A cationic lipid compound, its preparation method, composition and application

Abstract

The present invention discloses a cationic lipid compound, a preparation method thereof, a composition and an application. When the cationic lipid compound is used for preparing LNP, it has a good particle size (<200 nm) and a uniform particle distribution (PDI<0.25), can achieve excellent nucleic acid delivery effects both in vitro and in vivo, and at the same time exhibits low toxicity, having significant application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a cationic lipid compound, a preparation method thereof, and an application thereof. Background Art

[0002] The development of nucleic acid drugs has brought new hopes for the treatment of various diseases, such as genetic diseases, cancers, and some refractory infectious diseases. However, the effective delivery of nucleic acid drugs in vivo has always been a key problem hindering their wide application. Lipid nanoparticles (LNPs), as important carriers for delivering nucleic acids, have received great attention in recent years. Among the components of LNPs, cationic lipids play an irreplaceable core role. It forms an electrostatic complex with negatively charged nucleic acids to achieve the encapsulation and protection of nucleic acids, and at the same time helps LNPs escape from endosomes and release nucleic acids into the cytoplasm to play a role.

[0003] Existing cationic lipids have many limitations. From a structural perspective, most cationic lipid structures are relatively simple and difficult to precisely meet complex delivery requirements. For example, the structure of its polar head is single, resulting in poor binding ability with nucleic acids and stability in different physiological environments, and it cannot ensure the efficient and stable transportation of nucleic acids in vivo. In the design of the hydrophobic tail, there is a lack of fine regulation of lipid fluidity and biomembrane interaction, which affects the fusion efficiency of LNPs with target cells and the release rate of nucleic acids.

[0004] In terms of delivery performance, the nucleic acid-liposome complexes prepared from existing cationic lipids do not perform ideally. In in vitro experiments, their transfection efficiency often fails to reach the expected level, resulting in a large amount of nucleic acids being unable to effectively enter cells to play a role, causing waste of drugs and reduction of therapeutic effects. In in vivo experiments, these cationic lipids face more severe challenges. For example, during blood circulation, they are easily recognized and cleared by the immune system, making it difficult to achieve effective extrahepatic delivery, which limits their application in the treatment of systemic diseases. At the same time, when increasing the dosage of cationic lipids to improve the delivery efficiency, it will inevitably bring higher cytotoxicity and have a negative impact on the physiological functions of normal cells, severely restricting the clinical application of nucleic acid drugs.

[0005] In addition, safety issues and the difficulty of synthesis processes further limit the wide clinical application of cationic lipids.

[0006] Therefore, there is still an unmet need for cationic lipid compounds with novel structures that can be used to achieve higher delivery effects. Summary of the Invention

[0007] In order to overcome the problems that the cationic lipid structure for delivering nucleic acids in the prior art is single and the delivery effect of the prepared nucleic acid-liposome complex is poor, the present invention provides a cationic lipid compound, thereby achieving efficient nucleic acid delivery. In addition, the cationic lipid compound provided by the present invention can also achieve efficient delivery of mRNA and expression of target proteins.

[0008] In one aspect, the present invention provides a cationic lipid compound, its stereoisomer or its pharmaceutically acceptable salt having the structure shown in formula (I), wherein,

[0009]

[0010] R1 is H or OH.

[0011] In another aspect, the present invention provides a method for preparing the cationic lipid compound as described in any one of the embodiments herein, the method comprising the step of reacting a compound shown in formula (II) as a reactant, wherein,

[0012]

[0013] R2 is H or OH.

[0014] In one or more embodiments, the method comprises the step of reacting (13R,15Z)-13-hexyl-11-oxo-10,12-dioxacyclopent-15-en-7-yn-24-yl prop-2-enoate with the compound shown in formula (II); wherein, R2 is selected from H or OH.

[0015] Preferably, the method comprises the following steps:

[0016] S1: Synthesis of (10Z,12R)-octadec-9-ene-1,12-diol;

[0017] S2: Synthesis of (10Z,12R)-12-hydroxyoctadec-9-en-1-yl prop-2-enoate;

[0018] S3: Synthesis of 4-nitrophenyl {[(10z)-18-[(1-oxo-2-enyl)oxy]octadec-9-en-7-yl]oxy} formate;

[0019] S4: Synthesis of (13R,15Z)-13-hexyl-11-oxo-10,12-dioxacyclopent-15-en-7-yn-24-yl prop-2-enoate;

[0020] S5: Synthesis of the cationic lipid compound.

[0021] More preferably, in step S1, ricinoleic acid is dissolved in anhydrous tetrahydrofuran, and lithium aluminum hydride is added portionwise at 0 °C under nitrogen protection. After the addition is complete, the temperature is raised to room temperature and the mixture is reacted for 2.5 to 3.5 hours. After quenching the reaction solution by adding sodium sulfate decahydrate portionwise at 0 °C, the liquid is filtered, and the filtrate is concentrated and purified to obtain (10Z,12R)-octadec-9-ene-1,12-diol.

[0022] More preferably, in step S2, (10Z,12R)-octadec-9-ene-1,12-diol obtained in step S1 and N,N-diisopropylethylamine are dispersed in dichloromethane, and acryloyl chloride is added dropwise at 0 °C under nitrogen protection. After the addition is complete, the mixture is reacted at room temperature for 2.5 to 3.5 hours. The reaction solution is quenched by adding water, and extracted with dichloromethane. The organic layer is washed with saturated brine, dried over anhydrous sodium sulfate, the obtained liquid is filtered, and the filtrate is concentrated and purified to obtain (10Z,12R)-12-hydroxyoctadec-9-en-1-yl prop-2-enoate.

[0023] More preferably, in step S3, (10Z,12R)-12-hydroxyoctadec-9-en-1-yl prop-2-enoate obtained in step S2 is dissolved in dichloromethane, and 4-nitrophenyl chloroformate is added at 0 °C under nitrogen protection. After the addition is complete, piperazine is slowly added dropwise. After the addition is complete, the mixture is kept at a certain temperature and reacted for 1.5 to 2.5 hours. The reaction solution is quenched by adding water, and extracted with dichloromethane. The organic layer is washed with saturated brine, dried over anhydrous sodium sulfate, the obtained liquid is filtered, and the filtrate is concentrated and purified to obtain 4-nitrophenyl {[(10Z)-18-[(1-oxo-2-enyl)oxy]octadec-9-en-7-yl]oxy} formate.

[0024] More preferably, in step S4, 4-nitrophenyl {[(10Z)-18-[(1-oxo-2-enyl)oxy]octadec-9-en-7-yl]oxy} formate obtained in step S3 and non-2-yn-1-ol are dissolved in dichloromethane, and N,N-diisopropylethylamine and 4-dimethylaminopiperazine are added at 0 °C under nitrogen protection. After the addition is complete, the mixture is kept at a certain temperature and reacted for 20 to 40 minutes, and then the temperature is raised to room temperature and reacted for 15 to 17 hours. The reaction solution is quenched by adding water, and extracted with dichloromethane. The organic layer is washed with saturated brine, dried over anhydrous sodium sulfate, the obtained liquid is filtered, and the filtrate is concentrated and purified to obtain (13R,15Z)-13-hexyl-11-oxo-10,12-dioxacyclopent-15-ene-7-yne-24-yl prop-2-enoate.

[0025] More preferably, in step S5, the (13R,15Z)-13-hexyl-11-oxo-10,12-dioxol-15-ene-7-yne-24-yl prop-2-enoate obtained in step S4, hydroxyethylpiperazine and methanol are dissolved in dichloromethane, and the reaction is carried out at 65-70 °C under nitrogen protection for 7-9 hours; the reaction solution is concentrated and purified to obtain (13R,15Z)-13-hexyl-11-oxo-10,12-dioxol-15-ene-7-yne-24-yl 3-[4-(2-hydroxyethyl)piperazin-1-yl]propionate.

[0026] More preferably, in step S5, the (13R,15Z)-13-hexyl-11-oxo-10,12-dioxol-15-ene-7-yne-24-yl prop-2-enoate obtained in step S4, 1-ethylpiperazine hydrochloride, N,N-diisopropylethylamine and methanol are dissolved in dichloroethane, and the reaction is carried out at 65-70 °C under nitrogen protection for 15-17 hours; the reaction solution is concentrated and purified to obtain 3-(4-ethylpiperazin-1-yl)propionic acid-(13R,15Z)-13-hexyl-11-oxo-10,12-dioxatetradec-15-ene-7-yne-24-yl ester.

[0027] Further preferably, the synthesis of the 1-ethylpiperazine hydrochloride comprises the following steps:

[0028] S1: Synthesis of 2-methylprop-2-yl 4-ethylpiperazine-1-carboxylate;

[0029] S2: Synthesis of 1-ethylpiperazine hydrochloride;

[0030] Among them, in step S1, N-BOC-piperazine, bromoethane and potassium carbonate are dispersed in N,N-dimethylformamide, and the reaction is carried out at room temperature under nitrogen protection for 15-17 hours. The reaction solution is concentrated and purified to obtain 2-methylprop-2-yl 4-ethylpiperazine-1-carboxylate;

[0031] In step S2, 2-methylprop-2-yl 4-ethylpiperazine-1-carboxylate is dispersed in a 1,4-dioxane solution of hydrogen chloride, and the reaction is carried out at room temperature under nitrogen protection for 15-17 hours. The reaction solution is filtered, the obtained filter cake is taken, washed with 1,4-dioxane, and the washed filter cake is dried to obtain 1-ethylpiperazine hydrochloride.

[0032] In another aspect, the present invention provides the use of the cationic lipid compound as described in any one of the embodiments herein as a surfactant.

[0033] Preferably, the cationic lipid compound is used as an emulsifier, suspending agent, dispersing agent, solubilizer, lubricant, thickening agent, bacteriostatic agent or preservative.

[0034] In another aspect, the present invention provides the use of a cationic lipid compound as described in any embodiment herein in the preparation of a lipid composition, and the lipid composition is used as a drug delivery carrier.

[0035] Preferably, the drug is a small molecule drug, nucleic acid or protein. More preferably, the drug is a nucleic acid, and the nucleic acid is DNA or RNA. Further preferably, the DNA is linear DNA or circular DNA. Further preferably, the circular DNA is a plasmid. Further preferably, the RNA is siRNA, ASO, mRNA, saRNA, dsRNA or shRNA. Further preferably, the RNA is siRNA or mRNA.

[0036] In another aspect, the present invention provides a lipid composition, the lipid component of the lipid composition includes a cationic lipid compound as described in any embodiment herein, the lipid component of the lipid composition further contains other lipids, and the other lipids include phospholipids, sterol compounds and PEG lipids; the molar ratio of the cationic lipid compound to the other lipid components is 30:70 to 60:40; the active ingredient of the lipid composition includes a therapeutic agent and / or a prophylactic agent; the mass ratio of the lipid component to the active ingredient is 7.5:1 to 30:1.

[0037] Preferably, the molar ratio of the ionizable cationic lipid compound to the other lipid components is 40:60 to 55:45. More preferably, the molar ratio of the ionizable cationic lipid compound to the other lipid components is 45:55 to 50:50.

[0038] Preferably, the molar ratio of the cationic lipid compound to phospholipids, sterol compounds and PEG lipids is 30% - 60%:5% - 40%:10% - 60%:0.1% - 15%. More preferably, the molar ratio of the cationic lipid compound to phospholipids, sterol compounds and PEG lipids is 30% - 60%:5% - 30%:20% - 60%:1% - 10%. Further preferably, the molar ratio of the cationic lipid compound to phospholipids, sterol compounds and PEG lipids is 40% - 55%:10% - 15%:30% - 50%:1% - 5%, such as 50:10:38.5:1.5, 50:10:35.5:4.5, 40:10:48.5:1.5, 45:10:43.5:1.5, 50:10:38.5:1.5, or 55:10:33.5:1.5.

[0039] Preferably, the mass ratio of the lipid component to the active ingredient is 7.5:1 to 30:1. More preferably, the mass ratio of the lipid component to the active ingredient is 10:1 to 20:1.

[0040] In one or more embodiments, the therapeutic and / or prophylactic agent is selected from one or more combinations of small molecule drugs, nucleic acids, and proteins.

[0041] Preferably, the therapeutic and / or prophylactic agent is a nucleic acid, and the nucleic acid is DNA or RNA. Further preferably, the DNA is linear DNA or circular DNA. Further preferably, the circular DNA is a plasmid. Further preferably, the RNA is siRNA, ASO, mRNA, saRNA, dsRNA, shRNA. Further preferably, the RNA is siRNA or mRNA.

[0042] In another aspect, the present invention provides the use of the lipid composition in the preparation of a drug for preventing or treating inflammation, infectious diseases, cancer, proliferative diseases, genetic diseases, autoimmune diseases, or metabolic diseases.

[0043] In another aspect, the present invention provides a pharmaceutical composition comprising the lipid composition as described in any one of the embodiments herein and a pharmaceutically acceptable carrier, solvent, or excipient.

[0044] The present invention provides a novel cationic lipid, which has at least one of the following advantages compared with the cationic lipids disclosed in the prior art:

[0045] 1. The chemical structure is different from all the cationic lipids disclosed in the prior art and is a completely new compound.

[0046] 2. The RNA-LNP composition prepared by adding the cationic lipid of the present invention (30 mol% - 60 mol%) has a good particle size (<200 nm) and a uniform particle distribution (PDI < 0.25).

[0047] 3. When used for preparing LNP, it can achieve excellent nucleic acid delivery effects both in vivo and in vitro, and at the same time exhibits low toxicity.

[0048] 4. A piperazine group is introduced into the polar head, which shows low toxicity while carrying a positive charge; in addition, different structural head R1 groups can be used to meet the delivery requirements of different biomolecules.

[0049] Therefore, the cationic lipid provided by the present invention has significant application value. Description of the Drawings

[0050] Figure 1It is the synthetic route diagram of Compound 1.

[0051] Figure 2 It is the nuclear magnetic resonance spectrum of Compound 1. Among them, 2A is the 1 1H NMR spectrum of Compound 1, and 2B is the 13 13C NMR spectrum of Compound 1.

[0052] Figure 3 It is the synthetic route diagram of Compound 2.

[0053] Figure 4 It is the nuclear magnetic resonance spectrum of Compound 2. Among them, 4A is the 1 1H NMR spectrum of Compound 1, and 4B is the 13 13C NMR spectrum of Compound 1.

[0054] Figure 5 It is the expression of GFP in cells after in vitro delivery of pGFP-LNP.

[0055] Figure 6 It is the relative expression level of TNFα and cell viability in cells after delivering siRNA-LNP containing Compound 1 or Compound 2 at different molar ratios. Among them, 6A is the relative expression level of TNFα and cell viability in cells after delivering siRNA-LNP containing Compound 1 at different molar ratios; 6B is the relative expression level of TNFα and cell viability in cells after delivering siRNA-LNP containing Compound 2 at different molar ratios. Detailed implementation manners

[0056] In the present invention, the preparation method of the citrate phosphate buffer is preferably as follows:

[0057] S1: Weigh 21.014 g of citric acid (C6H8O 7· ·H2O), dissolve it with distilled water and make up the volume to 1000 mL to obtain a 0.1 mol / L citric acid solution;

[0058] S2: Weigh 71.632 g of disodium hydrogen phosphate (Na2HPO4·12H2O), dissolve it with distilled water and make up the volume to 1000 mL to obtain a 0.2 mol / L disodium hydrogen phosphate solution;

[0059] S3: Measure the 0.1 mol / L citric acid solution and 0.2 mol / L disodium hydrogen phosphate solution according to the volume in Table 1 below, mix them and add distilled water to make up the volume to 200 mL to obtain the citrate-phosphate buffer with the corresponding pH value.

[0060] Table 1: Formulas of citrate-phosphate buffers with different pH values

[0061]

[0062] Example 1: Synthesis of Compound 1

[0063] As Figure 1 shown, the synthesis route of Compound 1 includes 5 steps (step 1 to step 5), which are specifically as follows:

[0064] S1 (step 1): Synthesis of (10Z,12R)-octadec-9-ene-1,12-diol (Compound 1-1)

[0065] Dissolve ricinoleic acid (5.00 g, 16.75 mmol) in anhydrous tetrahydrofuran (250 mL). Under nitrogen protection at 0 °C, add lithium aluminum hydride (LAH, 826 mg, 21.78 mmol) in batches. After addition, raise the temperature to room temperature and react for 3 hours. According to TLC (petroleum ether / ethyl acetate = 1 / 1, v / v, R f = 0.3) indicating that the raw material reaction is complete, add sodium sulfate decahydrate (30.00 g) to the reaction solution in batches at 0 °C for quenching, filter the liquid, and concentrate the filtrate. The crude product is purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1, v / v) to obtain a colorless oily compound (Compound 1-1, 3.56 g, yield 74.7%).

[0066] According to mass spectrometry determination, the main ion peak characteristics of Compound 1-1 are: MS m / z (ESI, electrospray ionization): 267.2 [M - H2O + H] + .

[0067] S2 (step 2): Synthesis of (10Z,12R)-12-hydroxyoctadec-9-en-1-yl 2-propenoate (Compound 1-2)

[0068] Disperse Compound 1-1 (3.56 g, 12.51 mmol) and N,N-diisopropylethylamine (4.00 g, 31.28 mmol) in dichloromethane (30 mL). Under nitrogen protection at 0 °C, dropwise add acryloyl chloride (1.13 g, 12.51 mmol). After addition, react at room temperature for 3 hours. According to TLC (petroleum ether / ethyl acetate = 2 / 1, v / v, R f(= 0.7) After indicating that the raw material reaction was completed, water (50 mL) was added to the reaction solution to quench it, and it was extracted with dichloromethane (50 mL × 2); the organic layer was washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, the obtained liquid was filtered, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 7 / 1, v / v) to obtain a colorless oily compound (Compound 1-2, 2.69 g, yield 60.7%).

[0069] S3 (step 3): Synthesis of 4-nitrophenyl {[(10z)-18-[(1-oxo-2-enyl)oxy]octadec-9-en-7-yl]oxy} formate (Compound 1-3)

[0070] Compound 1-2 (2.69 g, 7.59 mmol) was dissolved in dichloromethane (50 mL). Under nitrogen protection at 0 °C, 4-nitrophenyl chloroformate (3.20 g, 15.94 mmol) was added. After addition, piperazine (3.00 g, 37.95 mmol) was slowly added dropwise. After the addition was complete, the reaction was kept warm for 2 hours. According to TLC (petroleum ether / ethyl acetate = 5 / 1, v / v, R f (= 0.7) After indicating that the raw material reaction was completed, water (50 mL) was added to the reaction solution to quench it, and it was extracted with dichloromethane (50 mL × 2); the organic layer was washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, the obtained liquid was filtered, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 8 / 1, v / v) to obtain a colorless oily compound (Compound 1-3, 3.92 g, crude product), which was directly used for the next step.

[0071] S4 (step 4): Synthesis of (13R,15Z)-13-hexyl-11-oxo-10,12-dioxacyclopentene-15-ene-7-yne-24-yl prop-2-enoate (Compound 1-4)

[0072] The crude Compound 1-3 (3.92 g, crude product) obtained in S3 and non-2-yn-1-ol (1.60 g, 11.39 mmol) were dissolved in dichloromethane (50 mL). Under nitrogen protection at 0 °C, N,N-diisopropylethylamine (2.45 g, 18.98 mmol) and 4-dimethylaminopiperazine (927 mg, 7.59 mmol) were added. After addition, the reaction was kept warm for 30 minutes and then raised to room temperature and reacted for 16 hours. According to TLC (petroleum ether / ethyl acetate = 5 / 1, v / v, R fAfter the reaction of the raw materials was completed as indicated by = 0.6), water (50 mL) was added to the reaction solution to quench it, and it was extracted with dichloromethane (50 mL×2); the organic layer was washed with saturated brine (50 mL×2), dried over anhydrous sodium sulfate, and then the obtained liquid was filtered, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 12 / 1, v / v) to obtain a colorless oily compound (Compound 1-4, 2.53 g, two-step yield 66.0%).

[0073] S5 (step 5): Synthesis of (13R,15Z)-13-hexyl-11-oxo-10,12-dioxacyclopentadec-15-en-7-yn-24-yl 3-[4-(2-hydroxyethyl)piperazin-1-yl]propionate (Compound 1)

[0074] Compound 1-4 (600 mg, 1.19 mmol), 2-(piperazin-1-yl)ethanol (165 mg, 1.25 mmol) and methanol (10 mL) were dissolved in dichloromethane (10 mL), and the reaction was carried out at 68 °C under nitrogen protection for 8 hours. According to TLC (petroleum ether / ethyl acetate = 2 / 1, v / v, R f = 0.4, 0.5% NH3) indicating that the reaction of the raw materials was completed, the reaction solution was concentrated. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1, v / v) to obtain a colorless oily compound (Compound 1, 640 mg, 1.01 mmol, yield 84.9%).

[0075] According to mass spectrometry determination, the main ion peak characteristics of Compound 1 were: MS m / z (ESI): 635.5 [M+H] + , and the molecular formula of Compound 1 was C 35 H 62 N2O6, with a molecular weight of 606.9.

[0076] The nuclear magnetic resonance spectrum of Compound 1 was as Figure 2 shown, and its 1 1H NMR characteristics were: 11H NMR (400 MHz, DMSO-d6) δ 5.53 - 5.40 (m, 1H), 5.37 - 5.23 (m, 1H), 4.73 - 4.66 (m, 2H), 4.66 - 4.58 (m, 1H), 4.33 (t, J = 8.0 Hz, 1H), 3.99 (t, J = 8 Hz, 2H), 3.51 - 3.41 (m, 2H), 2.55 - 2.50 (m, 2H), 2.47 - 2.13 (m, 16H), 2.05 - 1.92 (m, 2H), 1.62 - 1.48 (m, 4H), 1.47 - 1.38 (m, 2H), 1.37 - 1.17 (m, 24H), 0.87 - 0.81 (m, 6H).

[0077] Example 2: Synthesis of Compound 2

[0078] As Figure 3 shown, the synthetic route of Compound 2 includes 3 steps (step 1 - step 3), which are specifically as follows:

[0079] S1 (step 1): Synthesis of 2 - methylpropan - 2 - yl 4 - ethylpiperazine - 1 - carboxylate (Compound 2 - 1)

[0080] Disperse N - BOC - piperazine (10.00 g, 53.69 mmol), bromoethane (7.02 g, 64.43 mmol) and potassium carbonate (18.50 g, 0.13 mol) in N,N - dimethylformamide (30 mL), and react at room temperature under nitrogen protection for 16 hours. According to TLC (dichloromethane / methanol = 30 / 1, 1% NH3, R f = 0.7) indicating the completion of the reaction of the starting materials, filter the reaction solution, and concentrate the filtrate to obtain a yellow oily compound (Compound 2 - 1, 11.1 g, crude product), which is directly used for the next step.

[0081] S2 (step 2): Synthesis of 1 - ethylpiperazine hydrochloride (Compound 2 - 2)

[0082] Disperse the above crude Compound 2 - 1 in a solution of hydrogen chloride in 1,4 - dioxane (30 mL, 4M), and react at room temperature under nitrogen protection for 16 hours. According to TLC (dichloromethane / methanol = 50 / 1, 1% NH3, R f(= 0.2) After indicating that the raw material reaction was completed, the reaction solution was filtered, and the obtained filter cake was washed with 1,4-dioxane (100 mL). The washed filter cake was dried to obtain a white solid compound (Compound 2-2, 14.31 g, crude product), which was directly used for the next step.

[0083] S3 (step 3): Synthesis of (13R,15Z)-13-hexyl-11-oxo-10,12-dioxatetracosa-15-en-7-yn-24-yl 3-(4-ethylpiperazin-1-yl)propionate (Compound 2)

[0084] Compound 1-4 (600 mg, 1.19 mmol), Compound 2-2 (234 mg, crude product), N,N-diisopropylethylamine (461 mg, 3.57 mmol) and methanol (10 mL) were dissolved in dichloroethane (12 mL), and the reaction was carried out at 68 °C under nitrogen protection for 16 hours. According to TLC (petroleum ether / ethyl acetate = 5 / 1, v / v, R f = 0.4, 0.5% NH3) After indicating that the raw material reaction was completed, the reaction solution was concentrated to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v) to obtain a pale yellow oily compound (Compound 2, 563 mg, three-step yield 76.5%).

[0085] According to mass spectrometry determination, the main ion peak characteristics of Compound 2 were: MS m / z (ESI): 619.5 [M+H] + Compound 2 had the molecular formula C 35 H 62 N2O5, with a molecular weight of 590.9.

[0086] The nuclear magnetic resonance spectrum of Compound 2 was as Figure 4 shown, and its 1 1H NMR characteristics were: 1 1H NMR (400 MHz, CDCl3) δ 5.59-5.42 (m, 1H), 5.42-5.24 (m, 1H), 4.81-4.59 (m, 3H), 4.07 (t, J = 6.8 Hz, 2H), 2.71 (t, J = 7.2 Hz, 2H), 2.65-2.27 (m, 13H), 2.25-2.15 (m, 2H), 2.09-1.94 (m, 2H), 1.68-1.44 (m, 6H), 1.43-1.17 (m, 25H), 1.08 (t, J = 7.2 Hz, 3H), 0.96-0.77 (m, 6H).

[0087] Example 3: Preparation and Parameter Characterization of siRNA-LNP Complexes

[0088] Dissolve the cationic lipid (Compound 1 or Compound 2), DSPC, cholesterol, and DMG-PEG2000 in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 to obtain a lipid mixture; and dissolve the siRNA lyophilized powder in a 25 mM sodium acetate (pH 5.0) solution to obtain a siRNA sodium acetate solution.

[0089] Set the total flow rate to 12 - 30 mL / min, the flow rate ratio of the lipid mixture to the siRNA sodium acetate solution to 1:3, and the weight ratio of the lipid mixture to siRNA to 20:1, and prepare the siRNA-LNP complex using a microfluidic device. Replace the buffer of the encapsulated siRNA-LNP with Tris buffer or phosphate buffer at pH 7.4 through a tangential flow filtration system for buffer exchange and concentration to obtain the concentrated siRNA-LNP, then add a 50% sucrose solution to obtain a sucrose solution containing 8.7% (w / v) siRNA-LNP. Finally, filter the siRNA-LNP sucrose solution through a 0.2 μm sterile filter.

[0090] Table 2: Exemplary siRNA-LNP Parameter Characterization

[0091]

[0092] Determine the average particle size, PDI, and Zeta potential of the lipid nanoparticles by dynamic light scattering using a Malvern Zetasizer Nano ZS (purchased from Malvern UK). According to the instructions, use the Quant-iT™ RiboGreen® RNA Reagent and Kit (purchased from Invitrogen) to determine the encapsulation efficiency of the lipid nanoparticles.

[0093] The in vivo delivery efficiency of siRNA is generally negatively correlated with the apparent pKa of LNP. Use 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) fluorescence analysis to determine the apparent pKa of LNP. Prepare LNP containing different cationic lipids according to the above method, dissolve each LNP in a citrate-phosphate buffer in the pH range of 3 - 9, and add 2-(p-toluidino)-6-naphthalenesulfonic acid respectively. After vortex mixing, measure the fluorescence intensity at an excitation wavelength of 325 nm and an emission wavelength of 435 nm using a multifunctional microplate reader at room temperature. Fit the fluorescence intensity values and pH values into a curve. It can be known that at a specific pH value, all ionizable groups are charged and the fluorescence value reaches the maximum, and the pKa value is the pH value that produces half of the maximum fluorescence intensity.

[0094] The parameters of the siRNA-LNP containing Compound 1 or Compound 2 are shown in Table 2. The particle size is good (<200 nm), the particle distribution is uniform (PDI < 0.25), and the encapsulation efficiency can reach more than 90%.

[0095] Example 4: Detection of in vitro delivery effect of siRNA-LNP

[0096] Referring to the siRNA-LNP preparation method in Example 3 and according to the lipid formulation in Table 3 below, siRNA-LNP was prepared. Among them, the siRNA targets TNF-α (GenBank: MH180383.1), and the sequence of the siRNA can be referred to CN114410627B. The HaCat cells were passaged into a 12-well plate to ensure that the cell density reached about 50% of the well plate area the day before transfection. During transfection, the siRNA-LNP preparation containing Compound 1 or Compound 2 was added dropwise to each well; the siRNA-Lip3000 preparation, which replaced the LNP in the siRNA-LNP preparation with Lip3000 (i.e., Lipofectamine 3000, purchased from Thermo Fisher Scientific, catalog number L3000015), was used as a control; among them, the preparation method of siRNA-Lip3000 was: 100 pmol of siRNA was added to 50 μL of DMEM medium as tube A, and 5 μL of lip3000 was added to 50 μL of DMEM medium as tube B. After mixing tubes A and B thoroughly, they were left standing at room temperature for 15 min and then used for transfecting cells.

[0097] At 48 h after transfection, the cell RNA was extracted using the RNA prep Pure Cell / Bacteria Kit (purchased from Tiangen Biotech Co., Ltd., catalog number DP430), and cDNA was obtained by reverse transcription. The mRNA expression level of the target gene was detected by Q-PCR.

[0098] Table 3: Lipid formulation and parameter characterization of siRNA-LNP

[0099]

[0100] As can be seen from Table 3, when transfecting 100 nM of siRNA, the siRNA-LNP preparations with Compound 1 or Compound 2 as cationic lipids can both effectively knockdown the target gene by more than 50%, showing excellent delivery effects, which are better than the effects achieved by Lip3000 delivery at the same transfection concentration.

[0101] Example 5: Detection of in vitro delivery effect of pGFP-LNP

[0102] In this example, by preparing and delivering pGFP-LNP in vitro, the delivery ability of LNP was further verified.

[0103] Referring to the preparation method of siRNA-LNP in Example 3, replace the siRNA therein with an equal mass of pGFP plasmid to obtain pGFP-LNP; wherein, the pGFP plasmid has the nucleotide sequence shown in SEQ ID NO: 1; the formulation of the lipid complex of LNP is cationic lipid:DSPC:cholesterol:DMG-PEG2000 (molar ratio) = 50:10:38.5:1.5, and the specific grouping is shown in Table 4. The experimental process is as follows:

[0104] S1: Cell passage: Passage HaCat cells into a 12-well plate to ensure that the cell density reaches about 50% of the well plate area before transfection the next day;

[0105] S2: Plasmid dilution: Add 125 μL of Opti-MEM serum-free medium and 1000 ng of GFP plasmid into a 1.5 mL EP tube, and gently mix to dilute the plasmid;

[0106] S3: Preparation of transfection mixture reagent: Add 5 μL of P3000™ reagent into another EP tube, then add 125 μL of Opti-MEM serum-free medium, and gently mix; immediately add 3 μL of Lip3000 reagent into the above mixture containing P3000™ reagent and Opti-MEM, and gently mix, and incubate at room temperature for 5 minutes to obtain the transfection mixture reagent;

[0107] S4: Preparation of transfection complex: Add the diluted plasmid solution in S2 into the transfection mixture reagent obtained in S3, gently mix, and incubate at room temperature for 20 minutes to form a transfection complex;

[0108] S5: Transfect cells: Aspirate the old medium in the culture well, gently rinse the cells once with 1 mL of Opti-MEM serum-free medium, and then add 500 μL of fresh Opti-MEM serum-free medium to each well. Dropwise add the prepared transfection complex into the cell culture well; directly add the pGFP-LNP preparation containing Compound 1 or Compound 2 dropwise to the cell well, and gently shake the culture plate to make it evenly distributed;

[0109] S6: Fluorescence detection: Place the cells in an incubator at 37 °C and 5% CO2 for culture. After 4 - 6 hours, change the medium to complete medium and continue to culture. After 48 hours of transfection, observe the fluorescence signal intensity in each group of cells under a fluorescence microscope.

[0110] Table 4: Grouping for detecting the in vitro delivery effect of pGFP-LNP

[0111]

[0112] It can be seen from Figure 5 that in the cells of the Lip3000+GFP plasmid group, fluorescence signals of normal intensity appeared; in the cells of the compound 1 group and the compound 2 group, no obvious fluorescence was observed; in the cells of the compound 1+GFP plasmid group and the compound 2+GFP plasmid group, strong fluorescence signals were observed, and the effect was better than that of the Lip3000+GFP plasmid group.

[0113] Example 6: Median effective dose (ED50) and cytotoxicity of siRNA-LNP in vitro

[0114] Referring to the siRNA-LNP preparation method in Example 3, and according to the lipid formulation in Table 5 below, siRNA-LNP was prepared; among them, the molar ratios (mol%) of the cationic lipids (compound 1 or compound 2) used were 30%, 35%, 40%, 45%, 50%, 55% or 60% respectively; the siRNA targeted TNF-α (GenBank: MH180383.1), and the sequence of the siRNA can refer to CN114410627B. The HaCat cells were passaged into a 12-well plate to ensure that the cell density reached about 50% of the well plate area the day before transfection. At the time of transfection, the LNP preparation containing compound 1 or compound 2 was directly added dropwise to each well. At 48 h after transfection, the cell RNA was extracted using the RNAprep Pure Cell / Bacteria Kit (purchased from Tiangen Biotech Co., Ltd., product number DP430), and cDNA was obtained by reverse transcription. The mRNA expression level of the target gene was detected by Q-PCR. In addition, the cell viability level was detected using the Cell Counting Kit-8 (purchased from Beyotime, product number C0037) at 24 h after transfection.

[0115] Table 5: Lipid formulation of siRNA-LNP

[0116]

[0117] It can be seen from Figure 6 that when the molar ratio is between 40% and 50%, the siRNA-LNP with compound 1 as the cationic lipid has the relatively best target gene knockdown effect, which can achieve the effect of knocking down more than 50% of the target gene, and the cell viability is close to or greater than 80%; when the molar ratio is between 40% and 55%, the siRNA-LNP with compound 2 as the cationic lipid has the relatively best target gene knockdown effect, which can achieve the effect of knocking down more than 50% of the target gene, and the cell viability is above 75%. In summary, when the molar ratio of this type of cationic lipid is between 45% and 50%, the effect of delivering RNA is relatively the best, and the impact on cell growth is relatively small.

[0118] Example 7: Determination of mRNA-LNP in vivo delivery effect

[0119] Referring to the siRNA-LNP preparation method of Example 3, Fluc mRNA was used instead of siRNA to prepare Fluc mRNA-LNP; wherein the formula of the lipid complex of LNP was cationic lipid: DSPC: cholesterol: DMG-PEG2000 (molar ratio) = 50:10:38.5:1.5.

[0120] The C57bl / 6 mice used in this example are commercially available.

[0121] C57bl / 6 mice were anesthetized with aflototin, and the surface hair was simply shaved. Fluc mRNA was injected into the tail vein at doses of 10 mg / kg, 50 mg / kg and 100 mg / kg, respectively. The animals were euthanized 5 h and 24 h after injection, respectively, and the livers were collected and ground into homogenate using a cryo-grinder at -20 °C. 50 mg was weighed into a new sterile 1.5 ml centrifuge tube, and the relative fluorescence intensity (RLU) of each sample was detected using the Firefly Luciferase Reporter Gene Assay Kit (Biyuntian, Cat. No. RG009S) according to the instructions.

[0122] As shown in Table 6, the Fluc mRNA-LNPs with the two compounds as cationic lipids can be effectively delivered into the body, and the fluorescence intensity has a good dose-dependent relationship with the delivered Fluc mRNA-LNP.

[0123] Table 6: Luciferase assay values in mouse liver homogenate

[0124]

[0125] In summary, the cationic lipids provided by the present invention, such as Compound 1 and Compound 2, have at least one of the following advantages compared to the cationic lipids disclosed in the prior art:

[0126] 1. The chemical structure is different from the cationic lipids disclosed in the prior art and is a completely new compound.

[0127] 2. The RNA-LNP composition prepared by adding the cationic lipid of the present invention (30 mol% to 60 mol%) has a good particle size (<200 nm) and a uniform particle distribution (PDI<0.25).

[0128] 3. When used for preparing LNP, it can achieve excellent nucleic acid delivery effects both in vitro and in vivo. In vitro, it can achieve high siRNA cell transfection efficiency and low cytotoxicity; in animals, it can achieve good expression levels and expression durations of the target proteins corresponding to mRNA, and at the same time show low toxicity.

[0129] 4. A piperazine group is introduced into the polar head. While carrying a positive charge, it shows low toxicity; in addition, different structural head R1 groups can be used to introduce groups with different charged or lipophilic properties, thereby further improving the properties of the lipid to meet the delivery requirements of different biomolecules.

Claims

1. A cationic lipid compound having a structure as shown in formula (I) or a pharmaceutically acceptable salt thereof, characterized in that, Among them, R1 is H or OH.

2. A method for preparing the cationic lipid compound as described in claim 1, characterized in that, The method includes a step of reacting with the compound shown in formula (Ⅱ), where R2 is H or OH.

3. The method according to claim 2, wherein The method includes a step of reacting (13R,15Z)-13-hexyl-11-oxo-10,12-dioxacyclopentene-15-en-7-yn-24-yl prop-2-enoate with the compound shown in formula (Ⅱ); where R2 is H or OH.

4. Use of the cationic lipid compound according to claim 1 in the preparation of a lipid composition, characterized in that, The lipid composition is used as a drug delivery carrier.

5. A lipid composition, characterized in that, The lipid component of the lipid composition includes the cationic lipid compound as described in claim 1, the lipid component of the lipid composition further contains other lipids, and the other lipids include phospholipids, sterol compounds and PEG lipids; the molar ratio of the cationic lipid compound to other lipid components is 30:70 to 60:40; the active ingredient of the lipid composition includes a therapeutic agent and / or a prophylactic agent; the mass ratio of the lipid component to the active ingredient is 7.5:1 to 30:

1.

6. The lipid composition according to claim 5, characterized in that, The therapeutic agent and / or prophylactic agent is selected from one or more combinations of small molecule drugs, nucleic acids and proteins.

7. Use of the lipid composition according to claim 5 or 6 in the preparation of a drug for preventing or treating inflammation.

8. A pharmaceutical composition, characterized in that, Comprising the lipid composition according to claim 5 or 6 and a pharmaceutically acceptable carrier, solvent or excipient.

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