Ionizable lipid compound as well as preparation method and application thereof
By preparing the Ugi four-component reaction of ionizable lipid compounds, the problem of excessive accumulation of LNPs in the liver was solved, and specific expression in the spleen was achieved without the need for targeted ligand modification. This simplified the preparation process and improved efficacy and safety.
Patent Information
- Application Number
- CN202511790005.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-01
- Publication Date
- 2026-02-27
AI Technical Summary
Existing lipid nanoparticles (LNPs) have the problem of excessive accumulation in the liver when used as delivery carriers. This leads to non-specific distribution, which weakens the therapeutic effect on target organs and increases the metabolic burden on the liver. It is difficult to achieve tissue-specific delivery, especially in spleen-targeted delivery, where there are risks of immunogenicity and complex ligand-carrier coupling processes.
Liposome formulations targeting the spleen were prepared using ionizable lipid compounds via a Ugi four-component reaction. Their unique structure enables the specific expression of bioactive components in the spleen without the need for targeting ligand modification, avoiding off-target effects in non-target organs such as the liver. The targeting effect was detected using a PerkinElmer IVIS Lumina in vivo imaging system.
This method enables the specific expression of bioactive components in the spleen without the need for targeted ligand modification, avoiding off-target effects in non-target organs such as the liver, simplifying the preparation process, and improving efficacy and safety.
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Figure CN121574066A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of medicine, and particularly relates to an ionizable lipid compound and a preparation method and application thereof. BACKGROUND
[0002] In recent years, messenger RNA (mRNA) technology has shown significant application potential in the fields of vaccine development, tumor immunotherapy and genetic disease treatment. Its core advantage lies in being able to directly guide the host cells to synthesize functional proteins without integrating into the host genome, thereby avoiding the potential genomic insertion risk in traditional gene therapy. However, the existing lipid nanoparticles (LNPs) as delivery carriers still face an important challenge in actual application: how to achieve tissue-specific delivery to improve the therapeutic effect and reduce systemic toxicity. The traditional LNPs are mediated by apolipoprotein E (ApoE) to target liver cells, resulting in more than 80% of mRNA accumulating in the liver. Although this feature has advantages in the treatment of liver-related diseases (such as genetic metabolic diseases), in vaccines or systemic immunotherapy, non-specific distribution may weaken the therapeutic effect of the target organ and increase the metabolic burden of the liver. Therefore, developing an LNP delivery system that can actively target the immune core organ, the spleen, has become an important research direction for optimizing mRNA therapy.
[0003] The spleen, as the largest secondary lymphoid organ in the human body, plays a core role in immune response, and its targeted delivery is of great significance to improve the efficacy of vaccines and treat immune-related diseases. The anatomical structure and immune function of the spleen provide a unique biological basis for mRNA delivery: the large intercellular space of the marginal sinus endothelial cells in the red pulp allows nanoparticles to be passively retained; and the dense distribution of high-activity immune cells such as dendritic cells, macrophages and B cells in the white pulp is a key target for initiating adaptive immune response. Studies have shown that compared with local immunization methods such as intramuscular injection, the targeted delivery of mRNA vaccines to the spleen can significantly enhance the immune response and effectively initiate long-lived antigen-specific CD8 + T cell response, which is particularly important in inducing anti-tumor immune memory.
[0004] However, existing spleen targeting strategies mostly rely on the modification of antibodies or sugar ligands to achieve active targeting. Such methods not only face the risk of immunogenicity, but also are difficult to achieve large-scale production due to the complexity of ligand-carrier coupling process. SUMMARY
[0005] The purpose of this invention is to provide an ionizable lipid compound, its preparation method, and its application. The ionizable lipid compound, its stereoisomers, tautomers, solvent compounds, or pharmaceutically acceptable salts provided by this invention can achieve specific expression of bioactive components (including mRNA) in the spleen without relying on any targeted ligand modification, effectively avoiding off-target effects in non-target organs such as the liver. Furthermore, the preparation process of the spleen-targeting liposome formulation is simple and easy to implement, and it has significant scientific value and application prospects.
[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides an ionizable lipid compound and its stereoisomers, tautomers, solvent compounds, or pharmaceutically acceptable salts, wherein the ionizable lipid compound has the structure shown in formula (I): Formula (I); In formula (I): R1 is selected from , or ; R2 is selected from ; R3 is selected from or ; R4 is selected from , , , , , , , , , , , , , , , , , , , or .
[0007] This invention provides an ionizable lipid compound and its stereoisomers, tautomers, solvent compounds, or pharmaceutically acceptable salts, wherein the ionizable lipid compound has the structure shown in Formula (I). The ionizable lipid compound of Formula (I) and its stereoisomers, tautomers, solvent compounds, or pharmaceutically acceptable salts provided by this invention can achieve specific expression of bioactive components (e.g., mRNA) in the spleen without the need for targeting ligand modification, effectively avoiding off-target effects in non-target organs such as the liver. Based on current research advancements in organ-targeted LNPs, this invention uses a highly sensitive PerkinElmer IVIS Lumina in vivo imaging system to detect the specific expression of mRNA in the spleen-targeting liposome formulation provided by this invention in the spleen, while no expression is observed in other organs. Attached Figure Description
[0008] Figure 1 The results of bioluminescence in mice in vivo and in vitro in various organs of the LNP-3 / mFluc formulation; Figure 2 The bioluminescence results of LNP-19 / mFluc formulation in mice in vivo and in vitro in various organs; Figure 3 The results of bioluminescence in mice and in vitro in various organs of the LNP-20 / mFluc formulation; Figure 4 The hydrogen NMR spectrum of ionizable lipid number 3; Figure 5 The hydrogen NMR spectrum of ionizable lipid number 19; Figure 6 The hydrogen NMR spectrum of ionizable lipid number 20; Figure 7 Particle size and potential diagrams for LNP-3, LNP-19, and LNP-20; Figure 8 A flowchart illustrating the preparation process of the ionizable lipid compounds provided by this invention; Figure 9 This is a flowchart illustrating the preparation of the ionizable lipid compound No. 1 in Example 1 of the present invention; Figure 10 This is a flowchart illustrating the preparation process of the amine compounds used in the embodiments of the present invention; Figure 11 This is a flowchart illustrating the preparation process of the carboxylic acid compounds used in the embodiments of the present invention; Figure 12 This is a flowchart illustrating the preparation process of the isonitrile compounds used in the embodiments of the present invention. Detailed Implementation
[0009] This invention provides an ionizable lipid compound and its stereoisomers, tautomers, solvent compounds, or pharmaceutically acceptable salts, wherein the ionizable lipid compound has the structure shown in formula (I): Formula (I); In formula (I): R1 is selected from , or ; R2 is selected from ; R3 is selected from or ; R4 is selected from , , , , , , , , , , , , , , , , , , , or .
[0010] In this invention, the ionizable lipid compound is selected from any one of the compounds numbered 1 to 22 in Table 1.
[0011] Table 1. Compounds numbered 1–22
[0012] This invention provides a method for preparing the ionizable lipid compound described above, comprising the following steps: The ionizable lipid compound is obtained by mixing aldehydes, amines, isonitriles, carboxylic acids, and organic solvents and carrying out a Ugi-4-component reaction (Ugi-4CR). The aldehyde compound has the structure shown in formula a, the amine compound has the structure shown in formula b, the isonitrile compound has the structure shown in formula c, and the carboxylic acid compound has the structure shown in formula d. Formula a; Formula b; Formula c; Formula d.
[0013] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.
[0014] In this invention, the aldehyde compounds are selected from... In this invention, Commercially available products are preferred.
[0015] In this invention, the amine compound is selected from... or The aforementioned Commercially available products are preferred. Preferred products are self-made. Preferred according to Figure 10 The preparation process shown is followed.
[0016] In this invention, the isonitrile compound is selected from... , or The isonitrile compound is preferably a self-made product. Preferred Figure 12 The preparation process shown is followed to prepare the product. , Preferred Figure 12 The preparation process is similar to that shown, except that the preparation is carried out by a method that ... Figure 12 The raw materials in the preparation process shown are replaced with or .
[0017] In this invention, the carboxylic acid compounds are selected from... , , , , , , , , , , , , , , , , , , , or The aforementioned Commercially available products are preferred. Preferred Figure 11The preparation process shown is followed to prepare the product. , , , , , , , , , , , , , , , , , or Preferred Figure 11 The preparation process is similar to that shown, except that the preparation is carried out by a method that ... Figure 11 The raw materials in the preparation process shown can be replaced with the corresponding acids and alcohols.
[0018] The organic solvent used in the Ugi four-component reaction is preferably methanol. This invention does not have special requirements on the amount of the organic solvent used, as long as the Ugi four-component reaction proceeds smoothly.
[0019] In this invention, the molar ratio of the aldehyde compound, amine compound, isonitrile compound and carboxylic acid compound is preferably (0.1~1.5):(0.1~1.5):(0.1~1.5):(0.1~1.5), and in the examples it can be 1:1:1:1.
[0020] In this invention, the preferred temperature for the Ugi four-component reaction is 25-40°C, and in the examples, it can be 40°C; the preferred time is 12-72 hours, and in the examples, it can be 24 hours. The Ugi four-component reaction is preferably carried out under stirring conditions.
[0021] In this invention, after the Ugi four-component reaction is completed, a Ugi four-component reaction solution is obtained. Preferably, the Ugi four-component reaction solution is desolventized and then purified by column chromatography to obtain the ionizable lipid compound. In this invention, the desolventization is preferably performed by vacuum distillation. The column chromatography purification is preferably performed using a 200-300 mesh silica gel column. The eluent used for the column chromatography purification is preferably dichloromethane (DCM) and methanol (MeOH), and the volume ratio of DCM:MeOH is preferably 20:1.
[0022] This invention provides the application of the ionizable lipid compounds and their stereoisomers, tautomers, solvent compounds or pharmaceutically acceptable salts described in the above technical solutions in the preparation of spleen-targeting drugs.
[0023] This invention provides a spleen-targeting liposome formulation comprising ionizable lipids, neutral lipids, structural lipids, PEG-lipids, and bioactive components. The ionizable lipids are one or more of the ionizable lipid compounds described above, their stereoisomers, tautomers, solvent compounds, and pharmaceutically acceptable salts.
[0024] In this invention, the spleen-targeting liposome formulation can specifically be spleen-targeting lipid nanoparticles.
[0025] In this invention, the bioactive ingredient is selected from bioactive ingredients with negative charge or bioactive ingredients that are hydrophobic.
[0026] In this invention, the preferred molar ratio of the ionizable lipids, neutral lipids, structured lipids, and PEG-lipids is (0~50):(5~80):(5~50):(0.1~10), and the ionizable lipids are not zero, more preferably (10~50):(5~60):(10~40):(0.5~5), and in the examples it can be 50:10:38.5:1.5. In this invention, the neutral lipids preferably include one or more of 1,2-distearate-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and sphingomyelin (SM).
[0027] In this invention, the structural lipids preferably include one or more of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, and rapeseed sterol.
[0028] In this invention, the PEG-lipid preferably includes one or more of the following: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (DMG-PEG), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG), PEG-disterol glycerol (DSG-PEG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearate, PEG-diacylglycerol amide (DAG-PEG), PEG-dipalmitoylphosphatidylethanolamine (DPPE-PEG), PEG-phosphatidylethanolamine (PE-PEG), PEG-succinate diacylglycerol, PEG-ceramide (PEG-cer), PEG dialkoxypropyl carbamate, and PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA). The DMG-PEG used in the embodiments of the present invention can be DMG-PEG2000.
[0029] In this invention, the bioactive ingredient preferably includes one or more of nucleic acid molecules, protein molecules, polypeptide molecules, and other small molecule drugs. In embodiments of this invention, the bioactive ingredient may be messenger RNA.
[0030] In this invention, the particle size of the targeted spleen lipid nanoparticles is preferably 50-150 nm.
[0031] This invention provides a method for preparing the spleen-targeting liposome formulation described above, comprising the following steps: Ionizable lipids, neutral lipids, structured lipids, PEG-lipids, and ethanol are mixed to obtain the ethanol phase; The bioactive ingredient was dissolved in a buffer salt solution to obtain an aqueous phase; The ethanol phase and the aqueous phase are mixed to obtain the initial formulation; The initial formulation was sequentially dialyzed and concentrated to obtain the spleen-targeting liposome formulation.
[0032] This invention mixes ionizable lipids, neutral lipids, structural lipids, PEG-lipids, and ethanol to obtain an ethanol phase. In this invention, the molar ratio of the ionizable lipids, neutral lipids, structural lipids, and PEG-lipids is (0~50):(5~80):(5~50):(0.1~10), and the amount of ionizable lipids is not zero. This invention does not have specific requirements regarding the amount of ethanol used.
[0033] This invention dissolves the bioactive ingredient in a buffer salt solution to obtain an aqueous phase. In this invention, the buffer salt solution is preferably a citrate buffer solution. The pH value of the citrate buffer solution is preferably 4. The molar concentration of the citrate buffer solution is preferably 50 mM.
[0034] After obtaining the ethanol phase and the aqueous phase, the present invention mixes the ethanol phase and the aqueous phase to obtain a preliminary formulation. In the present invention, the mixing is preferably performed in a microfluidic instrument. The volume ratio of the ethanol phase to the aqueous phase during mixing is preferably 1:3. The flow rate of the ethanol phase is preferably 3 mL / min, and the flow rate of the aqueous phase is preferably 9 mL / min.
[0035] After obtaining the initial formulation, the present invention sequentially dialyzes and concentrates the initial formulation to obtain the spleen-targeting liposome formulation. In this invention, the dialysis is preferably performed in a PBS buffer solution. The pH of the PBS buffer solution is preferably 7.4. The PBS buffer solution is preferably prepared with RNase-free water. The dialysis time is preferably 8 hours. The dialysis preferably removes ethanol from the initial formulation. The present invention does not have specific requirements for the specific implementation method of the concentration.
[0036] The present invention provides a pharmaceutical composition comprising the spleen-targeting liposome formulation described above and a pharmaceutically acceptable carrier.
[0037] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0038] The following embodiments are in accordance with Figure 8 The preparation process shown is followed.
[0039] Example 1 The preparation process of compound number 1 provided in this embodiment is as follows: Figure 9 As shown: The synthesis steps for compound number 1 are as follows: Carboxylic acid (0.5 mmol), amine (0.5 mmol), isonitrile (0.5 mmol), and aldehyde (0.5 mmol) were dissolved in MeOH (2 mL) and stirred at 40 °C for 24 h. After the reaction was complete, the solvent was removed under reduced pressure, and the mixture was purified by column chromatography (200-300 mesh silica gel column, eluent: DCM:MeOH volume ratio = 20:1), yielding compound number 1. The synthesized product was dissolved in CDCl3 and purified by... 1 The structure of the product was analyzed by HNMR.
[0040] Compounds numbered 2 to 22 were synthesized according to the above synthetic method, and their proton NMR spectra are as follows: Compound numbered 1: 1 H NMR (400 MHz, Chloroform- d ) δ 5.39 – 5.29 (m, 2H), 4.86 – 4.78 (m, 1H), 3.33 – 3.18 (m, 4H), 2.70 – 2.58 (m, 4H), 2.50 (t, J = 6.8Hz, 2H), 2.43 – 2.34 (m, 6H), 2.02 – 1.91 (m, 4H), 1.78 – 1.68 (m, 2H), 1.54 – 1.45 (m, 5H), 1.30 – 1.20 (m, 48H), 0.85 (t, J = 6.4 Hz, 15H). Compound number 2: 1 H NMR (400 MHz, Chloroform- d ) δ 5.38 – 5.27 (m, 2H), 4.85 – 4.78 (m, 1H), 3.31 – 3.18 (m, 4H), 2.68 – 2.59 (m, 4H), 2.48 (t, J = 7.3Hz, 2H), 2.40 – 2.34 (m, 6H), 2.06 – 1.92 (m, 4H), 1.79 – 1.68 (m, 2H), 1.54 – 1.44 (m, 5H), 1.32 – 1.21 (m, 52H), 0.85 (t, J = 6.1 Hz, 15H). Compound number 3: 1 H NMR (400 MHz, Chloroform- d ) δ 5.38 – 5.27 (m, 2H), 4.86 – 4.78 (m, 1H), 3.30 – 3.18 (m, 4H), 2.72 – 2.54 (m, 4H), 2.50 (t, J= 6.5Hz, 2H), 2.42 – 2.35 (m, 6H), 2.01 – 1.92 (m, 4H), 1.77 – 1.68 (m, 2H), 1.53 – 1.46 (m, 5H), 1.32 – 1.22 (m, 56H), 0.85 (t, J = 6.7 Hz, 15H). Compound number 4: 1 H NMR (400 MHz, Chloroform- d ) δ 5.37 – 5.27 (m, 2H), 4.85 – 4.77 (m, 1H), 3.30 – 3.18 (m, 4H), 2.73 – 2.53 (m, 4H), 2.49 (t, J = 6.5Hz, 2H), 2.42 – 2.36 (m, 6H), 2.00 – 1.92 (m, 4H), 1.77 – 1.69 (m, 2H), 1.53 – 1.46 (m, 5H), 1.33 – 1.22 (m, 52H), 0.85 (t, J = 6.7 Hz, 15H). Compound number 5: 1 H NMR (400 MHz, Chloroform- d ) δ 5.40 – 5.29 (m, 2H), 4.90 – 4.80 (m, 1H), 3.31 – 3.15 (m, 4H), 2.46 – 2.17 (m, 13H), 2.01 – 1.92(m, 6H), 1.75 – 1.62 (m, 2H), 1.52 – 1.45 (m, 5H), 1.32 – 1.23 (m, 48H), 0.88 – 0.83 (m, 15H). Compound number 6: 1 H NMR (400 MHz, Chloroform- d) δ 5.37 – 5.24 (m, 2H), 4.88 – 4.79 (m, 1H), 3.31 – 3.11 (m, 4H), 2.51 – 2.37 (m, 3H), 2.36 – 2.15(m, 10H), 2.02 – 1.88 (m, 6H), 1.72 – 1.61 (m, 2H), 1.50 – 1.41 (m, 5H), 1.29 – 1.20 (m, 52H), 0.83 (t, J = 6.4 Hz, 15H). Compound number 7: 1 H NMR (400 MHz, Chloroform- d ) δ 5.40 – 5.26 (m, 2H), 4.89 – 4.79 (m, 1H), 3.31 – 3.14 (m, 4H), 2.56 – 2.49 (m, 2H), 2.43 – 2.17(m, 11H), 2.05 – 1.90 (m, 6H), 1.80 – 1.67 (m, 2H), 1.53 – 1.43 (m, 5H), 1.29 – 1.20 (m, 56H), 0.85 (t, J = 6.7 Hz, 15H). Compound number 8: 1 H NMR (400 MHz, ) δ 5.38 – 5.25 (m, 2H), 4.90 – 4.80 (m, 1H), 3.31 – 3.12 (m, 4H), 2.55 – 2.48 (m, 2H), 2.44 – 2.18 (m, 11H), 2.03– 1.90 (m, 6H), 1.78 – 1.65 (m, 2H), 1.54 – 1.43 (m, 5H), 1.30 – 1.20 (m,52H), 0.85 (t, J = 6.7 Hz, 15H). Compound number 9: 1 H NMR (400 MHz, Chloroform- d ) δ 5.35 – 5.26 (m, 2H), 4.86 – 4.77 (m, 1H), 3.30 – 3.12 (m, 4H), 2.46 (t, J0.83 (t, J = 6.5 Hz, 15H). Compound number 10: 1 H NMR (400 MHz, Chloroform- d ) δ 5.37 – 5.26 (m,2H), 4.87 – 4.77 (m, 1H), 3.28 – 3.10 (m, 4H), 2.46 (t, J = 7.1 Hz, 2H), 2.40 –2.13 (m, 11H), 2.02 – 1.90 (m, 4H), 1.74 – 1.60 (m, 6H), 1.53 – 1.41 (m, 5H), 1.27 – 1.20 (m, 52H), 0.84 (t, J = 6.5 Hz, 15H). Compound number 11: 1 H NMR (400 MHz, Chloroform- d ) δ 5.39 – 5.26 (m,2H), 4.88 – 4.79 (m, 1H), 3.31 – 3.14 (m, 4H), 2.45 (t, J = 7.1 Hz, 2H), 2.40 –2.16 (m, 11H), 2.03 – 1.92 (m, 4H), 1.74 – 1.61 (m, 6H), 1.52 – 1.44 (m, 5H), 1.31 – 1.22 (m, 56H), 0.85 (t, J = 6.6 Hz, 15H). Compound number 12: 1 H NMR (400 MHz, Chloroform- d ) δ 5.37 – 5.25 (m,2H), 4.86 – 4.80 (m, 1H), 3.33 – 3.15 (m, 4H), 2.44 (t, J= 7.1 Hz, 2H), 2.39 –2.15 (m, 11H), 2.02 – 1.92 (m, 4H), 1.75 – 1.60 (m, 6H), 1.52 – 1.44 (m, 5H), 1.30 – 1.20 (m, 52H), 0.85 (t, J = 6.6 Hz, 15H). Compound number 13: 1 H NMR (400 MHz, Chloroform- d ) δ 5.37 – 5.29 (m,2H), 4.88 – 4.78 (m, 1H), 3.31 – 3.15 (m, 4H), 2.57 – 2.21 (m, 13H), 2.03 –1.94 (m, 4H), 1.78 – 1.69 (m, 2H), 1.68 – 1.59 (m, 4H), 1.53 – 1.43 (m, 5H), 1.31 – 1.22 (m, 50H), 0.84 (t, J = 6.7 Hz, 15H). Compound number 14: 1 H NMR (400 MHz, Chloroform- d ) δ 5.39 – 5.30 (m,2H), 4.89 – 4.81 (m, 1H), 3.30 – 3.17 (m, 4H), 2.47 – 2.25 (m, 13H), 2.05 –1.96 (m, 4H), 1.74 – 1.62 (m, 6H), 1.54 – 1.46 (m, 5H), 1.40 – 1.25 (m, 54H), 0.87 (t, J = 6.7 Hz, 15H). Compound number 15: 1 H NMR (400 MHz, Chloroform- d ) δ 5.41 – 5.29 (m,2H), 4.93 – 4.77 (m, 1H), 3.29 – 3.15 (m, 4H), 2.36 – 2.22 (m, 13H), 2.04 –1.93 (m, 4H), 1.70 – 1.60 (m, 6H), 1.52 – 1.45 (m, 5H), 1.29 – 1.22 (m, 58H), 0.87 (t,J = 6.4 Hz, 15H). Compound number 16: 1 H NMR (400 MHz, Chloroform- d ) δ 5.38 – 5.26 (m,2H), 4.87 – 4.79 (m, 1H), 3.30 – 3.16 (m, 4H), 2.41 (t, J = 7.0 Hz, 2H), 2.32 –2.23 (m, 11H), 2.02 – 1.94 (m, 4H), 1.70 – 1.58 (m, 6H), 1.51 – 1.44 (m, 5H), 1.30 – 1.22 (m, 52H), 0.85 (t, J = 6.1 Hz, 15H). Compound number 17: 1 H NMR (400 MHz, Chloroform- d ) δ 5.41 – 5.26 (m,2H), 4.90 – 4.78 (m, 1H), 3.30 – 3.12 (m, 4H), 2.41 (t, J = 7.1 Hz, 2H), 2.35 –2.19 (m, 11H), 2.02 – 1.91 (m, 4H), 1.70 – 1.56 (m, 6H), 1.52 – 1.43 (m, 5H), 1.31 – 1.23 (m, 56H), 0.85 (t, J = 6.2 Hz, 15H). Compound number 18: 1 H NMR (400 MHz, Chloroform- d ) δ 5.42 – 5.27 (m,2H), 4.87 – 4.77 (m, 1H), 3.29 – 3.13 (m, 4H), 2.40 (t, J = 7.4 Hz, 2H), 2.36 –2.17 (m, 11H), 2.02 – 1.89 (m, 4H), 1.71 – 1.56 (m, 6H), 1.52 – 1.42 (m, 5H), 1.33 – 1.19 (m, 60H), 0.84 (t, J = 6.6 Hz, 15H). Compound number 19: 1 H NMR (400 MHz, Chloroform- d ) δ 5.42 – 5.25 (m,8H), 3.35 – 3.14 (m, 3H), 2.84 – 2.61 (m, 5H), 2.53 (s, 4H), 2.41 – 2.20 (m,3H), 2.11 – 1.96 (m, 8H), 1.93 – 1.75 (m, 1H), 1.71 – 1.48 (m, 3H), 1.39 –1.16 (m, 46H), 0.93 – 0.78 (m, 12H). Compound number 20: 1 H NMR (400 MHz, Chloroform- d ) δ 5.42 – 5.24 (m,2H), 4.89 – 4.77 (m, 1H), 3.33 – 3.06 (m, 4H), 2.46 – 2.18 (m, 13H), 2.02 –1.88 (m, 4H), 1.71 – 1.57 (m, 6H), 1.54 – 1.35 (m, 5H), 1.33 – 1.20 (m, 56H), 0.88 – 0.81 (m, 15H). Compound number 21: 1 H NMR (400 MHz, Chloroform- d ) δ 5.39 – 5.26 (m,8H), 3.27 – 3.04 (m, 3H), 2.74 (t, J = 6.4 Hz, 3H), 2.69 – 2.59 (m, 2H), 2.49(s, 4H), 2.37 – 2.19 (m, 2H), 2.06 – 1.94 (m, 7H), 1.67 – 1.58 (m, 3H), 1.55– 1.41 (m, 3H), 1.39 – 1.04 (m, 48H), 0.92 – 0.68 (m, 12H). Compound number 22: 1 H NMR (400 MHz, Chloroform- d) δ 5.41 – 5.27 (m,8H), 3.37 – 3.28 (m, 1H), 3.24 – 3.14 (m, 3H), 2.99 (q, J = 7.1 Hz, 4H), 2.89(t, J = 7.9 Hz, 2H), 2.80 – 2.69 (m, 5H), 2.39 – 2.19 (m, 3H), 2.08 – 1.92 (m,10H), 1.66 – 1.36 (m, 4H), 1.33 – 1.17 (m, 46H), 0.90 – 0.69 (m, 12H). The example used in this embodiment The synthesis flowchart is as follows Figure 10 As shown: Step I: Figure 10 The starting compound 1 (20 mmol) was dissolved in DCM (20 mL) and stirred at 0 °C. After adding DMF (2 mmol), oxalyl chloride (30 mmol) was added dropwise, and the reaction continued for 4 h. Triethylamine (60 mmol) and ammonia (30 mmol) were added sequentially, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was quenched with water, extracted with ethyl acetate, and the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was purified by column chromatography (silica gel column, eluent PE / EA = 50 / 1) to obtain... Figure 10 Intermediate compound 2 was obtained with a yield of 82%.
[0041] Step II: Figure 10 Intermediate compound 2 was dissolved in THF (40 mL) under a nitrogen atmosphere and stirred at 0 °C. After adding LAH (1 M in THF, 30 mmol), the reaction was raised to 50 °C and stirred overnight, monitored by TLC. After the reaction was complete, the reaction was quenched, extracted with ethyl acetate, and the combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain... The yield was 82%.
[0042] This embodiment uses The synthesis flowchart is as follows Figure 11 As shown: In a round-bottom flask, add the following ingredients in sequence: Figure 11 Middle dicarboxylic acid compound 1 (20 mmol). Figure 11The middle alcohol compound 2 (10 mmol), DMAP (5 mmol), DCM (20 mL), and EDCI (15 mmol) were stirred overnight at room temperature and monitored by TLC. After the reaction was complete, the solvent was removed under reduced pressure. The mixture was then purified by column chromatography (silica gel column, eluent PE / EA v / v = 5 / 1) to obtain... The yield was 85%. All carboxylic acid ester structures used in the examples can be prepared according to the above synthesis method.
[0043] The example used in this embodiment Synthesis methods such as Figure 12 As shown: Will Figure 12 10 mmol of the middle primary amine compound was added to ethyl formate (200 mmol), stirred overnight at 70 °C, and the ethyl formate was removed under reduced pressure. The system was then protected with nitrogen, and 20 mL of DCM and 100 mmol of triethylamine were added, with stirring at 0 °C. Phosphorus oxychloride (13 mmol) was slowly added dropwise using a constant pressure dropping funnel, and the reaction was stirred at room temperature for 4 h, monitored by TLC. After the reaction was complete, the reaction was quenched with 10 wt% potassium carbonate aqueous solution and stirred for 30 min. Finally, the mixture was extracted with ethyl acetate, the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was purified by column chromatography (silica gel column, eluent DCM / MeOH v / v = 10 / 1) to obtain... The yield was 36%. All isonitrile structures used in the examples can be prepared according to the above synthesis method.
[0044] Example 2: Preparation, particle size and potential determination of lipid nanoparticles: In this embodiment, firefly luciferase (Flu) was selected as the reporter gene marker mRNA, namely Flu mRNA. LNPs / mFluc (LNPs loaded with Flu mRNA) were prepared, and their formulation properties such as particle size, potential, and encapsulation efficiency were investigated to evaluate their formulation properties.
[0045] The preparation method of LNPs / mFluc is as follows: (1) Solution preparation: The ionizable lipid compound, DOPE, Chol, and DMG-PEG2000 (molar ratio 50:10:38.5:1.5) were dissolved in 0.5 mL of anhydrous ethanol to form the ethanol phase. At the same time, Fluc mRNA was dissolved in 1.5 mL of 50 mM (pH=4) citrate buffer solution to form the aqueous phase.
[0046] (2) LNP preparation: LNPs were prepared using a microfluidic instrument. The ethanol phase and aqueous phase obtained in step (1) were mixed at a volume ratio of 1:3 to obtain the initial LNPs / mFluc formulation. The flow rate of the ethanol phase was 3 mL / min and the flow rate of the aqueous phase was 9 mL / min.
[0047] (3) Ultrafiltration: The initial LNPs / mFluc formulation obtained in step (2) was placed in a dialysis bag and dialyzed in PBS buffer solution (prepared with RNase-free water) at pH 7.4 for 8 hours to collect the ethanol-free LNPs / mFluc formulation. The dialyzed formulation was then concentrated by ultrafiltration to obtain the final LNPs / mFluc formulation.
[0048] Pharmaceutical properties of LNPs / mFluc: Particle size, potential, and PDI determination: The particle size and potential of LNPs / mFluc were determined using a Malvern laser particle size analyzer. A certain volume of freshly prepared LNPs / mFluc solution was diluted 10 times with PBS, and its particle size, potential, and PDI were determined using a Malvern laser particle size analyzer. Each sample was measured in triplicate at a temperature of 25℃. The results are shown in Table 2.
[0049] Encapsulation efficiency testing: using Quant-iT TM RiboGreen TM The kit was used to test the encapsulation efficiency of each LNP / mFluc formulation. Representative LNP test results are shown in Table 2.
[0050] Table 2. Parameters of nanoparticles formed by encapsulating Fluc mRNA with lipid nanoparticles
[0051] As shown in Table 2, the average particle size of all lipid nanoparticles is in the range of 90 to 130 nm, the PDI is around 0.1, the particle size distribution is uniform, the potential is negative, and the encapsulation efficiency is above 80%.
[0052] Example 3: In vivo expression of LNPs / mFluc Furthermore, the ability of LNPs prepared from representative ionizable lipids of this invention to deliver mRNA in vivo was investigated. This invention selected firefly luciferase as the reporter gene marker mRNA, namely Fluc mRNA, and then prepared LNPs / mFluc (LNP-3, LNP-19, and LNP-20 prepared in Example 2) according to the method of Example 2. The ability of the LNPs / mFluc formulation system prepared from the compounds of this invention to express mRNA in vivo was investigated via tail vein administration.
[0053] LNP formulation containing 0.5 mg / kg Fluc-mRNA was injected into C57 mice via tail vein injection. Six hours after administration, the mice were intraperitoneally injected with fluorescein potassium substrate, and the bioluminescent signal in vivo was detected using an IVIS small animal in vivo imaging system. The results of the mouse in vivo imaging are shown in the figure below. Figure 1 The left side of the image Figure 2 The left side of the image and Figure 3 The left image is shown in the figure. Mice were euthanized, and their organs—heart, liver, spleen, lungs, and kidneys—were precisely isolated. The bioluminescence intensity of each isolated organ was detected using an IVIS instrument. The results are shown in the figure. Figure 1 The right side of the image Figure 2 The right side of the diagram and Figure 3 The right-hand side of the image.
[0054] In vivo expression studies via tail vein injection showed that, based on the total bioluminescence amount in the tail vein, the ionizable lipids of this invention can promote higher mRNA expression in mice. Furthermore, LNP-3 / mFluc, LNP-19 / mFluc, and LNP-20 / mFluc, after tail vein injection, can deliver mRNA to the spleen, where it is almost not expressed in the liver and other organs, achieving spleen-specific targeting. Among these, LNP-19 and LNP-20 showed better spleen-targeting effects, while LNP-3 was the most effective.
[0055] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. An ionizable lipid compound and its stereoisomers, tautomers, solvent compounds, or pharmaceutically acceptable salts, characterized in that, The ionizable lipid compound has the structure shown in formula (I): Formula (I); In formula (I): R1 is selected from , or ; R2 is selected from ; R3 is selected from or ; R4 is selected from , , , , , , , , , , , , , , , , , , , or .
2. The method for preparing the ionizable lipid compound according to claim 1, characterized in that, Includes the following steps: The ionizable lipid compound is obtained by mixing aldehydes, amines, isonitriles, carboxylic acids and organic solvents and carrying out a Ugi four-component reaction. The aldehyde compound has the structure shown in formula a, the amine compound has the structure shown in formula b, the isonitrile compound has the structure shown in formula c, and the carboxylic acid compound has the structure shown in formula d. Formula a; Formula b; Formula c; Formula d.
3. The preparation method according to claim 2, characterized in that, The molar ratio of the aldehydes, amines, isonitriles, and carboxylic acids is (0.1~1.5):(0.1~1.5):(0.1~1.5):(0.1~1.5).
4. The preparation method according to claim 2 or 3, characterized in that, The reaction of the four components of Ugi is carried out at a temperature of 25-40°C for 12-72 hours.
5. The use of the ionizable lipid compound of claim 1 and its stereoisomers, tautomers, solvent compounds or pharmaceutically acceptable salts in the preparation of spleen-targeting drugs.
6. A liposome formulation targeting the spleen, characterized in that, It includes ionizable lipids, neutral lipids, structured lipids, PEG-lipids, and bioactive components, wherein the ionizable lipids are one or more of the ionizable lipid compounds of claim 1, their stereoisomers, tautomers, solvent compounds, and pharmaceutically acceptable salts.
7. The spleen-targeting liposome formulation according to claim 6, characterized in that, The molar ratio of the ionizable lipid, neutral lipid, structural lipid, and PEG-lipid is (0~50):(5~80):(5~50):(0.1~10), and the ionizable lipid is not 0.
8. The spleen-targeting liposome formulation according to claim 6 or 7, characterized in that, The neutral lipids include one or more of the following: 1,2-distearatel-sn-glycero-3-phosphocholine, 1,2-dispalmitoyl-sn-glycero-3-phosphocholine, 1,2-dispalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and sphingomyelin. The structural lipids include one or more of cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, and rapeseed sterol; The PEG-lipids include one or more of 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide, 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)], PEG-disterol glycerol, PEG-dipalmitoyl, PEG-dioleoyl, PEG-distearate, PEG-diacylglycerol amide, PEG-dipalmitoylphosphatidylethanolamine, PEG-phosphatidylethanolamine, PEG-succinate diacylglycerol, PEG-ceramide, PEG-dialkoxypropylcarbamate, and PEG-1,2-dimyristoyloxypropyl-3-amine.
9. The spleen-targeting liposome formulation according to claim 6, characterized in that, The bioactive components include one or more of nucleic acid molecules, protein molecules, polypeptide molecules, and other small molecule drugs.
10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the spleen-targeting liposome formulation as described in any one of claims 6 to 9 and a pharmaceutically acceptable carrier.