Cationic lipid compounds, methods of making and using the same

Abstract

The application relates to the field of drug carriers, and specifically discloses a cationic lipid compound and a preparation method and application thereof, the cationic lipid compound comprising a compound shown in formula I; in the formula I, n=1, 2, 3 or 4; R1 and R2 are each independently selected from a secondary long-chain alkyl ester shown in formula II; in the formula II, n1=2, 4 or 6; n2=5, 7, 9 or 11; n3=5, 7, 9 or 11. The cationic lipid compound is combined with a phospholipid, cholesterol and a polyethylene glycol lipid to form an LNP carrier, and then a nucleic acid drug is encapsulated to form a corresponding drug. The cationic lipid compound has the advantages of easy production raw material, simple synthesis steps, good drug delivery effect, efficient entry into a receptor cell and activation of an immune response of the body, and practical application value.

Description

Technical Field

[0001] This application relates to the technical field of drug carriers, and more specifically, to cationic lipid compounds, their preparation methods, and applications. Background Technology

[0002] Gene therapy, a new technology born from the combination of modern medicine and molecular biology, involves introducing a target gene into a patient's body to correct or compensate for diseases caused by defective or abnormal genes, thereby achieving therapeutic effects. As a novel approach to disease treatment, gene therapy has already seen some successful applications, and further scientific breakthroughs will continue to drive its development into mainstream medicine. Common gene therapies include plasmid DNA (pDNA), antisense oligonucleotides (antisense ODN), small interfering RNA (siRNA), small hairpin RNA (shRNA), and messenger RNA (mRNA). mRNA is a type of single-stranded ribonucleic acid containing specific genetic information. It can deliver this genetic information to ribosomes within cells, where it serves as a template to guide the body in synthesizing specific target proteins. Compared to traditional protein drugs that struggle to cross cell membranes, mRNA's mechanism of action allows it to deliver transmembrane or intracellular proteins, making the treatment or prevention of more diseases possible. These advantages of mRNA also make it a trend in future drug development.

[0003] The key to gene therapy lies in delivering gene drugs to target cells within the body to exert their effects. However, directly introducing exogenous genes into the body will result in degradation by nucleases, breaking them down into small nucleotide molecules before they reach the target cells, thus rendering them ineffective. Therefore, the crucial element for achieving gene therapy is the development of an efficient and safe gene delivery system.

[0004] Gene vectors undergo several complex processes when delivering genes: reaching target cells via blood circulation, cellular uptake, endosome escape, intracellular movement, and release of gene material from the vector. The main obstacles are extracellular barriers caused by the complex blood environment and intracellular barriers caused by lysosomal enzyme degradation. Therefore, finding suitable gene vectors that enable target genes to reach their target sites and exert their effects is a pressing problem for gene vector researchers.

[0005] Currently, gene delivery vector systems are mainly divided into two categories: viral vector systems and non-viral vector systems. Viral vectors are a natural vector resource; viral genomes have simple structures, high transfection efficiency, and strong target cell specificity. However, they have poor targeting, low carrying capacity, and possess immunogenicity and potential tumorigenicity, making them difficult to meet the requirements for clinical application. Therefore, non-viral vector systems, which are diverse, non-immunogenic, and easy to produce, have received much attention in recent years and have been applied in many therapeutic areas.

[0006] The most commonly used non-viral vector system is the lipid carrier. Lipid carriers typically contain positively charged cationic lipids, which bind to negatively charged gene drugs through electrostatic interactions, thereby concentrating and packaging the gene material into smaller particles, forming lipid nanoparticles (LNPs). LNPs have shown unparalleled advantages over other types of liposomes in the preparation of gene delivery vectors and cell transfection. The smaller particle size of the complex reduces the chance of recognition, phagocytosis, and clearance by macrophages in vivo, improving the in vivo bioavailability of the drug. Simultaneously, for tumor tissues, the smaller particle size of the complex makes it easier to permeate through the intercellular spaces of vascular endothelial cells into the tumor parenchyma through osmosis and retention effects, increasing drug accumulation in tumor tissues. In terms of transfection, because the cell surface is slightly negatively charged, positively charged liposomes are more easily adsorbed onto the cell surface and enter the cell through mechanisms such as endocytosis, greatly increasing the transfection capacity of liposomes.

[0007] Currently, low-level viral vectors (LNPs) have become the most widely used non-viral vectors due to their simple structure, ease of operation, and high biocompatibility. However, the preparation process of most LNPs is complex and not easily scaled up for mass production. Therefore, providing a non-viral vector system with a simple preparation method and good therapeutic effect has become an urgent problem to be solved. Summary of the Invention

[0008] To improve drug delivery efficiency of gene vectors and simplify their synthesis, this application provides cationic lipid compounds, their preparation methods, and applications. These cationic lipid compounds are positively charged, making them more readily absorbed into cells, thus delivering drugs more effectively and producing better therapeutic effects. Furthermore, they are simple to synthesize, easy to prepare, and have practical application value.

[0009] In the first aspect, this application provides a cationic lipid compound, employing the following technical solution:

[0010] Cationic lipid compounds, including compounds represented by Formula I;

[0011]

[0012] In formula I, n = 1, 2, 3 or 4;

[0013] R1 and R2 are each independently selected from secondary long-chain alkyl esters represented by Formula II;

[0014]

[0015] In Formula II, n1 = 2, 4 or 6; n2 = 5, 7, 9 or 11; n3 = 5, 7, 9 or 11.

[0016] In this application, an amphiphilic derivative obtained by combining 3-((2-(dimethylamino)ethane)(methyl)amino)propionic acid with a secondary long-chain alkyl ester is used as a cationic lipid component in a gene drug carrier system. This positively charged derivative can better deliver gene drugs, resulting in better gene therapy efficacy. The synthesis of such cationic lipid compounds is simple, the raw materials are readily available, and large-scale production is possible.

[0017] In this application, the cationic lipid compound contains three ester groups. The spatial position of these three ester groups directly affects the function of the cationic lipid compound, while the number of carbon atoms on the alkyl chain affects the spatial structure of the ester group. Experimental verification shows that when n = 1, 2, 3 or 4, n1 = 2, 4 or 6, n2 = 5, 7, 9 or 11, and n3 = 5, 7, 9 or 11, the cationic compound has the best loading and delivery effect on gene drugs, thereby achieving the best therapeutic effect.

[0018] As a preferred technical solution, the cationic lipid compounds described in this application include compounds represented by formula III, IV, V, VI or VII.

[0019]

[0020]

[0021]

[0022]

[0023]

[0024] Secondly, this application provides a method for preparing a cationic lipid compound, which adopts the following technical solution: A method for preparing a cationic lipid compound, the method comprising the following steps:

[0025] Using brominated esters as raw materials, alkylation and reduction reactions are carried out sequentially.

[0026] The compound is then subjected to a condensation reaction with an acyl chloride compound, followed by a two-step substitution reaction and a single hydrolysis reaction. Finally, it is subjected to an esterification reaction with a monohydric secondary alcohol compound to obtain the cationic lipid compound.

[0027] As a preferred technical solution, the preparation method of the cationic lipid compound includes the following steps:

[0028] (1) Bromoesters react with TosMIC (p-toluenesulfonyl isonitrile) and NaH to form intermediate 2;

[0029] (2) Intermediate product 2 reacts under acidic conditions to generate intermediate product 3;

[0030] (3) Intermediate product 3 undergoes a reduction reaction with NaBH4 to generate intermediate product 4;

[0031] (4) Intermediate product 4 undergoes a condensation reaction with acyl chloride compounds to generate intermediate product 5;

[0032] (5) Intermediate product 5 undergoes a substitution reaction with NaI to generate intermediate product 6;

[0033] (6) Intermediate product 6 undergoes a substitution reaction with N,N,N'-trimethylethylenediamine to generate intermediate product 7;

[0034] (7) Intermediate product 7 undergoes hydrolysis reaction with LiOH to generate intermediate product 8;

[0035] (8) Intermediate product 8 undergoes esterification with a monohydric secondary alcohol to obtain the cationic lipid compound.

[0036] In this application, the raw material in step (1) is a brominated ester compound. In the actual production process, the specific raw material can be selected according to the structure of the specific cationic lipid compound synthesized.

[0037] Preferably, the brominated ester compounds include ethyl 8-bromooctanoate, ethyl 6-bromohexanoate, or ethyl 4-bromobutyrate.

[0038] Preferably, in step (1), TBAI (tetrabutylammonium iodide) is used as a catalyst for the reaction.

[0039] Preferably, in step (1), the solvent for the reaction includes a DMSO (dimethyl sulfoxide) solution.

[0040] Preferably, in step (1), the reaction is carried out at room temperature.

[0041] Preferably, in step (2), the solvent for the reaction includes a mixed solution of DCM (dichloromethane) and concentrated sulfuric acid, with a volume ratio of DCM:concentrated sulfuric acid = (3.5-8):1.

[0042] In this application, concentrated sulfuric acid provides an acidic environment for the reaction in step (2).

[0043] Preferably, in step (2), the reaction is carried out at room temperature.

[0044] In this application, intermediate 3 is a symmetrical ketone compound.

[0045] Preferably, in step (3), the solvent for the reaction includes a mixed solution of THF (tetrahydrofuran) and ethanol, with a volume ratio of THF:ethanol = (2-4):1.

[0046] Preferably, in step (3), the reaction is carried out at room temperature.

[0047] In this application, intermediate product 4 is an alcohol compound.

[0048] In this application, the acyl chloride compounds in step (4) are a general term for a class of compounds containing different numbers of carbon atoms and whose ends are not substituted with halogens. In the actual production process, they can be specifically selected according to the structure of the specific cationic lipid compound synthesized.

[0049] Preferably, the acyl chloride compound includes 3-chloropropionyl chloride, 2-chloroacetyl chloride, 4-chlorobutyryl chloride, or 5-chlorovaleryl chloride.

[0050] Preferably, in step (4), the solvent for the reaction includes a DCM solution containing pyridine, wherein the concentration of pyridine is 73-134 mM.

[0051] Preferably, in step (4), the reaction is carried out at room temperature.

[0052] Preferably, in step (5), the solvent for the reaction includes an acetone solution.

[0053] Preferably, in step (5), the reaction is carried out at 68-72°C. The reaction temperature can be, for example, 68°C, 70°C, 72°C, 68-70°C, 68-72°C, or 70-72°C.

[0054] Preferably, in step (6), the solvent for the reaction includes a DCM solution.

[0055] Preferably, in step (6), the reaction is carried out at room temperature.

[0056] Preferably, in step (7), the solvent for the reaction includes a mixed solution of ethanol and water, with a volume ratio of ethanol:water = 1:(1.5-3).

[0057] Preferably, in step (7), the reaction is carried out at room temperature.

[0058] Preferably, in step (8), DMAP (4-dimethylaminopyridine) and DCC (dicyclohexylcarbodiimide) are used as catalysts for the reaction.

[0059] Preferably, in step (8), the solvent for the reaction includes a DCM solution.

[0060] Preferably, in step (8), the reaction is carried out at room temperature.

[0061] In this application, in step (8), the monohydric secondary alcohol compound is a monohydric alcohol containing different numbers of carbon atoms, and the hydroxyl group is not at position 1. In the actual production process, it can be specifically selected according to the structure of the specific cationic lipid compound synthesized.

[0062] Preferably, the monohydric secondary alcohol compound includes 13-eicosalicol, 11-eicosalicol, 9-heptadecylol, 7-pentadecanol, or 9-eicosalicol.

[0063] Thirdly, this application provides an LNP vector, which adopts the following technical solution:

[0064] An LNP carrier comprising any one or a combination of at least two of the cationic lipid compounds described in the first aspect.

[0065] In this application, the cationic lipid compound in the LNP carrier can be a single cationic lipid compound or a combination of multiple cationic lipid compounds.

[0066] Preferably, the cationic lipid compound in the LNP carrier is 15% to 70% in molar fraction, for example, it can be 15%, 30%, 45%, 60%, 70%, 15% to 30%, 15% to 45%, 15% to 60%, 15% to 70%, 30% to 45%, 30% to 60%, 30% to 70%, 45% to 60%, 45% to 70%, or 60% to 70%, etc.

[0067] In this application, when the cationic lipid compound in the LNP carrier is a single compound, its molar fraction in the LNP carrier is 15% to 70%; when the cationic lipid compound in the LNP carrier is a combination of multiple cationic lipid compounds, the total molar fraction of the combination of multiple cationic lipid compounds in the LNP carrier is 15% to 70%.

[0068] Preferably, the LNP carrier further comprises, by mole fraction, 8%–30% phospholipids, 15%–65% cholesterol, and 1.5%–3% polyethylene glycol lipids.

[0069] Preferably, the molar fraction of the phospholipid in the LNP carrier is 8% to 30%, for example, it can be 8%, 10%, 15%, 20%, 25%, 30%, 8% to 10%, 8% to 15%, 8% to 20%, 8% to 25%, 8% to 30%, 10% to 15%, 10% to 20%, 10% to 25%, 10% to 30%, 15% to 20%, 15% to 25%, 15% to 30%, 20% to 25%, 20% to 30%, or 25% to 30%, etc.

[0070] Preferably, the molar fraction of cholesterol in the LNP carrier is 15% to 65%, for example, it can be 15%, 30%, 45%, 65%, 15% to 30%, 15% to 45%, 15% to 65%, 30% to 45%, 30% to 65%, or 45% to 65%, etc.

[0071] Preferably, the polyethylene glycol lipid in the LNP carrier has a molar fraction of 1.5% to 3%, for example, it can be 1.5%, 2%, 2.5%, 3%, 1.5% to 2%, 1.5% to 2.5%, 1.5% to 3%, 2% to 2.5%, 2% to 3%, or 2.5% to 3%, etc.

[0072] As a preferred technical solution, the LNP carrier comprises, by mole fraction, 15%–70% cationic lipid compounds, 8%–30% phospholipids, 15%–65% cholesterol, and 1.5%–3% polyethylene glycol lipids.

[0073] In this application, by optimizing the components of the LNP carrier and their respective addition amounts, a synergistic effect is achieved, resulting in a larger total amount of drug loaded, better adsorption to cells, and thus better drug loading, improved drug utilization, and better therapeutic effect.

[0074] Fourthly, this application provides a drug, which adopts the following technical solution:

[0075] A drug comprising the LNP carrier described in the third aspect.

[0076] Preferably, the drug further includes a nucleic acid drug.

[0077] Preferably, in the drug, the molar ratio (i.e. N / P molar ratio) of the nitrogen content of the cationic lipid compound in the LNP carrier to the phosphorus content in the nucleic acid drug is (2-6):1, for example, it can be 2:1, 3:1, 4:1, 5:1, 6:1, (2-3):1, (2-4):1, (2-5):1, (2-6):1, (3-4):1, (3-5):1, (3-6):1, (4-5):1, (4-6):1 or (5-6):1, etc.

[0078] In this application, the ratio of cationic lipid compounds to nucleic acid drugs directly affects the encapsulation efficiency of the drug and the subsequent drug release effect. Experimental verification shows that when the molar ratio of nitrogen content in the cationic lipid compounds in the LNP carrier to phosphorus content in the nucleic acid drug is (2-6):1, the encapsulation efficiency of the drug is the highest, the therapeutic effect is the best, and the utilization rate of raw materials can be improved, thus saving production costs.

[0079] Fifthly, this application provides a method for preparing the above-mentioned drug, which adopts the following technical solution:

[0080] A method for preparing a drug, the method comprising the following steps:

[0081] Weigh out all components except cationic lipid compounds, dissolve them, and prepare a stock solution;

[0082] A cationic lipid compound was added to the stock solution and mixed to obtain a lipid alcohol phase;

[0083] Dilute the nucleic acid drug to prepare an aqueous phase of nucleic acid;

[0084] The lipid alcohol phase and the nucleic acid aqueous phase are mixed to prepare a pharmaceutical intermediate, which is then dialyzed to obtain the drug.

[0085] Preferably, anhydrous ethanol is used as a solvent when dissolving phospholipids, cholesterol, and polyethylene glycol lipids.

[0086] Preferably, when diluting nucleic acid drugs, an acidic buffer solution is used for dilution.

[0087] In this application, a dialysis apparatus is used to perform dialysis in order to remove the solvent.

[0088] Sixthly, this application provides a vaccine, which adopts the following technical solution:

[0089] A vaccine comprising the LNP vector described in the third aspect.

[0090] Seventhly, this application provides the use of the cationic lipid compound described in the first aspect in the preparation of LNP carriers, drugs or vaccines, or the use of the LNP carrier described in the third aspect in the preparation of drugs and / or vaccines.

[0091] In summary, this application has the following beneficial effects:

[0092] 1. This application uses an amphiphilic derivative obtained by combining 3-((2-(dimethylamino)ethane)(methyl)amino)propionic acid and a secondary long-chain alkyl ester as a cationic lipid component in a gene drug carrier system. It carries a positive charge, which makes it easier to adsorb onto the cell surface and enter the cell, resulting in higher drug delivery efficiency and better therapeutic effect. The raw materials are readily available, the preparation method is simple, and the synthesis efficiency is high, which creates conditions for large-scale production.

[0093] 2. The LNP carrier prepared using the above-mentioned cationic lipid compounds has good drug loading and delivery efficiency, with an encapsulation rate of over 88%. After being formulated into drugs with nucleic acid molecules, it can accurately and efficiently enter recipient cells and express related proteins, thereby triggering the body's immune response. It can then be used as a therapeutic drug to treat diseases or as a vaccine to activate immunity, showing broad application prospects. Attached Figure Description

[0094] Figure 1 This is a schematic diagram of the synthetic route for the cationic lipid compound TM3 in this application.

[0095] Figure 2 This is a 1H NMR spectrum image of intermediate product 2 from preparation example 1 of this application.

[0096] Figure 3 This is a 1H NMR spectrum image of intermediate product 3 in Preparation Example 1 of this application.

[0097] Figure 4 This is a 1H NMR spectrum image of intermediate product 4 in Preparation Example 1 of this application.

[0098] Figure 5 This is a 1H NMR spectrum image of intermediate product 5 from Preparation Example 1 of this application.

[0099] Figure 6 This is a 1H NMR spectrum image of intermediate product 7 from Preparation Example 1 of this application.

[0100] Figure 7 This is a hydrogen spectrum image of the cationic lipid compound TM3 prepared in Example 1 of this application.

[0101] Figure 8 This is an image showing the detection results of the protein expression level of drug TP1 in 293T cells in Example 1 of this application.

[0102] Figure 9 This is an image showing the detection results of anti-S protein antibody titer in mouse serum in Example 2 of this application. Detailed Implementation

[0103] This application provides cationic lipid compounds, which include compounds represented by Formula I;

[0104]

[0105] In formula I, n = 1, 2, 3 or 4;

[0106] R1 and R2 are each independently selected from secondary long-chain alkyl esters represented by Formula II;

[0107]

[0108] In Formula II, n1 = 2, 4 or 6; n2 = 5, 7, 9 or 11; n3 = 5, 7, 9 or 11.

[0109] Specifically, the cationic lipid compound includes TM1 represented by Formula III, TM2 represented by Formula IV, TM3 represented by Formula V, TM4 represented by Formula VI, or TM5 represented by Formula VII.

[0110] Specifically, this application uses TM3 as an example to illustrate the preparation method of the cationic lipid compound, and its synthetic route is shown in the schematic diagram below. Figure 1 As shown, the specific process is as follows:

[0111] (1) Ethyl 8-bromooctanoate in DMSO solution, with TBAI as catalyst, undergoes alkylation reaction with TosMIC and NaH at room temperature to generate intermediate product 2: 18-22 mmol ethyl 8-bromooctanoate is dissolved in 55-65 mL of anhydrous DMSO, stirred at 8-15 °C for 5-10 min, 8-12 mmol TosMIC is added, stirred for 5-10 min, 23-28 mmol NaH is added in multiple portions, and finally 1.8-2.2 mmol TBAI is added, the temperature is slowly raised to room temperature, and stirred for 1-3 h.

[0112] The reaction progress was monitored by TLC. When the reaction was complete, the system was cooled in an ice-water bath and quenched with 145–155 mL of ice water. The mixture was then extracted three times with DCM, 90–110 mL each time. All organic phases were collected, washed with 90–110 mL of water, and then washed twice with saturated sodium bicarbonate, 145–155 mL each time. After drying and concentration, the mixture was purified by silica gel column chromatography to obtain intermediate 2.

[0113] (2) Intermediate product 2 reacts in an acidic mixed solution of DCM and concentrated sulfuric acid (volume ratio of (3.5–8):1) at room temperature to produce intermediate product 3:

[0114] Weigh 3.9–4.2 g of intermediate product 2 and dissolve it in 45–55 mL of DCM solution, stirring for 3–7 min. Add 7–12 mL of concentrated sulfuric acid and stir at room temperature for 2–5 h.

[0115] The reaction progress was monitored by TLC. After the reaction was complete, 45-55 mL of water was added to the system, mixed well, and allowed to stand for separation. The aqueous layer was extracted with 45-55 mL of DCM. All organic phases were combined and eluted with 45-55 mL of saturated sodium bicarbonate. After drying and concentration, the organic phases were purified by silica gel column chromatography to obtain intermediate product 3.

[0116] (3) Intermediate product 3 undergoes a reduction reaction with NaBH4 at room temperature in a mixed solution of THF and ethanol (volume ratio of (2-4):1) to generate intermediate product 4:

[0117] Weigh 3–5 mmol of intermediate 3 and dissolve it in 45–55 mL of a mixed solution of THF and ethanol (volume ratio of (2–4):1), and stir at 0–4 °C for 3–7 min. Slowly add 3–5 mmol of NaBH4 in several portions and react at room temperature for 3–5 h.

[0118] The reaction progress was monitored by TLC. After the reaction was complete, 90–110 mL of ice water was added to quench the reaction. The mixture was then extracted three times using DCM, with 90–110 mL extracted each time. All organic phases were combined and eluted with 45–55 mL of saturated sodium bicarbonate. After drying and concentration, the organic phase was purified by silica gel column chromatography to obtain intermediate product 4.

[0119] (4) Intermediate 4 undergoes a condensation reaction with 3-chloropropionyl chloride in a DCM solution containing pyridine (pyridine concentration of 73–134 mM) at room temperature to generate intermediate 5:

[0120] Weigh 4–6 mmol of intermediate 4 and dissolve it in 45–55 mL of DCM solution. Then add 4–6 mmol of pyridine and stir at 0–4 °C for 3–7 min. Slowly add 7–9 mmol of 3-chloropropionyl chloride and react at room temperature for 0.5–2 h.

[0121] The reaction progress was monitored by TLC. After the reaction was complete, 90-110 mL of ice water was added to quench the reaction. The mixture was then washed with 90-110 mL of water and then washed twice with 140-160 mL of saturated sodium bicarbonate solution each time. After drying and concentration, the mixture was purified by silica gel column chromatography to obtain intermediate product 5.

[0122] (5) Intermediate product 5 undergoes a substitution reaction with NaI in acetone solution at 68–72 °C to generate intermediate product 6:

[0123] Weigh 3-5 mmol of intermediate product 5 and dissolve it in 45-55 mL of acetone solution. Slowly add 3-6 mmol of NaI, raise the temperature to 68-72 °C, and react for 12-18 h.

[0124] The reaction progress was monitored by TLC. After the reaction was complete, the organic phase was eluted with 45-55 mL of saturated sodium thiosulfate, dried and concentrated, and then purified by silica gel column chromatography to obtain intermediate product 6.

[0125] (6) Intermediate product 6 undergoes a substitution reaction with N,N,N'-trimethylethylenediamine in DCM solution at room temperature to generate intermediate product 7:

[0126] Weigh 3–5 mmol of intermediate product 6 and dissolve it in 45–55 mL of DCM solution. Add 5–7 mmol of N,N,N'-trimethylethylenediamine and stir the mixture at room temperature for 1–3 h.

[0127] The reaction progress was monitored by TLC. After the reaction was complete, the organic phase was eluted with 45-55 mL of saturated sodium bicarbonate, dried and concentrated, and then purified by silica gel column chromatography to obtain intermediate product 7.

[0128] (7) Intermediate product 7 undergoes hydrolysis with LiOH at room temperature in a mixed solution of ethanol and water (volume ratio 1:(1.5~3)) to generate intermediate product 8:

[0129] Weigh 4–6 mmol of intermediate product 7 and 18–22 mmol of LiOH and dissolve them in 90–110 mL of a mixed solution of ethanol and water (volume ratio 1:(1.5–3)). Stir at room temperature for 4–7 h.

[0130] The reaction progress was monitored by TLC. After the reaction was complete, the ethanol was evaporated and the mixture was acidified to a pH of approximately 2 with 1.5–2.5 M hydrochloric acid. The mixture was then extracted three times with 90–110 mL of DCM each time. All organic phases were combined and eluted with 45–55 mL of saturated sodium bicarbonate. After drying and concentration, the organic phase was purified by silica gel column chromatography to obtain intermediate 8.

[0131] (8) Intermediate product 8 undergoes esterification with 9-heptadecyl alcohol at room temperature in DCM solution under the action of DMAP and DCC to generate the cationic lipid compound TM3:

[0132] Weigh 4–6 mmol of intermediate product 8, 14–16 mmol of DMAP and 16–20 mmol of 9-heptadecyl alcohol and dissolve them in 55–65 mL of DCM. Stir for 3–7 min, add 13–17 mmol of DCC, and stir overnight at room temperature.

[0133] The reaction progress was monitored by TLC. After the reaction was complete, the mixture was washed three times with water, 55–65 mL each time. It was then washed twice with saturated sodium bicarbonate, 45–55 mL each time. After drying and concentration, the mixture was purified by silica gel column chromatography to obtain the cationic lipid compound TM3.

[0134] This application also provides an LNP carrier, which, by mole fraction, comprises 15%–70% cationic lipid compound, 8%–30% phospholipid, 15%–65% cholesterol, and 1.5%–3% polyethylene glycol lipid.

[0135] This application also provides a drug comprising an LNP carrier and a nucleic acid drug, wherein the molar ratio of the nitrogen content of the cationic lipid compound in the LNP carrier to the phosphorus content in the nucleic acid drug is (2-6):1.

[0136] The drug is prepared by the following method:

[0137] Weigh 1.5–1.8 mg of phospholipids, 1.7–1.9 mg of cholesterol, and 0.8–1 mg of polyethylene glycol lipids, and dissolve them in 0.8–1.5 mL of anhydrous ethanol at 55–65 °C to prepare a stock solution;

[0138] 13–15 mg of a cationic lipid compound was added to the stock solution at room temperature and mixed to obtain a lipid alcohol phase.

[0139] The nucleic acid drug was diluted with an acidic buffer solution to a final concentration of 0.5–0.7 mg / mL, mixed thoroughly, and a nucleic acid aqueous phase was prepared. The lipid alcohol phase and the nucleic acid aqueous phase were mixed at a volume ratio of 1:(2–5) at room temperature using a mixer to prepare a drug intermediate. The intermediate was then dialyzed to remove the solvent and replace it with a Tris solution containing 0.2%–0.5% sodium chloride and 4%–6% sucrose at a concentration of 18–22 mM to obtain the drug.

[0140] The following is in conjunction with the appendix Figures 1-9 Preparation Examples 1-2 and Examples 1-2 further illustrate this application in detail.

[0141] Preparation Example

[0142] Preparation Example 1

[0143] This preparation example provides a cationic lipid compound TM3, the structural formula of which is shown in Formula V.

[0144]

[0145] The cationic lipid compound TM3 was prepared by the following method, and the synthetic route is shown in the figure below. Figure 1 As shown:

[0146] (1) Ethyl 8-bromooctanoate reacted with TosMIC and NaH in DMSO solution with TBAI as a catalyst at room temperature to produce intermediate 2:

[0147] In a 500 mL single-necked flask, weigh 5 g of ethyl 8-bromooctanoate (20 mmol) and dissolve it in 60 mL of anhydrous DMSO. Stir at 10 °C for 7 min, add 1.9 g of TosMIC (10 mmol), stir for 7 min, add 1 g of NaH (25 mmol) in several portions, and finally add 0.7 g of TBAI (2 mmol). Slowly raise the temperature from 10 °C to room temperature and stir for 2 h.

[0148] The reaction progress was monitored by TLC. Once the reaction was complete, the system was cooled in an ice-water bath and quenched with 150 mL of ice water. The mixture was then extracted three times with 100 mL of DCM each time. All organic phases were collected, washed with 100 mL of water, and then washed twice with saturated sodium bicarbonate (150 mL each time). After drying and concentration, the mixture was purified by silica gel column chromatography to obtain intermediate 2. The 1H NMR spectrum of intermediate 2 is shown below. Figure 2 As shown.

[0149] (2) Intermediate product 2 reacts in an acidic mixed solution of DCM and concentrated sulfuric acid (volume ratio 5:1) at room temperature to produce intermediate product 3:

[0150] Weigh 4.1 g of intermediate product 2 into a 250 mL single-necked flask, dissolve it in 50 mL of DCM solution, and stir for 5 min. Add 10 mL of concentrated sulfuric acid and stir for 3 h at room temperature.

[0151] The reaction progress was monitored by TLC. After the reaction was complete, 50 mL of water was added to the system, mixed, and allowed to stand for phase separation. The aqueous layer was extracted with 50 mL of DCM, and all organic phases were combined. The organic phase was eluted with 50 mL of saturated sodium bicarbonate, dried, concentrated, and purified by silica gel column chromatography to obtain intermediate 3. The 1H NMR spectrum of intermediate 3 is shown below. Figure 3 As shown.

[0152] (3) Intermediate product 3 undergoes a reduction reaction with NaBH4 at room temperature in a mixed solution of THF and ethanol (volume ratio 3:1) to generate intermediate product 4:

[0153] In a 250 mL single-necked flask, 1.5 g of intermediate product 3 (4 mmol) was weighed and dissolved in 50 mL of a mixed solution of THF and ethanol (volume ratio 3:1), and stirred at 0 °C for 5 min. 0.15 g of NaBH4 (4 mmol) was slowly added in several portions, and the reaction was carried out at room temperature for 4 h.

[0154] The reaction progress was monitored by TLC. After the reaction was complete, 100 mL of ice water was added to quench the reaction. The mixture was then extracted three times using DCM, 100 mL each time. All organic phases were combined, eluted with 50 mL of saturated sodium bicarbonate, dried, concentrated, and purified by silica gel column chromatography to obtain intermediate 4. The 1H NMR spectrum of intermediate 4 is shown below. Figure 4 As shown.

[0155] (4) Intermediate 4 undergoes a condensation reaction with 3-chloropropionyl chloride at room temperature in a DCM solution containing pyridine (pyridine concentration of 100 mM) to generate intermediate 5:

[0156] In a 250 mL single-necked flask, 1.8 g of intermediate product 4 (5 mmol) was weighed and dissolved in 50 mL of DCM solution. Then, 0.4 g of pyridine (5 mmol) was added, and the mixture was stirred at 0 °C for 5 min. 1 g of 3-chloropropionyl chloride (8 mmol) was slowly added, and the mixture was reacted at room temperature for 1 h.

[0157] The reaction progress was monitored by TLC. After the reaction was complete, 100 mL of ice water was added to quench the reaction, followed by washing with 100 mL of water, and then washing twice with 150 mL of saturated sodium bicarbonate solution each time. After drying and concentration, the product was purified by silica gel column chromatography to obtain intermediate product 5. The 1H NMR spectrum of intermediate product 5 is shown below. Figure 5 As shown.

[0158] (5) Intermediate product 5 undergoes a substitution reaction with NaI in acetone solution at 70°C to generate intermediate product 6:

[0159] In a 250 mL single-necked flask, 1.8 g of intermediate product 5 (4 mmol) was weighed and dissolved in 50 mL of acetone solution. 0.8 g of NaI (5 mmol) was slowly added, and the temperature was raised to 70 °C. The reaction was allowed to proceed for 16 h.

[0160] The reaction progress was monitored by TLC. After the reaction was complete, the organic phase was eluted with 50 mL of saturated sodium thiosulfate, dried and concentrated, and then purified by silica gel column chromatography to obtain intermediate product 6.

[0161] (6) Intermediate product 6 undergoes a substitution reaction with N,N,N'-trimethylethylenediamine in DCM solution at room temperature to generate intermediate product 7:

[0162] In a 250 mL single-necked flask, 2.2 g of intermediate product 6 (4 mmol) was weighed and dissolved in 50 mL of DCM solution. 0.6 g of N,N,N'-trimethylethylenediamine (6 mmol) was added, and the mixture was stirred at room temperature for 2 h.

[0163] The reaction progress was monitored by TLC. After the reaction was complete, the organic phase was eluted with 50 mL of saturated sodium bicarbonate, dried, concentrated, and purified by silica gel column chromatography to obtain intermediate 7. The 1H NMR spectrum of intermediate 7 is shown below. Figure 6 As shown.

[0164] (7) Intermediate product 7 undergoes hydrolysis with LiOH in a mixed solution of ethanol and water (volume ratio 1:2) at room temperature to produce intermediate product 8:

[0165] In a 250 mL single-necked flask, 3.8 g of intermediate product 7 (5 mmol) and 0.5 g of LiOH (20 mmol) were weighed and dissolved in 100 mL of a mixed solution of ethanol and water (volume ratio 1:2). The solution was stirred at room temperature for 5 h.

[0166] The reaction progress was monitored by TLC. After the reaction was complete, the ethanol was evaporated and the solution was acidified to a pH of approximately 2 with 2M hydrochloric acid. The solution was then extracted three times using DCM, 100 mL each time. All organic phases were combined and eluted with 50 mL of saturated sodium bicarbonate. After drying and concentration, the solution was purified by silica gel column chromatography to obtain intermediate 8.

[0167] (8) Intermediate product 8 undergoes esterification with 9-heptadecyl alcohol in DCM solution under the action of DMAP and DCC at room temperature to generate the cationic lipid compound TM3:

[0168] In a 250 mL single-necked flask, weigh 3.6 g of intermediate 8 (5 mmol), 1.8 g of DMAP (15 mmol) and 4.6 g of 9-heptadecyl alcohol (18 mmol) and dissolve them in 60 mL of DCM. Stir for 5 min, add 3.1 g of DCC (15 mmol), and stir overnight at room temperature.

[0169] The reaction progress was monitored by TLC. After the reaction was complete, the mixture was washed three times with water (60 mL each time). It was then washed twice with saturated sodium bicarbonate (50 mL each time). After drying and concentration, the mixture was purified by silica gel column chromatography to obtain the cationic lipid compound TM3. The 1H NMR spectrum of TM3 is shown below. Figure 7 As shown.

[0170] The statistical results of the quality and yield of the products obtained in each step of the above reaction are shown in Table 1.

[0171] Table 1. Statistical results of the quality and yield of the products obtained in each step.

[0172] product Mass (g) Yield (%) Intermediate product 2 4.7 94 Intermediate product 3 3.7 62 Intermediate product 4 1.0 67 Intermediate product 5 1.9 82 Intermediate product 6 1.8 81 Intermediate product 7 1.8 60 Intermediate product 8 3 83 TM3 3.3 70

[0173] Preparation Example 2

[0174] This preparation example provides a drug TP1, wherein the nucleic acid drug is mRNA encoding the spike glycoprotein (S protein) of SARS-CoV-2 virus, and is prepared by the following method:

[0175] Weigh 1.64 mg HSPC, 1.83 mg cholesterol and 0.89 mg DSPE-PEG2000, and dissolve them in 1 mL anhydrous ethanol at 60 °C to prepare a stock solution.

[0176] 13.41 mg TM3 was added to the stock solution at room temperature and mixed to obtain the lipid alcohol phase.

[0177] Dissolve 1.6 mL of 1 mg / mL mRNA in 1.4 mL of pH 4.0 D-citric acid solution, mix well, and prepare the nucleic acid aqueous phase.

[0178] 1 mL of lipid alcohol phase and 3 mL of nucleic acid aqueous phase were mixed at room temperature using a mixer to prepare a pharmaceutical intermediate.

[0179] The intermediate was subjected to dialysis to remove ethanol and citric acid, and then replaced with a 20 mM Tris solution containing 0.4% sodium chloride and 5% sucrose to prepare the drug TP1.

[0180] Performance testing

[0181] The drug TP1 prepared in Preparation Example 2 was subjected to performance tests, including particle size detection, dispersion detection and encapsulation efficiency detection.

[0182] Particle size and dispersion testing

[0183] The particle size and dispersion degree (PDI) of nanoparticles were determined using a laser scattering particle size analyzer (PCS, Dandong BeNano 180Zeta pro laser particle size analyzer). A 671nm solid-state laser was used as the incident light, and the kinetic light scattering experiment was conducted at 25℃. The scattering light path adopted a 173° backscattering detection method. The average value of three consecutive measurements was used as the detection data.

[0184] Encapsulation rate testing

[0185] Encapsulation efficiency was determined using a quantitative fluorescence method with the Quant-it™ RiboGreen RNA Assay Kit. After adding Triton to break the emulsion and release the nucleic acids from the formulation, the specific nucleic acid dye ribogreen was added. The absorbance of the sample was measured using an ALLSHENG Feyond-A300 microplate reader with excitation wavelength set to 470 nm and emission wavelength set to 525 nm. The total nucleic acid content was calculated using a standard curve. Furthermore, for LNP samples without Triton treatment, the specific nucleic acid dye ribogreen was added. The absorbance of the sample was measured using an ALLSHENG Feyond-A300 microplate reader with excitation wavelength set to 470 nm and emission wavelength set to 525 nm. The free nucleic acid content was calculated using a standard curve, as shown in the following formula:

[0186] Encapsulation efficiency = (Total nucleic acid content - Free nucleic acid content) / Total nucleic acid content × 100%.

[0187] The results of particle size, dispersion and encapsulation efficiency are shown in Table 2.

[0188] Table 2 shows the test results of TP1 particle size, dispersion, and encapsulation efficiency.

[0189]

[0190] As can be seen from Table 2, the drug TP1 prepared according to the formulation and method of Preparation Example 2 has a suitable particle size and uniform particle size distribution, as well as a high encapsulation efficiency, indicating that it has good drug loading capacity, which creates conditions for subsequent practical applications.

[0191] Example 1

[0192] This embodiment detects the expression level of drug TP1 prepared in Preparation Example 2 in in vitro cultured cells. The steps are as follows:

[0193] (1) Cell culture and collection:

[0194] The 293T cell line, a derivative of the human embryonic kidney cell line 293, was obtained through laboratory passage and cultured in RPMI-1640 medium containing 10% FBS, with passages performed 2–3 times per week. Cells at approximately 80% confluence were seeded and plated. After cell counting, the cell concentration was adjusted to 2 × 10⁶ cells / mL using culture medium. 5 / mL, add 0.5mL of the above cell culture to each well of a 48-well plate, that is, the number of cells per well is approximately 1×10. 5 Each group had 3 replicates, and the blank control group also had 3 replicates.

[0195] (2) Co-incubation of cells and drugs:

[0196] The total mRNA concentration was detected by fluorescence detection of mRNA. A drug containing 1 μg mRNA was added to each well and incubated for 18, 24, 48 and 72 h, respectively, and the expression level of protein was observed at different incubation times.

[0197] (3) Protein expression detection:

[0198] Cell culture plates were centrifuged at 800g for 5 min, and the supernatant was collected and diluted 10-fold, 50-fold, and 100-fold with ddH2O to prepare test samples. A standard curve was plotted using the SARS-CoV-2 virus spike glycoprotein (S protein) as a standard, and the samples were detected using ELISA.

[0199] (4) Results Analysis:

[0200] The absorbance of the standard curve is calibrated by subtracting the OD value of the blank control from the OD value of the test sample. A standard curve is plotted with the standard concentration on the x-axis, and the concentration of S protein corresponding to the sample OD value is calculated based on the standard curve.

[0201] The results of detecting the protein expression level of drug TP1 in 293T cells are as follows: Figure 8 As shown. By Figure 8 It can be seen that the drug TP1 can be effectively transferred into 293T cells and express the S protein. Furthermore, the expression level continuously increases with increasing incubation time, and the protein expression level is positively correlated with the dose. These results indicate that the LNP vector can transfer and release drugs into cells, thus having practical application value for the treatment of related diseases.

[0202] Example 2

[0203] This embodiment tests the immunogenicity of drug TP1 prepared in Preparation Example 2 in vivo, and the steps are as follows:

[0204] Six female BALB / c mice aged 6–8 weeks were injected intramuscularly (im) with drug TP1 on days 1 and 14, respectively. The titer of anti-S protein antibody in mouse serum was detected by ELISA on days 10, 21, 28, and 35 after the first administration.

[0205] The results of detecting anti-S protein antibody titers in mouse serum are as follows: Figure 9 As shown. By Figure 9It can be seen that antibodies were produced as early as day 10 after the first administration. After a booster injection on day 14, antibody expression increased significantly on day 21 and continued to be expressed over time, still present on day 35. These results indicate that the drug can effectively activate the immune response in animals, significantly increasing antibody titers after administration. Therefore, it can be used as a vaccine to stimulate the body to produce a corresponding immune response with significant efficacy.

[0206] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A cationic lipid compound, characterized in that, The cationic lipid compounds include those represented by Formula I; In formula I, n = 1, 2, 3 or 4; R1 and R2 are each independently selected from secondary long-chain alkyl esters represented by Formula II; In Equation II, n1 = 6; n2 = 7; n3 = 7.

2. The cationic lipid compound according to claim 1, characterized in that, The cationic lipid compounds include compounds represented by formula V; 3. The method for preparing the cationic lipid compound according to claim 1 or 2, characterized in that, The preparation method of the cationic lipid compound includes the following steps: Using brominated esters as raw materials, alkylation and reduction reactions are carried out sequentially. The compound is then subjected to a condensation reaction with an acyl chloride compound, followed by a two-step substitution reaction and a single hydrolysis reaction. Finally, it is subjected to an esterification reaction with a monohydric secondary alcohol compound to obtain the cationic lipid compound.

4. An LNP vector, characterized in that, The LNP carrier contains any one or a combination of at least two of the cationic lipid compounds of claim 1.

5. The LNP carrier according to claim 4, characterized in that, The LNP carrier comprises 15% to 70% cationic lipid compounds by mole fraction.

6. The LNP vector according to claim 4, characterized in that, The LNP carrier further comprises, by mole fraction, 8%–30% phospholipids, 15%–65% cholesterol, and 1.5%–3% polyethylene glycol lipids.

7. A drug, characterized in that, The drug comprises the LNP carrier according to any one of claims 4-6.

8. The medicament according to claim 7, characterized in that, The drugs mentioned also include nucleic acid drugs.

9. The medicament according to claim 8, characterized in that, In the drug, the molar ratio of nitrogen content in the cationic lipid compound in the LNP carrier to phosphorus content in the nucleic acid drug is (2-6):

1.

10. The method for preparing the drug according to any one of claims 7-9, characterized in that, The method for preparing the drug includes the following steps: Weigh out all components except cationic lipid compounds, dissolve them, and prepare a stock solution; A cationic lipid compound was added to the stock solution and mixed to obtain a lipid alcohol phase; Dilute the nucleic acid drug to prepare an aqueous phase of nucleic acid; The lipid alcohol phase and the nucleic acid aqueous phase are mixed to prepare a pharmaceutical intermediate, which is then dialyzed to obtain the drug.

11. A vaccine, characterized in that, The vaccine comprises the LNP vector as described in any one of claims 4-6.

12. The use of the cationic lipid compound as described in claim 1 or 2 in the preparation of LNP carriers, pharmaceuticals or vaccines, or the use of the LNP carrier as described in any one of claims 4-6 in the preparation of pharmaceuticals and / or vaccines.

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