CN117003808A - Cationic lipid compound and preparation method and application thereof - Google Patents

Abstract

The invention provides a cationic lipid compound, a preparation method and application thereof. The cationic lipid compound has a structure shown in a formula I. The cationic lipid compound provided by the invention has a simple chemical structure, is easy to synthesize, and can be used for preparing a drug carrier, in particular a nucleic acid drug carrier. The drug-loaded lipid nanoparticle containing the cationic lipid compound has small particle size and uniform particle size distribution, and has good loading effect and delivery effect on nucleic acid drugs.

Description

Cationic lipid compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a cationic lipid compound, a preparation method and application thereof.

Background

The biological medicine field mainly contains two kinds of therapeutic medicines and preventive medicines. Wherein, the therapeutic drugs mainly comprise micromolecular drugs, macromolecular protein drugs and the like; the preventive medicine mainly comprises various vaccines, such as inactivated vaccine, attenuated vaccine, split vaccine, recombinant protein vaccine, etc. The development of nucleic acid drugs (siRNA, mRNA, DNA, etc.) has greatly promoted the development of therapeutic drugs and preventive drugs, has an epoch-making significance, and has advanced the biomedical field to the gene therapy era. However, nucleic acid drugs also suffer from the disadvantages of instability, high innate immunogenicity, low in vivo delivery efficiency, ease of clearance, etc.

Lipid nanoparticle (Lipid Nanoparticles, LNP) delivery systems, which refer to nanoparticles formed from multiple lipid components by self-assembly for encapsulation and delivery of nucleic acid drugs, can effectively improve the stability of nucleic acid drugs, reduce the immunogenicity of nucleic acid drugs and improve the in vivo delivery efficiency of nucleic acid drugs. The first commercial application case of LNP was on pattro, approved by the united states and the european union in 2018 for the treatment of amyloidosis. From this point, LNP has received much attention as a nucleic acid delivery vehicle. In particular, since 2020, LNP delivery systems have been used for novel coronavirus mRNA vaccines by both Moderna and BioNtech nucleic acid pharmaceutical enterprises. LNP delivery systems generally comprise four lipid components [ Pickington, emily H et al, "From influenza to COVID-19:Lipid nanoparticle mRNA vaccines at the frontiers of infectious diseases." Acta biomaterialia vol.131 (2021): 16-40.Doi:10.1016/j. Actbio.2021.06.023]: 1) An ionizable cationic lipid for binding to negatively charged mRNA; 2) Cholesterol: mediate LNP endocytosis, stabilize LNP structure; 3) Neutral phospholipids: auxiliary lipid can accelerate mRNA release during endocytosis; 4) PEG phospholipid: prolonging metabolism time, improving LNP stability, and controlling particle size.

However, there is currently less of the class of lipid compounds that can be used for nucleic acid drug delivery, and there is a need to develop more effective lipid compounds to promote the development of the nucleic acid drug industry.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide a cationic lipid compound, and a preparation method and application thereof. The cationic lipid compound has a simple chemical structure, is easy to synthesize, can be used for preparing a drug carrier, has small particle size and uniform particle size distribution of drug-loaded lipid nanoparticles containing the cationic lipid compound, and has good loading effect and delivery effect on nucleic acid drugs.

To achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a cationic lipid compound having the structure of formula I:

in the formula I, R ₁ One selected from C1-C10 alkylene and C2-C10 alkenylene;

R ₂ is CH and R ₃ Selected from the group consisting ofOne of the following;

alternatively, R ₂ Is N and R ₃ Selected from the group consisting of ₁ -OH andone of the following;

R ₄ selected from C10-C20 alkyl, C10-C20 alkenyl,One of the following;

wherein L is ₁ Is a single bond or C1-C6 alkylene, L ₂ Is a single bond or methylene, L ₃ Is C1-C2 alkylene, L ₄ Is C3-C12 alkylene, L ₅ Is C4-C15 alkyl;

represents a bond of a group.

The alkenyl group in the present invention may contain one carbon-carbon double bond or may contain a plurality of carbon-carbon double bonds.

In some embodiments of the invention, the R ₁ One selected from the following groups:

wherein — represents a bond of a group.

In some embodiments of the invention, the R ₂ Is CH and R ₃ One selected from the following groups:

wherein — represents a bond of a group.

In some embodiments of the invention, the R ₂ Is N and R ₃ One selected from the following groups:

wherein — represents a bond of a group.

In some embodiments of the inventionIn the formula, R is ₄ One selected from the following groups:

wherein — represents a bond of a group.

In some embodiments of the invention, the cationic lipid compound is selected from the following compounds 1 to 5:

in a second aspect, the present invention provides a process for the preparation of a cationic lipid compound as described in the first aspect, when R ₂ In the case of N, the preparation method comprises the following steps:

the compound is preparedAdding imidazole into an organic solvent, and dropwise adding tert-butyl diphenyl chlorosilane (TBDPSCl) for reaction to generate an intermediate 1; dissolving the intermediate 1 in an organic solvent, adding sodium hydride, sodium iodide and the compound +.>Carrying out reaction to generate an intermediate 2; adding the intermediate 2 and lithium hydroxide to tetrahydrofuran, methanol and waterReacting in the mixed solution to generate an intermediate 3; reacting the intermediate 3 with cholesterol under the catalysis of N, N' -dicyclohexylcarbodiimide and 4-Dimethylaminopyridine (DMAP) to generate an intermediate 4; dissolving the intermediate 4 into a mixed solution of dichloromethane and trifluoroacetic acid for reaction to generate an intermediate 5; combining said intermediate 5 with a compound Br-R ₄ Reacting in an organic solvent containing potassium carbonate and sodium iodide to generate an intermediate 6; adding the intermediate 6 and triethylamine hydrogen fluoride into an organic solvent, adjusting the pH value to be alkaline, and reacting to generate the cationic lipid compound;

alternatively, when said R ₂ In the case of CH, the preparation method comprises the following steps:

the compound is preparedAdding the intermediate and imidazole into an organic solvent, and dropwise adding tert-butyl diphenyl chlorosilane (TBDPSCl) for reaction to generate an intermediate 7; dissolving the intermediate 7 in an organic solvent, and adding pyridinium chlorochromate for reaction to generate an intermediate 8; compound Br-L ₄ OBn is dissolved in an organic solvent, molecular sieve, mg and iodine are sequentially added in a nitrogen environment for reaction to generate an intermediate 9, and the intermediate 8 is added for reaction to generate an intermediate 10; the intermediate 10 and the compound R ₃ -H reaction to form intermediate 11; adding the intermediate 11 into a mixed solution of tetrahydrofuran, methanol and acetic acid, adding palladium carbon, and reacting in a hydrogen environment to generate an intermediate 12; intermediate 12 and compoundReacting under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) and 4-Dimethylaminopyridine (DMAP) to generate an intermediate 13; adding the intermediate 13 and triethylamine hydrogen fluoride into an organic solvent, and adjusting the pH to be alkalineCarrying out reaction to generate an intermediate 14; dissolving the intermediate 14 in acetone, and dropwise adding Jones reagent for reaction to generate an intermediate 15; reacting said intermediate 15 with cholesterol under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) and 4-Dimethylaminopyridine (DMAP) to form said cationic lipid compound.

In a third aspect, the present invention provides the use of a cationic lipid compound according to the first aspect for the preparation of a pharmaceutical carrier, preferably for the preparation of a nucleic acid pharmaceutical carrier.

In a fourth aspect, the present invention provides a lipid nanoparticle comprising a cationic lipid compound as described in the first aspect.

In some embodiments of the invention, the lipid nanoparticle further comprises a structural lipid (Structural Lipids), cholesterol, and a polymer modified lipid.

In some embodiments of the invention, the structural lipid is a neutral phospholipid. The neutral phospholipid is preferably selected from one or more of distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC) and distearoyl phosphatidylethanolamine (DSPE).

Neutral phospholipids are a class of structural lipids commonly used in the art, but the structural lipids of the present invention are not limited to neutral phospholipids, and other types of structural lipid compounds are equally useful in the present invention.

In some embodiments of the invention, the polymer modified lipid is a polyethylene glycol modified lipid.

In some embodiments of the invention, the lipid nanoparticle comprises the following components in mole percent: 30-50% of the cationic lipid compound of the first aspect, 5-25% of a structural lipid, 28.5-48.5% of cholesterol and 0.5-3% of a polyethylene glycol modified lipid.

Wherein the mole percent of the cationic lipid compound may be 30%, 32%, 35%, 38%, 40%, 42%, 45%, 48%, 50%, etc.

The mole percent of the structural lipid may be 5%, 8%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, etc.

The mole percent of cholesterol may be 28.5%, 30%, 32%, 35%, 38%, 40%, 42%, 45%, 47%, 48.5%, etc.

The mole percentage of the polyethylene glycol modified lipid may be 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8% or 3%, etc.

In a fifth aspect, the present invention provides a drug-loaded lipid nanoparticle comprising: the lipid nanoparticle of the fourth aspect and a nucleic acid drug loaded in the lipid nanoparticle.

In a sixth aspect, the present invention provides a method for preparing the drug-loaded lipid nanoparticle according to the fifth aspect, the method comprising the steps of:

preparing an oil phase containing the lipid nanoparticle composition components and a water phase containing the nucleic acid drug respectively, mixing the oil phase and the water phase by adopting microfluidic equipment, and self-assembling to form the drug-loaded lipid nanoparticle.

Compared with the prior art, the invention has the following beneficial effects:

the cationic lipid compound provided by the invention has a simple chemical structure and is easy to synthesize, and can be used for preparing a drug carrier, in particular a nucleic acid drug carrier. The drug-loaded lipid nanoparticle containing the cationic lipid compound has small particle size, uniform particle size distribution, good encapsulation efficiency and transfection efficiency, can well load nucleic acid drugs, and can be delivered to cells and animals for expression.

Drawings

FIG. 1 shows Compound 1 of the present invention ¹ H NMR spectrum;

FIG. 2 is a diagram of Compound 2 of the present invention ¹ H NMR spectrum;

FIG. 3 is a diagram of Compound 3 of the present invention ¹ H NMR spectrum;

FIG. 4 is a diagram of Compound 4 of the present invention ¹ H NMR spectrum;

FIG. 5 is a diagram of Compound 5 of the present invention ¹ H NMR spectrum;

FIG. 6 shows the structural lipids used in example 7 of the present invention ¹ H NMR spectrum;

FIG. 7 is a graph showing the results of cell transfection of drug-loaded lipid nanoparticles prepared in examples 6 and 7 of the present invention.

Detailed Description

The technical scheme of the invention is further described below by the specific embodiments with reference to the accompanying drawings. It should be apparent to those skilled in the art that the detailed description is merely provided to aid in understanding the invention and should not be taken as limiting the invention in any way.

Example 1

This example provides compound 1, the synthetic route of which is as follows:

the preparation method comprises the following steps:

synthesis of Compound 1-1: the compound tert-butyl N- (3-hydroxypropyl) carbamate (9 g,51.362mmol,1 eq) was dissolved in dichloromethane (180 mL) and imidazole (6.99 g,101.724mmol,2 eq) was added. The mixture was cooled to about 0℃and TBDPSCl (15.53 g,56.498mmol,1.1 eq) was added dropwise and reacted at room temperature for 16 hours. After the reaction was completed, the excess TBDPSCl was quenched with water and extracted with dichloromethane. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, volume ratio 1:0-30:1) and the eluent was concentrated to give the product as a yellow oil (20 g, 94% yield). MS M/z [ M+Na] ⁺ (ESI):436.25.

Synthesis of Compounds 1-2: compound 1-1 (20 g,48.352mmol,1 eq) was dissolved in N, N-dimethylformamide (200 mL), cooled to 0deg.C, sodium hydride (2.9 g,72.528mmol,1.5 eq) was added in portions, sodium iodide (724.8 mg,4.835mmol,0.1 eq) and ethyl 8-bromooctoate (18.22 g,75.528mmol,1.5 eq) were added in sequence, and stirred at 40deg.C for 1 hour. After the reaction, the reaction mixture was quenched with a saturated aqueous ammonium chloride solution. Then extracted with dichloromethane, the organic phases were combined and washed successively with water and saturated aqueous sodium chloride, dried over anhydrous sodium sulfate and concentrated. Purifying the crude product by a silica gel column (petroleum ether/ethyl acetate, volume ratio is 1:0-1:9) The eluate was concentrated to give the product as a yellow oil (17 g, yield 54%). MS M/z [ M+Na] ⁺ (ESI):606.45.

Synthesis of Compounds 1-3: compound 1-2 (17 g,29.115mmol,1 eq) was dissolved in a tetrahydrofuran/methanol/water mixture (volume ratio 2:1:1,340 mL) and then lithium hydroxide (1.05 g,43.672mmol,1.5 eq) was added and reacted overnight at room temperature. After the reaction, the pH of the system was adjusted to 4 with 1mol/L hydrochloric acid and extracted three times with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to give the crude product as a colorless oil (16 g, yield 99%). MS M/z [ M+Na] ⁺ (ESI):578.30.

Synthesis of Compounds 1-4: compounds 1-3 (16 g,28.786mmol,1 eq) were dissolved in dichloromethane (320 mL) and cholesterol (13.36 g, 34.803 mmol,1.2 eq), N, N' -dicyclohexylcarbodiimide (8.91 g, 43.178 mmol,1.5 eq) and 4-dimethylaminopyridine (1.06 g,8.636mmol,0.3 eq) were added sequentially and reacted overnight at room temperature. After the reaction was completed, the resultant solid was filtered off, and the filtrate was concentrated. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, volume ratio 1:0-40:1) to give the product as a yellow oil (13 g, yield 49%).

Synthesis of Compounds 1-5: compounds 1 to 4 (13 g,14.062mmol,1 eq) were dissolved in a dichloromethane/trifluoroacetic acid mixture (volume ratio=5:1, 260 mL) and reacted at room temperature for 10 min. After the reaction was completed, the mixture was extracted twice with methylene chloride. The organic phases were combined and washed successively with water and saturated sodium chloride, dried over anhydrous sodium sulfate and concentrated. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol, volume ratio 1:0-20:1) to give the product as a white solid (8.5 g, 73% yield). MS M/z [ M+H ]] ⁺ (ESI):824.60.

Synthesis of Compounds 1-6: the compound oleyl alcohol (3 g,11.174mmol,1 eq) was dissolved in dichloromethane (60 mL), carbon tetrabromide (4.45 g, 13.09 mmol,1.2 eq) was added, triphenylphosphine (3.52 g, 13.09 mmol,1.2 eq) was slowly added at 0deg.C, and the reaction was carried out at room temperature for 60 minutes. After the reaction was completed, n-heptane was added until a solid was produced, and concentrated to dryness. N-heptane was added, the solid was filtered off, and the filtrate was concentrated to give the product as a colorless oily body (2.8 g, yield 76%). MS M/z [ M+H ]] ⁺ (ESI):330.19.

Synthesis of Compounds 1-7: compounds 1 to 5 (1.5 g,1.820mmol,1 eq) were dissolved in N, N-dimethylformamide (30 mL), and anhydrous potassium carbonate (0.3 g,2.184mmol,1.2 eq) was added and reacted at 100℃for 60 minutes; after completion of the reaction, compounds 1 to 6 (0.72 g,2.184mmol,1.2 eq) and sodium iodide (0.03 g,0.182mmol,0.1 eq) were added and the reaction was carried out at room temperature for 6 hours. After the reaction was completed, the solid was filtered off with celite, and the filtrate was washed with water and then with sodium chloride solution, dried over anhydrous sodium sulfate and concentrated. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, volume ratio 1:0-8:1), and the eluent was concentrated to give the product as a yellow oily substance (1.0 g, yield 51%). MS M/z [ M+H ]] ⁺ (ESI):1074.80.

Synthesis of Compound 1: compounds 1 to 7 (1.0 g,0.930mmol,1 eq) were dissolved in dichloromethane (20 mL) and a solution of hydrogen fluoride in triethylamine (1.50 mg,9.300mmol,10 eq) was added and reacted at room temperature for 2 hours. Regulating pH to 8 with triethylamine, concentrating, and spin drying. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol 1:0-60:1 by volume) and the eluate was concentrated to give the product as a pale yellow semisolid (0.472 g, 61% yield). MS M/z [ M+H ] + (ESI): 836.85.

Compound 1 ¹ The H NMR spectrum is shown in FIG. 1, and the nuclear magnetic data is ¹ H NMR(300MHz,CD3OD-d4)：5.28-5.23(m,3H),δ4.75(s,1H),3.61-3.58(m,2H),3.21-3.13(m,2H),3.13-3.00(m,4H),2.22-2.19(m,4H),1.94-1.61(m,11H),1.54-1.50(m,13H),1.46-1.19(m,32H),1.19-1.01(m,7H),1.01-0.96(m,6H),0.96-0.77(m,12H),0.62(s,3H)。

Example 2

This example provides compound 2, which is synthesized as follows:

the preparation method comprises the following steps:

synthesis of Compound 2-1: the compound 6-bromo-1-hexanol (3 g,16.667mmol,1 eq), 2-ethyl-decanoic acid (4.3 g,16.797mmol,1 eq), EDCI (3.9 g,20.313mmol,1.2 eq) and DMAP (0.41 g,3.361mmol,0.2 eq) were dissolved in dichloromethane (60 mL) and DIEA (N, N-diisopropylethylamine, 8.6g,66.667mmol,4 eq) was added and reacted at room temperature for 16 hours. The reaction mixture was diluted with dichloromethane, washed once with saturated aqueous sodium chloride, the organic phase was concentrated by drying over anhydrous sodium sulfate and the crude product was purified by column on silica gel (dichloromethane: methanol volume ratio=1:0-50:1). The eluate was concentrated to give the product as a white solid (4 g, yield 59%).

Synthesis of Compound 2-2: compound 1-5 (1 g,1.214mmol,1 eq) was dissolved in DMF (20 mL) and anhydrous potassium carbonate (0.25 g,1.812mmol,1.5 eq) and Compound 2-1 (0.76 g,1.881mmol,1.5 eq) was added to react with sodium iodide (0.02 g,0.138mmol,0.1 eq) at 100deg.C for 12 hours. The reaction solution was diluted with dichloromethane and extracted once with saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude product. Purifying the crude product by a silica gel column: (petroleum ether: ethyl acetate volume ratio = 1:0-70:1). The eluate was concentrated to give the product as an off-white oil (1.2 g, yield 85%). MS M/z [ M+H ]] ⁺ (ESI):1162.85.

Synthesis of Compound 2: compound 2-2 (1 g,0.861mmol,1 eq) was dissolved in dichloromethane (200 mL) and 3HF. TEA (1.4 g,8.696mmol,10 eq) was added and reacted at room temperature for 16 hours. The reaction solution is regulated to pH 8 by triethylamine, and is directly decompressed and concentrated to obtain crude product, and the crude product is purified by a silica gel column (dichloromethane and methanol with volume ratio of 1:0-50:1). The eluate was concentrated to give the product as a yellow semisolid (505.5 mg, yield 63%, purity 98.7%). MS M/z [ M+H ]] ⁺ (ESI):924.75.

Compound 2 ¹ The H NMR spectrum is shown in FIG. 2, and the nuclear magnetic data is ¹ H NMR(300MHz,Chloroform-d)：δ5.30-5.28(m,1H),4.49-4.39(m,1H),4.00(t,J＝6Hz,2H),3.60(t,J＝6Hz,2H),3.18-3.13(m,2H),3.06-3.00(m,4H),2.29-2.18(m,5H),1.98-1.71(m,7H),1.69-1.39(m,17H),1.38-1.23(m,17H),1.24-1.14(m,21H),1.11-0.99(m,6H),0.96-0.93(m,4H),0.89-0.79(m,16H),0.63(s,3H).

Example 3

This example provides compound 3, which is synthesized as follows:

the preparation method comprises the following steps:

synthesis of Compound 3-1: tetradecanoic acid (3 g,13.158mmol,1 eq), EDCI (2.8 g,14.583mmol,1.1 eq) and DMAP (0.18 g,1.475mmol,0.1 eq) were dissolved in dichloromethane (60 mL), 3-bromo-1-propanol (1.7 g,12.319mmol,0.91 eq) was added and reacted at room temperature for 16 hours. The reaction mixture was diluted with dichloromethane, washed once with saturated aqueous sodium chloride, the organic phase was concentrated by drying over anhydrous sodium sulfate, and the crude product was purified by a silica gel column: (Petroleum ether and ethyl acetate, volume ratio 1:0-50:1). The eluate was concentrated to give the product as a white solid (3 g, yield 66%).

Synthesis of Compound 3-2: compound 1-5 (1 g,1.214mmol,1 eq) was dissolved in anhydrous potassium carbonate (0.25 g,1.812mmol,1.5 eq) to DMF (20 mL), compound 3-2 (0.63 g,1.82 mmol,1.5 eq) was added to react with sodium iodide (0.02 g,0.138mmol,0.1 eq) at 100℃for 12 hours, the pH of the reaction solution was adjusted to 7, the reaction solution was diluted with methylene chloride and extracted once with saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography on silica gel (petroleum ether and ethyl acetate, volume ratio 1:0-5:1). The eluate was concentrated to give the product as an off-white oil (1.2 g, 92% yield). MS M/z [ M+H ]] ⁺ (ESI):1092.75.

Synthesis of Compound 3: compound 3-2 (1 g,0.916mmol,1 eq) was dissolved in dichloromethane (20 mL), and triethylamine bifluoride (1.5 g,9.158mmol,10 eq) was added and reacted at room temperature for 16 hours. The PH of the reaction solution is regulated to 8 by triethylamine, the reaction solution is directly decompressed and concentrated to obtain a crude product, and the crude product is purified by a silica gel column: (dichloromethane and methanol in a volume ratio of 1:0-50:1). The eluate was concentrated to give the product as a yellow semisolid (572.8 mg, yield 70%, purity 99.345%). MS M/z [ M+H ]] ⁺ (ESI):854.65.

Compound 3 ¹ The H NMR spectrum is shown in FIG. 3, and the nuclear magnetic data is ¹ H NMR(400MHz,Chloroform-d)：δ5.39-5.38(m,1H),4.87-4.49(m,1H),4.16(t,J＝6Hz,2H),3.69(t,J＝5.6Hz,2H),3.18-3.10(m,4H),3.05-3.01(m,2H),2.36-2.28(m,6H),2.06-1.96(m,4H),1.93-1.81(m,5H),1.69-1.45(m,13H),1.39-1.26(m,30H),1.22-1.09(m,7H),1.07-0.98(m,6H),0.96-0.92(m,3H),0.95-0.81(m,9H),0.72(s,3H).

Example 4

This example provides compound 4, which is synthesized as follows:

the preparation method comprises the following steps:

synthesis of Compound 4-1: compound 1, 9-nonanediol (20 g,124.8mmol,1 eq) and imidazole (8.5 g,124.8mmol,1 eq) were dissolved in tetrahydrofuran (1250 mL), TBDPSCl (34.3 g,124.8mmol,1 eq) was added dropwise and reacted at room temperature for 3 hours. After completion of the reaction, the resulting solid was filtered off, and the filtrate was concentrated. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol, volume ratio 1:0-8:1), and the eluent was concentrated to give the product as a colourless oil (13 g, yield 26%). MS M/z [ M+Na ] + (ESI): 421.20.

Synthesis of Compound 4-2: compound 4-1 (5 g, 12.552 mmol,1 eq) was dissolved in methylene chloride (100 mL), and pyridinium chlorochromate (4.06 g,18.813mmol,1.5 eq) was added and reacted at room temperature for 3 hours. After the reaction was completed, the resultant solid was filtered off. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, volume ratio 1:0-50:1) and the eluent was concentrated to give the product as a colourless oil (4 g, 80% yield). MS M/z [ M+Na ] + (ESI): 419.10

Synthesis of Compound 4-3: the starting benzyl-6-bromohexylether (2 g,7.375mmol,1 eq) was dissolved in tetrahydrofuran (30 mL). A certain amount of the above solution was taken and added with molecular sieve (4A), mg (537.7 Mg,22.125mol,3 eq) and catalytic amount of elemental iodine in this order under nitrogen atmosphere, heated to 50℃and stirred until the solution turned from yellow to colorless. Slowly dripping the residual tetrahydrofuran solution of benzyl-6-bromohexyl ether into the reaction system, and continuing the reaction for 2 hours to obtain the compound 4-3.

Synthesis of Compound 4-4: compound 4-2 (2.34 g,8.587mmol,0.8 eq) was dissolved in tetrahydrofuran (50 mL) and molecular sieve (4A) was added under nitrogen. The tetrahydrofuran solution of the compound 4-3 is added into the reaction system in a dropwise manner, and the reaction is carried out for 2 hours at room temperature. The reaction solution was added to a saturated aqueous ammonium chloride solution, and extracted twice with methylene chloride. The organic phases are combined and washed with saturated sodium chloride, then dried over anhydrous sodium sulfate and dried. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, volume ratio 1:0-20:1) to give the product as a colorless oil (3.5 g, 40% yield). MS M/z [ M+Na ] + (ESI): 611.45.

Synthesis of Compounds 4-5: compound 4-4 (3.5 g,5.943mmol,1 eq) was dissolved in dichloromethane (70 mL) and N, N-dimethylglycine hydrochloride (1.24 g,8.915mmol,1.5 eq) and N, N-diisopropylethylamine (1.15 g,8.915mmol,1.5 eq) were added. 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.71 g,8.915mmol,1.5 eq) and 4-dimethylaminopyridine (217.8 mg,1.783mmol,0.3 eq) were added in sequence and reacted overnight at room temperature. After the completion of the reaction, the reaction solution was diluted with methylene chloride, washed with saturated sodium chloride, then dried over anhydrous sodium sulfate, filtered and spin-dried. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, volume ratio 1:0-3:1) to give the product as a colorless oil (3 g, 75% yield). MS M/z [ M+H ]] ⁺ (ESI):674.40.

Synthesis of Compounds 4-6: compound 4-5 (2.5 g,3.624mmol,1 eq) was dissolved in a tetrahydrofuran/methanol/acetic acid mixture (volume ratio 20:20:1, 100 mL) and anhydrous palladium on carbon (10%, 2.5g,100% wt) was added under nitrogen. The reaction system was then replaced with hydrogen (50 atm) and reacted overnight at room temperature. The pH of the system was adjusted to-8 with triethylamine and then filtered through celite. The filtrate was concentrated and the crude product was purified by column on silica gel (dichloromethane/methanol, volume ratio 1:0-10:1) and the eluent was concentrated to give the product as a colourless oil (1.1 g, 41% yield). MS M/z [ M+H ]] ⁺ (ESI):584.70.

Synthesis of Compounds 4-7: compound 4-6 (1.1 g,1.507mmol,1 eq) was dissolved in dichloromethane (22 mL), 2-hexyldecanoic acid (579.7 mg,2.260mmol,1.5 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (433.3 mg,2.260mmol,1.5 eq) and 4-dimethylaminopyridine (52.2 mg, 0.450 mmol,0.3 eq) were added sequentially and reacted overnight at room temperature. The reaction solution was diluted with dichloromethane, washed with saturated sodium chloride, then dried over anhydrous sodium sulfate, filtered and concentrated. MS M/z [ M+H ] + (ESI): 822.75.

Synthesis of Compounds 4-8: compounds 4-8 were dissolved in methylene chloride (30 mL),hydrogen fluoride triethylamine (2.43 g,15.070mmol,10 eq) was added and reacted at room temperature for 16 hours. The pH of the system was adjusted to 8 with triethylamine and concentrated. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol, volume ratio 1:0-30:1), and the eluent was concentrated to give the product as a colourless oil (700 mg, 80% yield). MS M/z [ M+H ]] ⁺ (ESI):584.55.

Synthesis of Compounds 4-9: compound 4-8 (700 mg,1.199mmol,1 eq) was dissolved in acetone (25 mL), jones reagent (2.672 mol/L,1.44mL,3.2 eq) was added dropwise and stirred at 0deg.C for 1 hour. Then 20mL of water was added and extracted twice with ethyl acetate. The organic phases were combined and washed with saturated sodium chloride, then dried over anhydrous sodium sulfate and concentrated. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol, volume ratio 1:0-20:1), and the eluent was concentrated to give the product as a white solid (450 mg, yield 63%). MS M/z [ M+H ] + (ESI): 599.00.

Synthesis of Compound 4: compound 4-9 (600 mg,1.003mmol,1 eq) was dissolved in dichloromethane (12 mL), cholesterol (552 mg,1.504mmol,1.5 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (288.5 mg,1.504mmol,1.5 eq) and 4-dimethylaminopyridine (36.8 mg,0.301mmol,0.3 eq) were added in sequence and reacted overnight at room temperature. The reaction solution was diluted with methylene chloride, washed with saturated sodium chloride, then dried over anhydrous sodium sulfate and dried. The crude product was purified by column chromatography on silica gel (n-hexane/ethyl acetate, volume ratio 1:0-5/1), and the eluent was concentrated to give the product as a milky white oil (578.5 mg, yield 59%). MS M/z [ M+H ] + (ESI): 966.70.

Compound 4 ¹ The H NMR spectrum is shown in FIG. 4, and the nuclear magnetic data is ¹ H NMR(400MHz,Chloroform-d)：δ5.37(d,J＝5.1Hz,1H),5.00-4.87(m,1H),4.66-4.54(m,1H),4.05(t,J＝6.6Hz,2H),3.17(s,2H),2.38(s,6H),2.34-2.23(m,5H),2.05-1.92(m,2H),1.90-1.77(m,3H),1.68-1.39(m,21H),1.38-1.19(m,38H),1.18-1.06(m,6H),1.04-0.94(m,5H),0.92-0.83(m,15H),0.68(s,3H).

Example 5

This example provides compound 5, the synthetic route of which is as follows:

the preparation method comprises the following steps:

synthesis of Compound 5-1: the compound linoleyl alcohol (1 g,3.753mmol,1 eq) was dissolved in dichloromethane (20 mL), carbon tetrabromide (1.49 g,4.504mmol,1.2 eq) was added, and triphenylphosphine (1.18 g,4.504mmol,1.2 eq) was added and reacted at room temperature for 1 hour. N-heptane was added until a solid developed, and the mixture was concentrated to dryness. N-heptane was added, the solid was filtered off, and the filtrate was concentrated to give the product as a colorless oily body (1 g, yield 81%).

Synthesis of Compound 5-2: compounds 1-5 (1.0 g,1.213mmol,1 eq) were dissolved in N, N-dimethylformamide (20 mL), and anhydrous potassium carbonate (0.25 g,1.820mmol,1.5 eq) was added and reacted at 100℃for 30 minutes. Compound 5-1 (0.60 g, 1.82mmol, 1.5 eq) was added and reacted at 100℃for 16 hours. Cooled to room temperature, the reaction solution was diluted with dichloromethane and the solid was filtered off with celite, and the filtrate was washed with water and then with sodium chloride solution, dried over anhydrous sodium sulfate and concentrated. The crude product was purified by column chromatography on silica gel (petroleum ether/ethyl acetate, volume ratio 1:0-8:1) and concentrated to give the product as a yellow oily substance (0.8 g, 61% yield). MS M/z [ M+H ] + (ESI): 1072.80.

Synthesis of Compound 5: compound 5-2 (0.8 g,0.746mmol,1 eq) was dissolved in methylene chloride (16 mL), and a solution of triethylamine hydrogen fluoride (1.2 g,7.460mmol,10 eq) was added and reacted at room temperature for 2 hours. Regulating pH to 8 with triethylamine, concentrating, and spin drying. The crude product was purified by column chromatography on silica gel (dichloromethane/methanol, volume ratio 1:0-30:1) and concentrated to give the product as pale yellow semi-solid (0.531 g, 85% yield). MS M/z [ M+H ] + (ESI): 834.70.

Compound 5 ¹ The H NMR spectrum is shown in FIG. 5, and the nuclear magnetic data is ¹ H NMR(300MHz,CD3OD-d4)：δ5.31-5.18(m,5H),4.49-4.48(m,1H),3.60(t,J＝6Hz,2H),3.22-3.19(m,2H),3.05-2.99(m,4H),2.68(t,J＝6Hz,2H),2.23-2.19(m,4H),1.99-1.94(m,5H),1.86-1.70(m,5H),1.63-1.39(m,13H),1.30-1.19(m,27H),1.14-1.01(m,7H),0.98-0.90(m,5H),0.86-0.77(m,13H),0.63(s,3H).

Example 6

The present example provides a series of drug-loaded lipid nanoparticles, which are prepared as follows:

(1) Cationic lipid compound, DSPC (distearoyl phosphatidylcholine), CHOL (cholesterol) and ALC-0159 (polyethylene glycol modified lipid, available from Xiaomenobang Biotechnology Co., ltd.) were dissolved in absolute ethanol at a molar ratio of 50:10:38.5:1.5 to prepare an oil phase having a total lipid concentration of 14.4 mmol/L;

wherein the cationic lipid compounds are respectively compound 1 to compound 5 and ALC-0315 (purchased from Xiaomenobang biotechnology Co., ltd.);

(2) mRNA or Luciferase-mRNA (Luciferase-labeled mRNA) was dissolved in a citric acid buffer (pH=4.5, 50 mM) to prepare an aqueous phase having an mRNA concentration of 0.133mg/mL (0.4 mmol/L);

(3) Introducing the oil phase and the water phase into a microfluidic nano manufacturing system, controlling the volume ratio of the oil phase to the water phase to be 1:3, so that the N/P (the molar ratio of nitrogen in the lipid to phosphorus in the mRNA) of the lipid to the mRNA is 6:1, the total flow rate is 12mL/min, the waste is discharged 0.2mL before, and the waste is discharged 0.1mL after, and preparing the colostrum; 3mL of colostrum was loaded into a 10kD dialysis card, the magnetic stirring rotation speed was set at 120rpm, and dialyzed in 500mL of PBS dialysate for 4 hours to obtain drug-loaded lipid nanoparticles (named mRNA-LNP or Luciferase-mRNA-LNP).

Example 7

The present example provides a series of drug-loaded lipid nanoparticles, which are prepared as follows:

(1) Cationic lipid compound, structural lipid, CHOL (cholesterol) and ALC-0159 (polyethylene glycol modified lipid, available from Xiaomenobang Biotechnology Co., ltd.) are dissolved in absolute ethanol according to a molar ratio of 50:10:38.5:1.5 to prepare an oil phase with a total lipid concentration of 14.4 mmol/L;

wherein the cationic lipid compounds are respectively compound 1 to compound 5 and ALC-0315 (purchased from Xiaomenobang biotechnology Co., ltd.);

(2) mRNA or Luciferase-mRNA (Luciferase-labeled mRNA) was dissolved in a citric acid buffer (pH=4.5, 50 mM) to prepare an aqueous phase having an mRNA concentration of 0.133mg/mL (0.4 mmol/L);

(3) Introducing the oil phase and the water phase into a microfluidic nano manufacturing system, wherein the volume ratio of the oil phase to the water phase is 1:3, so that the N/P (the molar ratio of nitrogen in the lipid to phosphorus in the mRNA) of the lipid and the mRNA is 6:1, the total flow rate is 12mL/min, the waste is discharged at 0.2mL before, and the waste is discharged at 0.1mL after, and the colostrum is prepared; 3mL of colostrum was loaded into a 10kD dialysis card, the magnetic stirring rotation speed was set at 120rpm, and dialyzed in 500mL of PBS dialysate for 4 hours to obtain drug-loaded lipid nanoparticles (named mRNA-LNP or Luciferase-mRNA-LNP).

In this embodiment, the structural lipid is:

the synthetic route and the preparation method are as follows:

synthesis of intermediate 1: cholic acid (30 g,73.529mmol,1 eq) was dissolved in N, N-dimethylformamide (600 mL), and dimethylaminoethanol (13.088 g,147.059mmol,2 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (21.066 g, 110.284 mmol,1.5 eq) and 4-dimethylaminopyridine (2.691 g,22.059mmol,0.3 eq) were added sequentially. The reaction was stirred at room temperature overnight, collected and purified by reverse phase column, and the eluate was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated to give the product as a white solid (30 g, yield 85%, purity 99%).

Synthesis of intermediate 2: intermediate 1 (3 g,6.254mmol,1 eq) was dissolved in dichloromethane (30 mL) and 4-dimethylaminopyridine (840.5 mg,6.879mmol,1.1 eq) was added. A solution of octadecanoyl chloride (2.084 g,6.879mmol,1.1 eq) in dichloromethane (30 mL) was slowly added dropwise to the reaction solution. The reaction was carried out at room temperature for 2 hours, and the reaction solution was extracted with methylene chloride. The organic phases were combined, dried over anhydrous sodium sulfate and concentrated. The crude product was purified by column chromatography on silica gel (dichloromethane: methanol volume ratio=20:1) to give the product as a white solid (1.6 g, yield 34%, purity 99%).

Synthesis of structural lipids: intermediate 2 (1.4 g,0.899mmol,1 eq) was dissolved in dichloromethane (23 mL) and methyl iodide (284 mg,3.752mmol,2 eq) was added. The reaction was carried out at room temperature for 16 hours, and the reaction mixture was concentrated, and the crude product was purified by silica gel column (dichloromethane: methanol volume ratio=10:1) to give the product as a yellow solid (1.1243 g, yield 66%, purity 98%).

The structural lipid ¹ The H NMR spectrum is shown in FIG. 6, and the nuclear magnetic data is ¹ H NMR(400MHz,DMSO-d6)：δ4.44(s,3H),4.12(dd,J＝19.8Hz,3.4Hz,2H),3.79(s,1H),3.72-3.56(m,3H),3.12(s,9H),2.46-2.32(m,2H),2.32-2.08(m,4H),2.04-1.91(m,1H),1.88-1.59(m,6H),1.59-1.43(m,5H),1.43-1.32(m,7H),1.23(s,28H),1.20-1.12(m,1H),1.02-0.92(m,5H),0.89-0.75(m,6H),0.59(s,3H).

Physical property test:

the particle size, PDI (dispersibility index) and encapsulation efficiency of the mRNA-LNP provided in examples 6 and 7 above were tested as follows:

particle size, PDI: 100. Mu.L of mRNA-LNP sample and 900. Mu.L of PBS were mixed uniformly and added into a sample cell, the sample cell was placed into a sample chamber of a Malvern Zetasizer Ultra nm particle size potentiometer, the sample type was selected to be liposomes, the equilibration time was 30 seconds, and the particle size and PDI were measured.

Encapsulation efficiency: encapsulation efficiency of mRNA was determined by Ribogreen fluorescence analysis. Two mRNA-LNP samples were taken, one sample was diluted 50-fold with 1 XTE buffer and combined with Ribogreen fluorescent dye, and the unencapsulated mRNA content was determined by means of a microplate reader (excitation wavelength 480nm and emission wavelength 520 nm) (F _free ) The method comprises the steps of carrying out a first treatment on the surface of the Another sample was demulsified with 2% Triton X-100 solution (polyethylene glycol octylphenyl ether), diluted to 1/50 of the original mRNA concentration, combined with Ribogreen fluorescent dye, and the total mRNA was measured by an enzyme-labeled instrument (excitation wavelength 480nm and emission wavelength 520 nm) (F) _total ) According to formula EE% = (F _total -F _free )/F _total The encapsulation efficiency of mRNA-LNP was calculated as X100%.

The results of the above tests are shown in table 1 below:

TABLE 1

As can be seen from the test results of Table 1, the mRNA-LNP prepared using the compounds 1 to 5 and the commercial lipid ALC-0315 had a particle size of 50 to 145nm, and a PDI of 0.02 to 0.31, and the particle size was small and uniform. In addition, the encapsulation rate of the mRNA-LNP prepared by adopting the compounds 1 to 5 is 93 to 98 percent, which is obviously higher than that of the commercial lipid ALC-0315, and the encapsulation effect of the cationic lipid compound provided by the invention is better.

Cell transfection efficiency test:

293T cells were added to 96-well plates at a cell density of 2X 10 ⁴ Each well has a volume of 100. Mu.L and a temperature of 37℃and 5% CO ₂ Is cultured overnight under the condition of (2); the Luciferase-mRNA-LNP prepared in example 6 and example 7 were added, respectively, and the mRNA concentration was controlled to 50 ng/well. After 18h of incubation, 100. Mu.L of luciferases fluorogenic substrate was added to each well and reacted for 5min; the fluorescence value of each well was measured using a fluoroenzyme-labeled instrument, and the results are shown in FIG. 7.

As can be seen from FIG. 7, the transfection efficiency of the Luciferase mRNA-LNP prepared with Compound 1, compound 2 and Compound 5 was similar to that of the commercial lipid ALC-0315, and the transfection efficiency of the Luciferase mRNA-LNP prepared with Compound 3 and Compound 4 was significantly higher than that of the commercial lipid ALC-0315. When structural lipids were used instead of DSPC, the transfection efficiency of Luciferase-mRNA-LNP was significantly improved.

While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. A cationic lipid compound, characterized in that the cationic lipid compound has a structure represented by formula I:

in the formula I, R ₁ One selected from C1-C10 alkylene and C2-C10 alkenylene;

R ₂ is CH and R ₃ Selected from the group consisting ofOne of the following;

alternatively, R ₂ Is N and R ₃ Selected from the group consisting of ₁ OH andone of the following;

R ₄ selected from C10-C20 alkyl, C10-C20 alkenyl,One of the following;

wherein L is ₁ Is a single bond or C1-C6 alkylene, L ₂ Is a single bond or methylene, L ₃ Is C1-C2 alkylene, L ₄ Is C3-C12 alkylene, L ₅ Is C4-C15 alkyl;

represents a bond of a group.

2. The cationic lipid compound of claim 1, wherein R ₁ One selected from the following groups:

wherein — represents a bond of a group.

3. The cationic lipid compound according to claim 1 or 2, wherein R ₂ Is CH and R ₃ One selected from the following groups:

alternatively, the R ₂ Is N and R ₃ One selected from the following groups:

wherein — represents a bond of a group.

4. A cationic lipid compound according to any one of claims 1-3, wherein R ₄ One selected from the following groups:

wherein — represents a bond of a group.

5. The cationic lipid compound according to any one of claims 1 to 4, wherein the cationic lipid compound is selected from the group consisting of compounds 1 to 5:

6. a kind of rightThe method for producing a cationic lipid compound according to any one of claims 1 to 5, wherein R is ₂ For N, the preparation method comprises the following steps:

the compound is preparedAdding imidazole into an organic solvent, and dropwise adding tert-butyl diphenyl chlorosilane for reaction to generate an intermediate 1; dissolving the intermediate 1 in an organic solvent, adding sodium hydride, sodium iodide and a compoundCarrying out reaction to generate an intermediate 2; adding the intermediate 2 and lithium hydroxide into a mixed solution of tetrahydrofuran, methanol and water for reaction to generate an intermediate 3; reacting the intermediate 3 with cholesterol under the catalysis of N, N' -dicyclohexylcarbodiimide and 4-dimethylaminopyridine to generate an intermediate 4; dissolving the intermediate 4 into a mixed solution of dichloromethane and trifluoroacetic acid for reaction to generate an intermediate 5; combining said intermediate 5 with a compound Br-R ₄ Reacting in an organic solvent containing potassium carbonate and sodium iodide to generate an intermediate 6; adding the intermediate 6 and triethylamine hydrogen fluoride into an organic solvent, adjusting the pH value to be alkaline, and reacting to generate the cationic lipid compound;

alternatively, the R ₂ For CH, the preparation method comprises the following steps:

the compound is preparedMiaowThe azole is added into an organic solvent, and tert-butyl diphenyl chlorosilane is added dropwise for reaction to generate an intermediate 7; dissolving the intermediate 7 in an organic solvent, and adding pyridinium chlorochromate for reaction to generate an intermediate 8; compound Br-L ₄ OBn is dissolved in an organic solvent, molecular sieve, mg and iodine are sequentially added in a nitrogen environment for reaction to generate an intermediate 9, and the intermediate 8 is added for reaction to generate an intermediate 10; the intermediate 10 and the compound R ₃ -H reaction to form intermediate 11; adding the intermediate 11 into a mixed solution of tetrahydrofuran, methanol and acetic acid, adding palladium carbon, and reacting in a hydrogen environment to generate an intermediate 12; intermediate 12 and the compound +.>Reacting under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to generate an intermediate 13; adding the intermediate 13 and triethylamine hydrogen fluoride into an organic solvent, adjusting the pH value to be alkaline, and reacting to generate an intermediate 14; dissolving the intermediate 14 in acetone, and dropwise adding Jones reagent for reaction to generate an intermediate 15; reacting said intermediate 15 with cholesterol under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to produce said cationic lipid compound.

7. Use of a cationic lipid compound according to any one of claims 1-5 for the preparation of a pharmaceutical carrier, preferably for the preparation of a nucleic acid pharmaceutical carrier.

8. A lipid nanoparticle comprising a cationic lipid compound according to any one of claims 1 to 5;

preferably, the lipid nanoparticle further comprises a structural lipid, cholesterol, and a polymer modified lipid;

preferably, the structural lipid is a neutral phospholipid;

preferably, the neutral phospholipid is selected from one or more of distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine and distearoyl phosphatidylethanolamine;

preferably, the polymer modified lipid is a polyethylene glycol modified lipid;

preferably, the lipid nanoparticle comprises the following components in mole percent: the cationic lipid compound of any one of claims 1-5, 30-50%, structural lipid 5-25%, cholesterol 28.5-48.5% and polyethylene glycol modified lipid 0.5-3%.

9. A drug-loaded lipid nanoparticle comprising: the lipid nanoparticle of claim 8 and a nucleic acid drug loaded in the lipid nanoparticle.

10. A method of preparing the drug-loaded lipid nanoparticle of claim 9, comprising the steps of:

preparing an oil phase containing the lipid nanoparticle composition components and a water phase containing the nucleic acid drug respectively, mixing the oil phase and the water phase by adopting microfluidic equipment, and self-assembling to form the drug-loaded lipid nanoparticle.