79 (m, 6H), 1.63 (dt, J=14.9, 7.6Hz, 9H), 1.46 (s, 4H), 1.36–1.25 (m, 41H), 0.88 (td, J=6.6, 2.7Hz, 12H). 1 HNMR results show that Figure 5 middle.

[0149] Example 6: Synthesis of compound 6 (i.e., formula (I6))

[0150] Compound 6 was synthesized according to the following route:

[0151] .

[0152] 1. Synthesis of Compound 6-1

[0153] 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) (6.2 g, 40.0 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 6-1 (5.88 g, yield 61.50%).

[0154] Compound 6-1 was analyzed by 1H NMR spectroscopy. 1 HNMR (400MHz, CDCl3) δ5.78 (ddt, J=16.9, 10.2, 6.7Hz, 1H), 5.10–4.75 (m, 3H), 3.41 (t, J=6.8Hz, 2H), 2. 35 (dt, J=14.8, 7.1Hz, 2H), 2.14–1.98 (m, 2H), 1.94–1.81 (m, 2H), 1.76–1.35 (m, 8H), 0.93–0.84 (m, 3H).

[0155] 2. Synthesis of Compound 6-2

[0156] At 25 °C, compound 6-1 (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 directly give the oily product 6-2 (3.00 g, yield 88.20%).

[0157] Compound 6-2 was analyzed by 1H NMR spectroscopy. 1 HNMR (400MHz, CDCl3) δ4.87 (p, J=6.3Hz, 1H), 3.39 (t, J=6.8Hz, 2H), 2.48–2.24 (m, 4 H), 1.94–1.80 (m, 2H), 1.69–1.58 (m, 2H), 1.57–1.09 (m, 6H), 0.88 (t, J=6.9Hz, 3H).

[0158] 3. Synthesis of compound 6-3

[0159] At 25°C, N,N'-dicyclohexylcarboimide (DCC) (2.50 g, 12.10 mmol) was added to 50 mL of dichloromethane solution containing compound 6-2 (3.10 g, 10.00 mmol), and the resulting reaction solution was stirred at room temperature for 10 min. Subsequently, 1-butyloctanol (1.70 g, 9.00 mmol) and 4-dimethylaminopyridine (DMAP) (50 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 6-3 (3.90 g, yield 72.20%).

[0160] Compound 6-3 was analyzed by 1H NMR spectroscopy. 1 HNMR (400MHz, CDCl3) δ4.95–4.79 (m, 1H), 4.06–3.90 (m, 2H), 3.47–3.34 (m, 2H), 2.46 –2.26 (m, 4H), 2.02–1.77 (m, 4H), 1.73–1.44 (m, 9H), 1.28 (s, 14H), 1.03–0.80 (m, 9H).

[0161] 4. Synthesis of Compound 6

[0162] In a 50 mL round-bottom flask, compound 6-3 (0.20 g, 0.44 mmol), ethanolamine (0.012 g, 0.20 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.20 mmol) were added sequentially. 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 a pale yellow oily compound 6 (0.078 g, 0.07 mmol, yield 36.1%).

[0163] Compound 6 was analyzed by ESI-MS [M+H]. + 854.71 and 1 HNMR confirmed. 1 HNMR (400MHz, CDCl3) δ4.91–4.79 (m, 2H), 3.96 (dd, J=11.1, 5.9Hz, 4H), 3.54 (t, J=5.3Hz, 2H), 2.59 (t, J=5.2Hz, 2H), 2.52–2.42 (m, 4H), 2 .38–2.24 (m, 8H), 1.93–1.82 (m, 4H), 1.63–1.54 (m, 8H), 1.47 (dt, J=15.1, 7.7Hz, 5H), 1.31 (dd, J=25.8, 5.0Hz, 37H), 0.97–0.78 (m, 18H). 1 HNMR results show that Figure 6 middle.

[0164] Example 7: Synthesis of compound 7 (i.e., formula (I7))

[0165] Compound 7 was synthesized according to the following route:

[0166] .

[0167] 1. Synthesis of Compound 7

[0168] In a 50 mL round-bottom flask, compound 3-3 (0.19 g, 0.44 mmol), 4-amino-1-butanol (0.18 g, 0.20 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.20 mmol) were added sequentially. 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 a pale yellow oily compound 7 (0.073 g, 0.07 mmol, yield 38.40%).

[0169] Compound 7 was analyzed by ESI-MS [M+H]. + 902.71 and 1 HNMR confirmed. 1 HNMR (400MHz, CDCl3) δ5.76 (dd, J=15.3, 5.4Hz, 2H), 5.60–5.54 (m, 2H), 5.38 (ddd, J=2 8.5, 10.8, 5.9Hz, 4H), 4.88 (d, J=12.1Hz, 2H), 4.54 (t, J=10.4Hz, 4H), 3.58 (s, 2H), 2.5 5 (d, J=49.0Hz, 6H), 2.42–2.21 (m, 8H), 2.10–1.98 (m, 5H), 1.94–1.82 (m, 9H), 1.62 (dd, J=15.3, 7.4Hz, 4H), 1.46 (d, J=6.0Hz, 4H), 1.28 (t, J=9.5Hz, 30H), 0.92–0.85 (m, 12H).

[0170] Example 8: Synthesis of compound 8 (i.e., formula (I8))

[0171] Compound 8 was synthesized according to the following route:

[0172] .

[0173] 1. Synthesis of Compound 8

[0174] Compound 4-1 (0.19 g, 0.44 mmol), 4-amino-1-butanol (0.18 g, 0.20 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.20 mmol) were placed in a 50 mL round-bottom flask, and 10 mL of acetonitrile was added. The mixture was stirred and refluxed at 85 °C for 4 hours. After the reaction was complete, the acetonitrile was removed by rotary evaporation, and the mixture was washed three times with dichloromethane and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography (ethyl acetate / methanol = 12 / 1) to give a pale yellow oily lipid compound 8 (0.068 g, yield 35.70%).

[0175] Compound 8 was analyzed by ESI-MS [M+H]. + 874.68 and 1 HNMR confirmed. 1 HNMR (400MHz, CDCl3) δ4.88 (t, J=12.4Hz, 2H), 4.76–4.57 (m, 4H), 3.55 (dd, J=12.4, 7.2Hz, 2H), 2.70–2.16 (m, 18H), 1.91–1.80 (m, 5H), 1.66 (dd, J=15.3, 7.6Hz, 6H), 1.52 (dt, J=14.3, 7.2Hz, 12H), 1.31 (ddd, J=22.7, 11.9, 6.5Hz, 29H), 0.92–0.82 (m, 12H).

[0176] Example 9: Synthesis of compound 9 (i.e., formula (I9))

[0177] Compound 9 was synthesized according to the following route:

[0178] .

[0179] 1. Synthesis of Compound 9

[0180] Compound 5-1 (0.19 g, 0.44 mmol), 4-amino-1-butanol (0.18 g, 0.20 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.20 mmol) were placed in a 50 mL round-bottom flask, and 10 mL of acetonitrile was added. The mixture was stirred and refluxed at 85 °C for 4 hours. After the reaction was complete, the acetonitrile was removed by rotary evaporation, and the mixture was washed three times with dichloromethane and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography (ethyl acetate / methanol = 20 / 1) to give a pale yellow oily lipid compound 9 (0.074 g, yield 38.90%).

[0181] Compound 9 was analyzed by ESI-MS [M+H]. + : 882.73 and 1 HNMR confirmed. 1 HNMR (400MHz, CDCl3) δ4.87 (t, J=12.4Hz, 2H), 4.06 (dt, J=13.6, 6.8Hz, 4H), 3.58 (t, J=5.0Hz, 2H), 2.52 (dd, J=30.8, 23.7Hz, 6H), 2 .38–2.24 (m, 8H), 1.86–1.76 (m, 6H), 1.64 (dt, J=14.9, 7.6Hz, 11H), 1.44 (s, 4H), 1.34–1.24 (m, 43H), 0.89 (td, J=6.6, 2.7Hz, 12H).

[0182] Example 10: Compound 10 (i.e., formula (I) 10 Synthesis of ))

[0183] Compound 10 was synthesized according to the following route:

[0184] .

[0185] 1. Synthesis of Compound 10

[0186] Compound 6-3 (0.20 g, 0.44 mmol), 4-amino-1-butanol (0.18 g, 0.20 mmol), K₂CO₃ (0.28 g, 2.00 mmol), and KI (0.03 g, 0.20 mmol) were placed in a 50 mL round-bottom flask, and 10 mL of acetonitrile was added. The mixture was stirred and refluxed at 85 °C for 4 hours. After the reaction was complete, the acetonitrile was removed by rotary evaporation, and the mixture was washed three times with dichloromethane and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography (ethyl acetate / methanol = 20 / 1) to give a pale yellow oily lipid compound 10 (0.072 g, yield 36.20%).

[0187] Compound 10 was analyzed by ESI-MS [M+H]. + 882.74 and 1 HNMR confirmed. 1HNMR (400MHz, CDCl3) δ4.89–4.80 (m, 2H), 3.95 (dd, J=11.1, 5.9Hz, 4H), 3.52 (t, J=5.3Hz, 2H), 2.60 (t, J=5.2Hz, 2H), 2.54–2.41 (m, 4H), 2 .36–2.22 (m, 8H), 1.95–1.83 (m, 4H), 1.65–1.53 (m, 10H), 1.45 (dt, J=15.1, 7.7Hz, 5H), 1.33 (dd, J=25.8, 5.0Hz, 39H), 0.96–0.75 (m, 18H).

[0188] Example 11: Preparation and Physicochemical Characterization of Lipid Nanoparticles

[0189] 1. Preparation of lipid nanoparticles

[0190] Compounds 1-10 prepared in Examples 1-10 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 centrifugal filter. The lipid nanoparticles prepared from compounds 1-10 and ALC-0135 were named LNP-1, LNP-2, LNP-3, LNP-4, LNP-5, LNP-6, LNP-7, LNP-8, LNP-9, LNP-10, and LNP-0315, respectively. The molecular structures of the lipid compounds are shown below. Figure 7 As shown.

[0191] 2. Determination of lipid nanoparticle size, polydispersity index and encapsulation efficiency

[0192] The particle size and polydispersity index (PDI) of the lipid nanoparticles LNP-1~LNP-10 and LNP-0315 obtained above were determined using a Malvern particle size analyzer (Malvern UK). The results are shown in Table 1.

[0193] 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-10 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), with excitation at 485 nm and emission at 528 nm.

[0194] To determine the total mRNA content in lipid nanoparticles, 10 μL of each of the LNP-1~LNP-10 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:

[0195] EE (%) = (Total mRNA concentration - Free mRNA concentration) / Total mRNA concentration × 100%.

[0196] The results are shown in Table 1.

[0197] Table 1 Physicochemical properties of LNP-1~LNP-10 and LNP-0315 lipid nanoparticles

[0198]

[0199] As shown in Table 1, the LNP-1 to LNP-10 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 130 nm, and their PDI values ​​are all less than 0.2. This indicates that all 11 types of lipid nanoparticles possess excellent physicochemical properties. Furthermore, the LNP-1 to LNP-10 lipid nanoparticles prepared in this invention and the LNP-0315 lipid nanoparticles prepared from commercially available ALC-0315 all exhibit high mRNA encapsulation efficiency.

[0200] Example 12: Cytotoxicity Experiment

[0201] 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-10 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 a CCK-8 assay kit. 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.

[0202] Table 2. In vitro toxicity data of LNP-1~LNP-10 and LNP-0315 lipid nanoparticles

[0203]

[0204] As shown in Table 2, the 11 lipid nanoparticles mentioned above all exhibit low cytotoxicity and have promising prospects for in vivo applications.

[0205] Example 13: Bioluminescence detection in vivo

[0206] To investigate the in vivo mRNA delivery of compounds 1-10 and commercially available ALC-0315, 33 female BALB / c mice weighing 16-18g were randomly divided into 11 groups of 3 mice each. Each group of mice was intramuscularly injected with 100μL of a solution containing LNP-1-LNP-10 and LNP-0315 lipid nanoparticles prepared in Example 11, along with 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-6 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 8 As shown, the liver bioluminescence effect of LNP-6 is significantly higher than that of LNP-0315. Furthermore, the average bioluminescence intensity of LNP-1~LNP-10 and LNP-0315 in the liver is shown in Table 3.

[0207] Table 3. In vivo bioluminescence data of LNP-1~LNP-10 and LNP-0315 lipid nanoparticles.

[0208]

[0209] As shown in Table 3, the lipid nanoparticles LNP-1~LNP-10 and LNP-0315 can effectively deliver mRNA in vivo and complete expression. The lipid nanoparticles prepared by compounds 1 and 6~10 in the embodiments of the present invention have better in vivo delivery effects than the lipid nanoparticles prepared by the commercially available ALC-0315 control group. The nucleic acid delivery efficiency of the lipid compounds in Examples 6 and 10 in the liver is 2.2 times and 2.5 times higher than that of the commercially available ALC-0315 control group, respectively, showing good application prospects.

[0210] 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 C1~C 10 Hydroxyalkyl; X is C1~C 20 Straight-chain or branched alkyl groups; Y is selected from any of the following structures: In this context, the dashed lines represent connection points.

2. 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: In this context, the dashed lines represent connection points.

3. The lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, X is selected from any of the following structures: In this context, 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 the following formula (I1) to formula (I2). 10 Any one of the structures shown: Equation (I1); Equation (I2); Equation (I3); Equation (I4); Equation (I6); Equation (I7); Equation (I8); Equation (I 10 ).

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 100~130nm 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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