Cationic lipid compound as well as preparation method, composition and application thereof

Abstract

The invention discloses a cationic lipid compound as well as a preparation method, a composition and application thereof, when the cationic lipid compound is used for preparing LNP, the particle size is good (1t, 200 nm), the particle distribution is uniform (PDilt, 0.25), an excellent nucleic acid delivery effect can be realized in vivo and in vitro, meanwhile, the toxicity is relatively low, and the cationic lipid compound has a remarkable application value.

Description

Technical Field

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

[0002] The development of nucleic acid drugs has brought new hope for the treatment of many diseases, such as genetic diseases, cancer, and some refractory infectious diseases. However, the effective delivery of nucleic acid drugs in vivo has always been a key problem that hinders their widespread application. Lipid nanoparticles (LNPs), as an important carrier for delivering nucleic acids, have received great attention in recent years. Among the components of LNPs, cationic lipids play an irreplaceable core role. They form electrostatic complexes with negatively charged nucleic acids to achieve the encapsulation and protection of nucleic acids, while helping LNPs escape from endosomes and release nucleic acids into the cytoplasm to exert their effects.

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

[0004] In terms of delivery performance, the performance of nucleic acid-liposome complexes prepared by existing cationic lipids is not ideal. In in vitro experiments, the transfection efficiency often fails to meet expectations, resulting in a large number of nucleic acids unable to effectively enter cells to exert their effects, causing drug waste and reduced therapeutic effects. In in vivo experiments, these cationic lipids face even 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, limiting their application in the treatment of systemic diseases. At the same time, increasing the dosage of cationic lipids in order to improve delivery efficiency will inevitably lead to higher cytotoxicity, which will have a negative impact on the physiological functions of normal cells, seriously restricting the clinical application of nucleic acid drugs.

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

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

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

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

[0009]

[0010] R1 is H or OH.

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

[0012]

[0013] R2 is H or OH.

[0014] In one or more embodiments, the method includes the step of reacting (13R, 15Z)-13-hexyl-11-oxo-10,12-dioxol-15-ene-7-yn-24-ylprop-2-enoate with a compound represented by formula (II) as reactants; 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-2-enoic acid propyl ester;

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

[0019] S4: Synthesis of (13R,15Z)-13-hexyl-11-oxo-10,12-dioxol-15-ene-7-yn-24-ylprop-2-enoate;

[0020] S5: Synthesis of cationic lipid compounds.

[0021] More preferably, the step S1 is to dissolve ricinoleic acid in anhydrous tetrahydrofuran, add lithium aluminum hydride in batches at 0°C under nitrogen protection, and after the addition, warm the temperature to room temperature for reaction for 2.5 to 3.5 hours; add sodium sulfate decahydrate in batches to the reaction solution at 0°C to quench, filter the liquid, and concentrate and purify the filtrate to obtain (10Z,12R)-octadec-9-ene-1,12-diol.

[0022] More preferably, the step S2 is to disperse the (10Z, 12R)-octadec-9-ene-1,12-diol and N,N-diisopropylethylamine obtained in step S1 in dichloromethane, add acryloyl chloride dropwise at 0°C under nitrogen protection, and react at room temperature for 2.5 to 3.5 hours; add water to the reaction solution to quench, and extract with dichloromethane; wash the organic layer with saturated brine, dry with anhydrous sodium sulfate, filter the obtained liquid, and concentrate and purify the filtrate to obtain (10Z, 12R)-12-hydroxyoctadec-9-ene-1-yl-2-enoic acid propyl ester.

[0023] More preferably, the step S3 is to dissolve the (10Z, 12R)-12-hydroxyoctadec-9-en-1-yl-2-enoic acid propyl ester obtained in step S2 in dichloromethane, add 4-nitrobenzene chloroformate at 0°C under nitrogen protection, slowly add piperazine dropwise after the addition, and keep the reaction warm for 1.5 to 2.5 hours; add water to the reaction solution to quench, and extract with dichloromethane; wash the organic layer with saturated brine, dry it with anhydrous sodium sulfate, filter the obtained liquid, and concentrate and purify the filtrate to obtain 4-nitrophenyl {[(10z)-18-[(1-oxo-2-enyl)oxy]octadec-9-en-7-yl]oxy} formate.

[0024] More preferably, the step S4 is to dissolve the 4-nitrophenyl {[(10z)-18-[(1-oxo-2-enyl)oxy]octadec-9-en-7-yl]oxy}carboxylate and non-2-yn-1-ol obtained in step S3 in dichloromethane, add N,N-diisopropylethylamine and 4-dimethylaminopiperazine at 0°C under nitrogen protection, and after the addition, keep the temperature to react for 20 to 40 minutes, then warm the temperature to room temperature to react for 15 to 17 hours; add water to the reaction solution to quench, and extract with dichloromethane; wash the organic layer with saturated brine, dry it with anhydrous sodium sulfate, filter the resulting liquid, and concentrate and purify the filtrate to obtain (13R, 15Z)-13-hexyl-11-oxo-10,12-dioxol-15-ene-7-yn-24-ylprop-2-enoate.

[0025] More preferably, the step S5 is to dissolve the (13R, 15Z)-13-hexyl-11-oxo-10,12-dioxol-15-ene-7-yn-24-yl prop-2-enoate obtained in step S4, hydroxyethylpiperazine and methanol in dichloromethane, and react at 65-70° C. under nitrogen protection for 7-9 hours; and concentrate and purify the reaction solution to obtain (13R, 15Z)-13-hexyl-11-oxo-10,12-dioxol-15-ene-7-yn-24-yl-3-[4-(2-hydroxyethyl)piperazine-1-yl]propanoate.

[0026] More preferably, the step S5 is to dissolve the (13R, 15Z)-13-hexyl-11-oxo-10,12-dioxol-15-ene-7-yn-24-yl prop-2-enoate obtained in step S4, 1-ethylpiperazine hydrochloride, N,N-diisopropylethylamine and methanol in dichloroethane, and react at 65 to 70° C. under nitrogen protection for 15 to 17 hours; and concentrate and purify the reaction solution to obtain 3-(4-ethylpiperazine-1-yl)propanoic acid-(13R, 15Z)-13-hexyl-11-oxo-10,12-dioxacyclopenta-15-ene-7-yn-24-yl ester.

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

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

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

[0030] Wherein, the step S1 is to disperse N-BOC-piperazine, ethyl bromide and potassium carbonate in N,N-dimethylformamide, and react at room temperature under nitrogen protection for 15 to 17 hours. The reaction solution is concentrated and purified to obtain 4-ethylpiperazine-1-carboxylic acid 2-methylprop-2-yl ester;

[0031] The step S2 is to disperse 2-methylpropane-2-yl 4-ethylpiperazine-1-carboxylate in a 1,4-dioxane solution of hydrogen chloride, and react for 15 to 17 hours at room temperature under nitrogen protection. The reaction solution is filtered, and the filter cake obtained by filtration is 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 a use of a cationic lipid compound as described in any embodiment herein as a surfactant.

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

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

[0035] Preferably, the drug is a small molecule drug, a nucleic acid or a 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 of the present invention, and the lipid component of the lipid composition also 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 preventive 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 other lipid components is 40:60 to 55:45. More preferably, the molar ratio of the ionizable cationic lipid compound to 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% to 60%: 5% to 40%: 10% to 60%: 0.1% to 15%. More preferably, the molar ratio of the cationic lipid compound to phospholipids, sterol compounds and PEG lipids is 30% to 60%: 5% to 30%: 20% to 60%: 1% to 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%, for example 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 agent and / or preventive agent is selected from a combination of one or more of small molecule drugs, nucleic acids and proteins.

[0041] Preferably, the therapeutic and / or preventive 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 a lipid composition in the preparation of a medicament 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 embodiment 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 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% to 60 mol%) has a good particle size (<200 nm) and a uniform particle distribution (PDI<0.25).

[0047] 3. When used to prepare LNP, it can achieve excellent nucleic acid delivery effects both in vivo and in vitro, while showing low toxicity.

[0048] 4. A piperazine group is introduced into the polar head, which exhibits lower toxicity while carrying a positive charge; in addition, head R1 groups with different structures can be used to adapt to the delivery requirements of different biological molecules.

[0049] Therefore, the cationic lipid provided by the present invention has significant application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1This is the synthetic route of compound 1.

[0051] Figure 2 is the NMR spectrum of compound 1. 1 H NMR spectrum, 2B is that of compound 1 13 C NMR spectrum.

[0052] Figure 3 This is the synthetic route of compound 2.

[0053] Figure 4 is the NMR spectrum of compound 2. Among them, 4A is the NMR spectrum of compound 1 1 H NMR spectrum, 4B is that of compound 1 13 C NMR spectrum.

[0054] Figure 5 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 of cells after delivery of siRNA-LNP containing different molar ratios of compound 1 or compound 2. Among them, 6A is the relative expression level of TNFα and cell viability of cells after delivery of siRNA-LNP containing different molar ratios of compound 1; 6B is the relative expression level of TNFα and cell viability of cells after delivery of siRNA-LNP containing different molar ratios of compound 2. DETAILED DESCRIPTION

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

[0057] S1: Weigh citric acid (C6H8O 7· H2O) 21.014 g, dissolved in distilled water and fixed to 1000 mL to obtain 0.1 mol / L citric acid solution;

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

[0059] S3: Take 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 to 200 mL to obtain a citric acid-phosphate buffer solution with the corresponding pH value.

[0060] Table 1: Citric acid-phosphate buffer formulations at different pH values

[0061]

[0062] Example 1: Synthesis of Compound 1

[0063] like Figure 1 As shown, the synthetic route of compound 1 includes 5 steps (step 1 to step 5), as follows:

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

[0065] Ricinoleic acid (5.00 g, 16.75 mmol) was dissolved in anhydrous tetrahydrofuran (250 mL). Lithium aluminum hydride (LAH, 826 mg, 21.78 mmol) was added in batches at 0°C under nitrogen protection. After the addition was completed, the mixture was heated to room temperature and reacted for 3 hours. According to TLC (petroleum ether / ethyl acetate = 1 / 1, v / v, R f = 0.3) indicated that the reaction of the raw materials was complete, sodium sulfate decahydrate (30.00 g) was added to the reaction solution in batches at 0°C to quench, the liquid was filtered, and the filtrate was concentrated. The crude product was 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, 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-enoic acid propyl ester (compound 1-2)

[0068] Compound 1-1 (3.56 g, 12.51 mmol) and N,N-diisopropylethylamine (4.00 g, 31.28 mmol) were dispersed in dichloromethane (30 mL), and acryloyl chloride (1.13 g, 12.51 mmol) was added dropwise at 0°C under nitrogen protection. After the addition was completed, the mixture was reacted at room temperature for 3 hours. According to TLC (petroleum ether / ethyl acetate = 2 / 1, v / v, R f= 0.7) indicated that the reaction of the raw materials was complete, 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 with anhydrous sodium sulfate, and the resulting 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}carboxylate (compound 1-3)

[0070] Compound 1-2 (2.69 g, 7.59 mmol) was dissolved in dichloromethane (50 mL), and 4-nitrobenzene chloroformate (3.20 g, 15.94 mmol) was added at 0°C under nitrogen protection. After the addition was completed, piperazine (3.00 g, 37.95 mmol) was slowly added dropwise. After the addition was completed, the reaction was kept warm for 2 hours. According to TLC (petroleum ether / ethyl acetate = 5 / 1, v / v, R f = 0.7) indicated that the reaction of the raw materials was complete, water (50 mL) was added to the reaction solution to quench, and it was extracted with dichloromethane (50 mL×2); the organic layer was washed with saturated brine (50 mL×2), dried with anhydrous sodium sulfate, and the resulting 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 in the next step.

[0071] S4 (step 4): Synthesis of (13R,15Z)-13-hexyl-11-oxo-10,12-dioxol-15-ene-7-yn-24-ylprop-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). N,N-diisopropylethylamine (2.45 g, 18.98 mmol) and 4-dimethylaminopiperazine (927 mg, 7.59 mmol) were added at 0°C under nitrogen protection. After the addition was completed, the mixture was kept warm for 30 minutes and then heated to room temperature for 16 hours. According to TLC (petroleum ether / ethyl acetate = 5 / 1, v / v, R f= 0.6) indicates that the reaction of the raw materials is complete, water (50 mL) is added to the reaction solution to quench, and it is extracted with dichloromethane (50 mL×2); the organic layer is washed with saturated brine (50 mL×2), dried with anhydrous sodium sulfate, and the resulting liquid is filtered and the filtrate is concentrated. The crude product is 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-dioxol-15-ene-7-yn-24-yl-3-[4-(2-hydroxyethyl)piperazin-1-yl]propanoate (Compound 1)

[0074] Compound 1-4 (600 mg, 1.19 mmol), hydroxyethylpiperazine (165 mg, 1.25 mmol) and methanol (10 mL) were dissolved in dichloromethane (10 mL) and reacted 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) indicated that the reaction of the raw materials was complete, and 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, the main ion peak characteristics of compound 1 are: MS m / z (ESI): 635.5 [M+H] + The molecular formula of compound 1 is C 35 H 62 N2O6, molecular weight is 606.9.

[0076] The NMR spectrum of compound 1 is as follows Figure 2 As shown, its 1 The characteristics of H NMR are: 1H 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] like Figure 3 As shown, the synthetic route of compound 2 includes three steps (step 1 to step 3), as follows:

[0079] S1 (step 1): Synthesis of 4-ethylpiperazine-1-carboxylic acid 2-methylpropan-2-yl ester (compound 2-1)

[0080] N-BOC-piperazine (10.00 g, 53.69 mmol), ethyl bromide (7.02 g, 64.43 mmol) and potassium carbonate (18.50 g, 0.13 mol) were dispersed in N,N-dimethylformamide (30 mL) and reacted at room temperature under nitrogen protection for 16 hours. According to TLC (dichloromethane / methanol = 30 / 1, 1% NH3, R f = 0.7) indicated that the reaction of the raw materials was complete, the reaction solution was filtered, and the filtrate was concentrated to obtain a yellow oily compound (compound 2-1, 11.1 g, crude product), which was directly used in the next step.

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

[0082] The crude compound 2-1 was dispersed in a solution of hydrogen chloride in 1,4-dioxane (30 mL, 4 M) and reacted for 16 hours at room temperature under nitrogen protection. According to TLC (dichloromethane / methanol = 50 / 1, 1% NH3, R f= 0.2) indicated that the reaction of the raw materials was completed, the reaction solution was filtered, and the filter cake obtained by filtration 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 in the next step.

[0083] S3 (step 3): Synthesis of 3-(4-ethylpiperazin-1-yl)propanoic acid-(13R,15Z)-13-hexyl-11-oxyde-10,12-dioxa-15-ene-7-yn-24-yl ester (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 reacted 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) indicated that the reaction of the raw material was complete, and 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 light yellow oily compound (compound 2, 563 mg, three-step yield 76.5%).

[0085] According to mass spectrometry, the main ion peak characteristics of compound 2 are: MS m / z (ESI): 619.5 [M+H] + The molecular formula of compound 2 is C 35 H 62 N2O5, molecular weight is 590.9.

[0086] The NMR spectrum of compound 2 is as follows Figure 4 As shown, its 1 The characteristics of H NMR are: 1 H 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 complex

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

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

[0090] Table 2: Exemplary siRNA-LNP parameter characterization

[0091]

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

[0093] The delivery efficiency of siRNA in vivo is generally negatively correlated with the apparent pKa of LNP. The apparent pKa of LNP was determined using 2-(p-toluidine)-6-naphthalenesulfonic acid (TNS) fluorescence analysis. LNPs containing different cationic lipids were prepared according to the above method, and each LNP was dissolved in a citric acid-phosphate buffer in the pH range of 3 to 9, and 2-(p-toluidine)-6-naphthalenesulfonic acid was added respectively. After vortex mixing, the fluorescence intensity at an excitation wavelength of 325nm and an emission wavelength of 435nm was measured using a multifunctional microplate reader at room temperature. The fluorescence intensity value and pH value were curve fitted, and it can be seen that at a specific pH value, all ionizable groups are charged, the fluorescence value reaches the maximum, and the pKa value is the pH value that produces half the maximum fluorescence intensity.

[0094] The parameters of 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: siRNA-LNP in vitro delivery effect detection

[0096] Referring to the siRNA-LNP preparation method of Example 3, siRNA-LNP was prepared according to the lipid formulation in Table 3 below. The siRNA targets TNF-α (GenBank: MH180383.1), and the sequence of siRNA can be found in CN114410627B. HaCat cells were passaged to 12-well plates to ensure that the cell density reached about 50% of the plate area before transfection the next day. During transfection, siRNA-LNP preparations containing compound 1 or compound 2 were added to each well; Lip3000 (i.e., Lipofectamine3000, purchased from Thermo Fisher Scientific, catalog number L3000015) was used as a control to replace the LNP in the siRNA-LNP preparation; wherein, the preparation method of siRNA-Lip3000 is as follows: 100 pmol of siRNA was added to 50 μL of DMEM culture medium as tube A, and 5 μL of lip3000 was added to 50 μL of DMEM culture medium as tube B. After tubes A and B were fully mixed, they were allowed to stand at room temperature for 15 minutes before being used to transfect cells.

[0097] At 48 h after transfection, RNA prep Pure Cell / Bacteria Kit (purchased from Tiangen, catalog number DP430) was used to extract cellular RNA and reverse transcribe it to obtain cDNA. The mRNA expression of the target gene was detected by Q-PCR.

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

[0099]

[0100] As shown in Table 3, when transfecting siRNA 100nM, the siRNA-LNP preparations with compound 1 or compound 2 as cationic lipids can achieve effective knockdown of the target by more than 50%, showing excellent delivery effect, which is better than the effect achieved by Lip3000 delivery at the same transfection concentration.

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

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

[0103] Referring to the siRNA-LNP preparation method of Example 3, the siRNA therein is replaced with a pGFP plasmid of equal mass to prepare pGFP-LNP; wherein the pGFP plasmid has a nucleotide sequence as shown in SEQ ID NO: 1; the formula 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 passaging: Passage HaCat cells into 12-well plates, ensuring that the cell density reaches about 50% of the well plate area before transfection the next day;

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

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

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

[0108] S5: Transfect cells: Aspirate the old culture medium in the culture wells, gently rinse the cells once with 1 mL of Opti-MEM reduced serum medium, and then add 500 μL of new Opti-MEM reduced serum medium to each well. Add the prepared transfection complex dropwise to the cell culture wells; add the pGFP-LNP preparation containing compound 1 or compound 2 directly to the cell wells, and gently shake the culture plate to evenly distribute it;

[0109] S6: Fluorescence detection: The cells were cultured in an incubator at 37°C and 5% CO2. After 4-6 hours, the culture medium was replaced with complete culture medium and continued to be cultured. After 48 hours of transfection, the fluorescence signal intensity of each group of cells was observed under a fluorescence microscope.

[0110] Table 4: Grouping of pGFP-LNP in vitro delivery effect detection

[0111]

[0112] Depend on Figure 5 It can be seen that in the cells of the Lip3000+GFP plasmid group, a fluorescence signal 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: ED50 and cytotoxicity of siRNA-LNP delivery in vitro

[0114] The siRNA-LNP preparation method of reference Example 3 and the lipid formulation of Table 5 below were used to prepare siRNA-LNP; wherein the molar ratio (mol%) of the cationic lipid (Compound 1 or Compound 2) used was 30%, 35%, 40%, 45%, 50%, 55% or 60% respectively; the siRNA targets TNF-α (GenBank: MH180383.1), and the sequence of siRNA can refer to CN114410627B. HaCat cells were passaged to 12-well plates to ensure that the cell density reached about 50% of the well plate area before transfection the next day. During transfection, the LNP preparation containing Compound 1 or Compound 2 was directly added to each well. At 48h after transfection, the RNAprep Pure Cell / Bacteria Kit (purchased from Tiangen Company, catalog number DP430) was used to extract cell RNA and reverse transcribe to obtain cDNA. The mRNA expression of the target gene was detected by Q-PCR. In addition, the cell viability level was detected 24 h after transfection using the Cell Counting Kit-8 kit (purchased from Beyotime, catalog number C0037).

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

[0116]

[0117] Depend on Figure 6 It can be seen that when the molar ratio is 40% to 50%, the target gene knockdown effect of siRNA-LNP with compound 1 as cationic lipid is relatively the best, 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 40% to 55%, the target gene knockdown effect of siRNA-LNP with compound 2 as cationic lipid is relatively the best, 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 45% to 50%, the effect of delivering RNA is relatively the best, and the effect on cell growth is 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 can be seen from Table 6, 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 to prepare LNP, it can achieve excellent nucleic acid delivery effects both in vivo and in vitro, and can achieve higher siRNA cell transfection efficiency and lower cytotoxicity in vitro; it can achieve better mRNA corresponding to the target protein expression level and expression duration in animals, while showing lower toxicity.

[0129] 4. A piperazine group is introduced into the polar head, which exhibits lower toxicity while carrying a positive charge. In addition, head R1 groups with different structures can be used to introduce groups with different charged or lipophilic properties, thereby further improving the performance of lipids to meet the delivery requirements of different biomolecules.

Claims

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

2. A method for preparing the cationic lipid compound according to claim 1, characterized in that: The method comprises the steps of carrying out a reaction using a compound represented by formula (II) as a reactant, wherein: R2 is H or OH.

3. The method according to claim 2, characterized in that The method comprises the steps of reacting (13R, 15Z)-13-hexyl-11-oxo-10,12-dioxol-15-ene-7-yn-24-ylprop-2-enoate with a compound represented by formula (II) as reactants; wherein R2 is H or OH.

4. Use of the cationic lipid compound as claimed in claim 1 as a surfactant.

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

6. A lipid composition, characterized in that The lipid component of the lipid composition includes the cationic lipid compound as described in claim 1, and the lipid component of the lipid composition also 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 preventive agent; the mass ratio of the lipid component to the active ingredient is 7.5:1 to 30:

1.

7. The lipid composition according to claim 6, wherein The therapeutic agent and / or preventive agent is selected from a combination of one or more of small molecule drugs, nucleic acids and proteins.

8. Use of the lipid composition as claimed in claim 6 or 7 in the preparation of a medicament for preventing or treating an inflammation, an infectious disease, a cancer, a proliferative disease, a hereditary disease, an autoimmune disease or a metabolic disease.

9. A pharmaceutical composition, characterized in that Comprising the lipid composition according to any one of claims 6 to 8 and a pharmaceutically acceptable carrier, solvent or excipient.

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