CN117003807A - Structural lipid compound and preparation method and application thereof - Google Patents

Abstract

The invention provides a structural lipid compound, a preparation method and application thereof. The structural lipid compound has a structure shown in a formula I. The structural lipid compound provided by the invention can replace traditional neutral phospholipid and is used for preparing drug-loaded lipid nano-particles. The drug-loaded lipid nanoparticle containing the lipid compound with the structure has small particle size and uniform particle size distribution, and has good loading effect and delivery effect on nucleic acid drugs.

Description

Structural 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 structural lipid compound and a preparation method and application thereof.

Background

After the outbreak of new crown epidemic, the mRNA vaccine developed by the pyro-BioNTech and Moderna is successfully commercialized in the United states, and mRNA drugs are also rapidly becoming a focus of attention in the biopharmaceutical field. Both vaccines were reported to have a protection rate against new coronavirus infection of more than 90% [ Pilkington, 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]. Compared with the traditional vaccine, the mRNA vaccine has the advantages of simple production process, high development speed, capability of rapidly coping with variant strains, capability of rapidly expanding production scale and the like; and mRNA is rapidly degraded in the cell without risk of integration into the host genome. However, mRNA also suffers from the disadvantages of instability, higher innate immunogenicity, low in vivo delivery efficiency, susceptibility to clearance [ Uddin, mohammad N, and Monzurul A Roni. "Challenges of Storage and Stability of mRNA-Based COVID-19 Vaccines." Vaccines vol.9,9 1033.17Sep.2021,doi:10.3390/Vaccines9091033].

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.

Neutral phospholipids can modulate the mobility of nanoparticles and enhance their delivery efficiency by promoting lipid phase changes that facilitate membrane fusion with endosomes. Common neutral phospholipids are DSPC, DOPC, DSPE, among others, DSPCHas been used for FDA approved SARS-CoV-2 vaccine mRNA-1273 and BNT162b2. Neutral phospholipids have important significance for the self-assembly process of LNP delivery systems, liposome stability, in vivo delivery, endocytosis, intracellular transfection release, etc.

However, there is less research associated with neutral phospholipids in LNP delivery systems and there is a serious patent protection barrier, so there is a need to develop more effective compounds with similar functions to promote the development of the nucleic acid pharmaceutical industry.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide a structural lipid compound, and a preparation method and application thereof. The structural lipid compound can replace traditional neutral phospholipid and is used for preparing drug-loaded lipid nano-particles. The drug-loaded lipid nanoparticle containing the lipid compound with the structure has small particle size and uniform particle size distribution, 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 structural lipid compound having a structure represented by formula I:

in the formula I, R ₁ 、R ₂ 、R ₃ Each independently selected from the group consisting of hydroxy, One of the following;

R ₄ selected from the group consisting ofOne of the following;

wherein L is ₁ Is C1-C19 alkyl or C9-C21 alkenyl, L ₂ Is C3-C13 alkylene, L ₃ Is C2-C14 alkyl, L ₄ Is a C2-C6 alkylene group,anions to satisfy chemical environment;

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 、R ₂ 、R ₃ Each independently selected from one of the following groups:

wherein — represents a bond of a group.

In some embodiments of the invention, the R ₁ 、R ₂ 、R ₃ Each independently selected from one of the following groups:

and R is ₁ 、R ₂ 、R ₃ Not all are selected from

Wherein — represents a bond of a group.

In some embodiments of the invention, theIs CF (CF) ₃ COO ^- 、I ^- Or Cl ^- Preferably CF ₃ COO ^- Or I ^- 。

In some embodiments of the invention, the structural lipid compound is selected from the following compounds 1-9 and 2-1:

in a second aspect, the present invention provides a method for preparing a structural lipid compound according to the first aspect, the method comprising the steps of:

combining cholic acid with a compoundDissolving in an organic solvent, and reacting under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) and 4-Dimethylaminopyridine (DMAP) to generate an intermediate 1; combining said intermediate 1 with R ₁ 、R ₂ And/or R ₃ To produce intermediate 2 as said structural lipid compound; or the intermediate 2 is further reacted with a compound M-CH ₃ Reacting to generate the structural lipid compound;

or comprises the following steps:

dissolving cholic acid in an organic solvent, adding potassium carbonate, and dropwise adding benzyl bromide for reaction to generate an intermediate 3; combining said intermediate 3 with R ₁ 、R ₂ And/or R ₃ Reacting the acid chloride compound of (a) to form intermediate 4; the intermediate 4 is reduced in a hydrogen environment under the catalysis of palladium-carbon to generate an intermediate 5; combining said intermediate 5 with a compoundDissolving in an organic solvent, and reacting under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to generate an intermediate 2 serving as the structural lipid compound; or the intermediate 2 is further reacted with a compound M-CH ₃ Reacting, producingTo form the structural lipid compound.

In the present invention, R is ₁ 、R ₂ 、R ₃ Refers to the acyl chloride compound of R ₁ 、R ₂ 、R ₃ A compound in which an acyloxy group is replaced with an acyl chloride group. For example, when R ₁ Is thatWhen in use, R is ₁ Acyl chloride compound of +.>

In a third aspect, the present invention provides the use of a structured 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 and a structural lipid compound as described in the first aspect.

In some embodiments of the invention, the lipid nanoparticle further comprises cholesterol and a polymer modified lipid.

In some embodiments of the invention, the cationic lipid is selected from one or more of methyl 4- (N, N-dimethylamino) butyrate (diiodo) methyl ester (abbreviated DLin-MC 3-DMA), [ (4-hydroxybutyl) azadialkyl ] bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (abbreviated ALC-0315) and heptadec-9-yl-8- [ (2-hydroxyethyl) (6-oxo-6- ((decyloxy) hexyl) amino) octanoate ] (abbreviated SM-102).

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 cationic lipid, 5-25% of structural lipid compound described in the first aspect, 28.5-48.5% of cholesterol and 0.5-3% of polyethylene glycol modified lipid.

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 structural lipid compound provided by the invention can replace traditional neutral phospholipids (such as DSPC and the like) and is used for preparing drug-loaded lipid nano-particles. The drug-loaded lipid nanoparticle containing the lipid compound with the structure has small particle size, uniform particle size distribution, good encapsulation efficiency and transfection efficiency, can well load nucleic acid drugs, and is delivered to cells and animal bodies for expression.

In addition, compared with the traditional neutral phospholipid (such as DSPC, etc.), the structural lipid compound provided by the invention has the advantages of simple synthesis process and high yield, and is beneficial to reducing the production cost.

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 is a diagram of Compound 6 of the present invention ¹ H NMR spectrum;

FIG. 7 is a diagram of Compound 7 of the present invention ¹ H NMR spectrum;

FIG. 8 is a diagram of Compound 8 of the present invention ¹ H NMR spectrum;

FIG. 9 is a drawing of compound 9 of the present invention ¹ H NMR spectrum;

FIG. 10 is a graph showing the result of cell transfection of drug-loaded lipid nanoparticles prepared in example 10 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 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 Compounds 1-2: compound 1-1 (3 g,7.255mmol,1 eq) and glutaric anhydride (927 mg,8.133mmol,1.3 eq) were dissolved in pyridine solution and stirred overnight at 80 ℃. The reaction solution was cooled to room temperature, concentrated, and pyridine was dried by spin-drying. The crude product was diluted with ethyl acetate and washed with saturated aqueous sodium chloride. The organic phase was dried over anhydrous sodium sulfate, filtered and dried by spin to give the product as a pale yellow oil (2 g, yield 54%).

Synthesis of Compounds 1-3: compound 1-2 (2 g,3.373mmol,1 eq) was dissolved in dichloromethane (20 mL). N-dodecanol (754 mg,4.384mmol,1.3 eq), EDCI (971 mg,5.057mmol,1.5 eq) and DMAP (123 mg.1.008mmol,0.3 eq) were added in this order and stirred at room temperature for 16 hours. The mixture was diluted with dichloromethane, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and dried. The crude product is purified by a silica gel column (the volume ratio of dichloromethane to methanol is 1:0-20:1). The eluate was concentrated to give the product as a yellow solid (1 g, yield 39%).

Synthesis of Compound 1: compounds 1-3 (1 g,1.314mmol,1 eq) were dissolved in dichloromethane (20 mL) and methyl iodide (560 mg,3.944mmol,3 eq) was added. Overnight at room temperature. Directly spin-drying, and purifying the crude product by a silica gel column (the volume ratio of dichloromethane to methanol is 1:0-10:1). The eluate was concentrated to give the product as a yellow solid (504.9 mg, yield 49%).

Compound 1 ¹ The H NMR spectrum is shown in FIG. 1, and the nuclear magnetic data is ¹ H NMR(400MHz,DMSO-d6)δ4.59-4.56(m,3H),4.14(s,2H),4.06-4.03(m,3H),3.90(s,1H),3.55(s,9H),2.83(s,4H),2.53-2.15(m,7H),1.97-1.87(m,5H),1.81-1.71(m,5H),1.64-1.43(m,9H),1.30-1.26(m,19H),1.14-1.02(m,4H),0.91-0.87(m,6H),0.70(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: compound 1-1 (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 Compound 2: compound 2-1 (1.4 g,0.899mmol,1 eq) was dissolved in dichloromethane (23 mL) and methyl iodide (284 mg, 3.751mmol, 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%).

Compound 2 ¹ The H NMR spectrum is shown in FIG. 2, 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).

Example 3

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

the preparation method comprises the following steps:

synthesis of Compound 3-1: compound 1-1 (10 g,20.846mmol,1 eq) was dissolved in tetrahydrofuran (200 mL), imidazole (2.84 g,41.692mmol,2 eq) was added, and tert-butyldiphenylchlorosilane (13.088 g,147.059mmol,2 eq) was slowly added. The reaction was carried out at room temperature for 4 hours, and the reaction solution was collected and diluted with methylene chloride. The organic phases were combined, concentrated and the crude product purified by silica gel column (dichloromethane: methanol volume ratio=20:1) and the eluent concentrated to give the product as a yellow oily body (9.0 g, yield 60%, purity 87%).

Synthesis of Compound 3-2: compound 3-1 (2.5 g,4.209mmol,1 eq) was dissolved in pyridine (40 mL) and octadecanoyl chloride (3.19 g,10.522mmol,2.5 eq) was added. The reaction mixture was stirred at 110℃for 3 hours, diluted with dichloromethane (100 mL), and the organic phases were combined and concentrated, the crude product was purified by column on silica gel (petroleum ether: ethyl acetate volume ratio=1:1), and the eluent was concentrated to give the product as a yellow oily body (2.0 g, yield 53%).

Synthesis of Compound 3-3: compound 3-2 (1.6 g,1.279mmol,1 eq) was dissolved in tetrahydrofuran (24 mL) and tetrabutylammonium fluoride (1.27 mg,8.953mmol,7 eq) was added. Stirring is carried out for 4 hours at 40℃under nitrogen. The reaction mixture was diluted with ethyl acetate (100 mL) and washed once with saturated aqueous sodium chloride (100 mL). The organic phases were combined and concentrated. The crude product obtained was purified by column chromatography on silica gel (dichloromethane: methanol volume ratio=20:1), and the eluent was concentrated to give the product as a colorless oil (1.0 g, yield 77%).

Synthesis of Compound 3: compound 3-3 (800 mg, 0.780 mmol,1 eq) was dissolved in methylene chloride (1.6 mL), methyl iodide (336.40 mg,2.370mmol,3 eq) was added, and the mixture was reacted at room temperature for 4 hours. After the reaction solution was concentrated, it was purified by a silica gel column (dichloromethane: methanol volume ratio=20:1), and the eluent was concentrated to give the product as a yellow oil (506.4 mg, yield 55%, purity 97%).

Compound 3 ¹ The H NMR spectrum is shown in FIG. 3, and the nuclear magnetic data is ¹ H NMR (300 MHz, chloroform-d) delta 5.10 (s, 1H), 4.94 (d, j=3.0 hz, 1H), 4.57 (d, j=4.5 hz, 2H), 4.14 (t, j=4.5 hz, 2H), 3.55-3.44 (m, 10H), 2.41-2.27 (m, 6H), 2.27-1.77 (m, 7H), 1.76-1.51 (m, 13H), 1.45-1.17 (m, 61H), 1.03-0.91 (m, 2H), 0.90-0.88 (m, 9H), 0.86-0.82 (m, 3H), 0.73 (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 1-2 (1 g,1.686mmol,1 eq) was dissolved in dichloromethane (20 mL) and then dodecylamine (406 mg,2.195mmol,1.3 eq), HATU (0.96 g,2.52mmol,1.5 eq) and DIEA (N, N-diisopropylethylamine, 650mg,5.04mmol,3 eq) were added sequentially. Stir at room temperature overnight. The reaction solution was diluted with dichloromethane, washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate and dried by spin-drying. The crude product is purified by a silica gel column (the volume ratio of dichloromethane to methanol is 1:0-6:1). The eluate was concentrated to give the product as a pale yellow oil (500 mg, yield 39%).

Synthesis of Compound 4: compound 4-1 (500 mg,0.657mmol,1 eq) was dissolved in methylene chloride (10 mL), methyl trifluoroacetate (252 mg,1.971mmol,3 eq) was added, triethylamine (7 mg,0.066mmol,0.1 eq) was added after 1 hour of reaction, and the mixture was stirred at room temperature for 2 hours. The reaction was concentrated and the crude product purified using a reverse phase column (C18 column). The eluate was concentrated to give the product as an off-white solid (258.0 mg, yield 50%).

Compound 4 ¹ The H NMR spectrum is shown in FIG. 4, and the nuclear magnetic data is ¹ H NMR(300MHz,DMSO-d6)δ7.73-7.71(m,1H),4.44(s,3H),3.79-3.63(m,4H),3.12(s,9H),3.05-2.97(m,2H),2.42-1.93(m,10H),1.82-1.62(m,8H),1.58-1.52(m,3H),1.46-1.15(m,29H),1.01-0.84(m,11H),0.60(s,3H).

¹⁹ F-NMR(400MHz,DMSO-d6)δ-74.38。

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: compound 1-1 (4 g,8.351mmol,1 eq) was dissolved in dichloromethane (40 mL), then dimethylaminopyridine (1.019 g,8.352mmol,1 eq) was added, and acid chloride (2.313 g,8.371mmol,1 eq) was added dropwise, stirred at room temperature for 2h, and extracted three times with dichloromethane (100 mL). The organic phases were combined, washed once with saturated aqueous sodium chloride (300 mL), the organic phases were collected and concentrated. The crude product was concentrated and purified by column chromatography on silica gel (dichloromethane: methanol volume ratio=20:1). After concentrating the eluate under reduced pressure, the product was obtained as an off-white oil (1.444 g, yield 23%, purity 95.6%).

Synthesis of Compound 5: compound 5-1 (1.3 g,1.747mmol,1 eq) was dissolved in methylene chloride (26 mL), methyl iodide (744 mg,5.239mmol,3 eq) was added, and the mixture was reacted at room temperature for 16 hours. The crude product was concentrated and purified by column chromatography on silica gel (dichloromethane: methanol volume ratio=20:1). The eluate was concentrated to give the product as a yellow solid (0.7098 g, yield 54%, purity 97%).

Compound 5 ¹ The H NMR spectrum is shown in FIG. 5, and the nuclear magnetic data is ¹ H NMR (400 MHz, chloroform-d) delta 5.38-5.30 (m, 2H), 4.61-4.54 (m, 2H), 4.15-4.13 (m, 2H), 4.01 (s, 1H), 3.88 (s, 1H), 3.56 (s, 9H),2.52-2.45(m,1H),2.39-2.17(m,10H),2.01-1.86(m,6H),1.80-1.70(m,4H),1.66-1.46(m,11H),1.35-1.25(m,20H),1.17-1.04(m,2H),1.00(d,J＝5.2Hz,3H),0.90-0.86(m,6H),0.70(s,3H).

Example 6

This example provides compound 6, the synthetic route of which is as follows:

the preparation method comprises the following steps:

synthesis of Compound 6-1: compound 1-1 (2 g,13.947mmol,1 eq) was dissolved in pyridine (20 mL), triethylbenzyl ammonium bromide (1.588 g,6.972mmol,2.5 eq) was added and the reaction was refluxed for 3h. The reaction was diluted with 100mL of dichloromethane, washed once with saturated aqueous sodium chloride (100 mL), the organic phase was collected and concentrated, and the crude product was purified by column on silica gel (dichloromethane: methanol volume ratio=10:1). After concentrating the eluate under reduced pressure, the product was obtained as a white solid (2 g, yield 58%).

Synthesis of Compound 6-2: compound 6-1 (2 g,1.605mmol,1 eq) was dissolved in tetrahydrofuran (20 mL), tetrabutylammonium fluoride (2.534 g,8.025mmol,5 eq) was added and stirred at 40℃for 16 hours. The reaction was diluted with dichloromethane (100 mL), washed once with saturated aqueous sodium chloride (100 mL), and the organic phase was collected and concentrated. The crude product was purified by column on silica gel (petroleum ether: ethyl acetate volume ratio=1:1). The eluate was concentrated under reduced pressure to give the product as an off-white oil (1 g, yield 62%, purity 92.8%).

Synthesis of Compound 6: compound 6-2 (1 g,0.992mmol,1 eq) was dissolved in methylene chloride (10 mL), methyl iodide (442 mg,2.976mmol,3 eq) was added, and the mixture was stirred at room temperature for 16 hours. The reaction solution was directly purified by silica gel column (dichloromethane: methanol volume ratio=30:1). After concentration of the eluate, the product was obtained as a yellow solid (505.5 mg, yield 49%, purity 95.043%).

Compound 6 ¹ The H NMR spectrum is shown in FIG. 6, and the nuclear magnetic data is ¹ H NMR (300 MHz, chloroform-d) delta 5.40-5.30 (m, 4H), 5.09 (s, 1H), 4.92 (s, 1H), 4.56 (s, 2H), 4.12 (s, 2H), 3.54-3.44 (m,10H),2.42-2.21(m,6H),2.08-1.94(m,11H),1.89-1.75(m,3H),1.71-1.56(m,10H),1.45-1.20(m,48H),1.05-0.97(m,3H),0.91-0.86(m,9H),0.80(d,J＝6.0Hz,3H),0.73(s,3H).

Example 7

This example provides compound 7, the synthetic route of which is as follows:

the preparation method comprises the following steps:

synthesis of Compound 7-1: compound 1-1 (3 g,6.254mmol,1 eq) was dissolved in pyridine (60 mL), benzyltriethylammonium chloride (427.3 mg,1.876mmol,0.3 eq) and oleoyl chloride (9.4 g,31.270mmol,5 eq) were added in sequence, and stirred at reflux for 3 hours. After cooling to room temperature, dichloromethane was added for dilution. The organic phase was collected and concentrated by washing with saturated sodium chloride. The crude product was purified by reverse phase column (40% -80% tetrahydrofuran, 30 min). The target eluate was collected and concentrated to give a colorless oil (2.5 g, yield 29%, purity 95%).

Synthesis of Compound 7: compound 7-1 (2.3 g,1.716mmol,1 eq) was dissolved in dichloromethane (46 mL), methyl iodide (487.2 mg,3.432mmol,2 eq) was added and reacted at room temperature for 16 hours. After the reaction solution was concentrated, the crude product was purified by a silica gel column (dichloromethane: methanol volume ratio=10:1) to give the product as a yellow solid (1.3042 g, yield 54%, purity 95%). MS M/z [ M ] + (ESI): 1287.05.

Compound 7 ¹ The H NMR spectrum is shown in FIG. 7, and the nuclear magnetic data is ¹ H NMR (300 MHz, chloroform-d) delta 5.48-5.24 (m, 6H), 5.10 (s, 1H), 4.94 (d, j=3.6 hz, 1H), 4.57 (d, j=5.5 hz, 3H), 4.12 (t, j=4.6 hz, 2H), 3.55 (s, 9H), 2.52-2.19 (m, 8H), 2.15-1.89 (m, 16H), 1.83-1.50 (m, 13H), 1.44-1.18 (m, 68H), 1.14-1.01 (m, 3H), 0.95-0.84 (m, 12H), 0.81 (d, j=6.3 hz, 3H), 0.74 (s, 3H).

Example 8

This example provides compound 8, which is synthesized as follows:

the preparation method comprises the following steps:

synthesis of Compound 8-1: the compound cholic acid (2 g, 4.292 mmol,1 eq) was dissolved in N, N-dimethylamide (40 mL), and dimethylamine butanol (746 mg,4.337mmol,1.3 eq), EDCI (1.4 g,7.292mmol,1.5 eq) and DMAP (719 mg,1.467mmol,0.3 eq) were added sequentially and stirred overnight at room temperature. The reaction was directly purified by reverse phase column (C18 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 (1.5 g, 60% yield).

Synthesis of Compound 8-2: compound 8-1 (1.5 g,2.959mmol,1 eq) and DMAP (433 mg,3.549mmol,1.2 eq) were dissolved in dichloromethane (15 mL). A solution of octadecanoyl chloride (983 mg,3.255mmol,1.1 eq) in dichloromethane (15 mL) was slowly added dropwise to the reaction solution at 0deg.C and reacted at room temperature for 2 hours. 100mL of 6wt% aqueous acetic acid was added and extracted three times with methylene chloride. The organic phases were combined, washed with saturated aqueous sodium chloride solution, then dried over anhydrous sodium sulfate and concentrated. The crude product is purified by a silica gel column (the volume ratio of dichloromethane to methanol is 1:0-20:1). The eluate was concentrated to give the product as a yellow solid (600 mg, yield 26%).

Synthesis of Compound 8: compound 8-2 (600 mg,0.775mmol,1 eq) was dissolved in dichloromethane (20 mL), methyl trifluoroacetate (297.6 mg,2.325mmol,3 eq) was added, and triethylamine (8 mg,0.078mmol,0.1 eq) was added after 1 hour of reaction. Stir at room temperature for 2 hours. The crude product was purified using reverse phase column (C18 column). The eluate was concentrated to give the product as an off-white semisolid (548.5 mg, 89% yield).

Compound 8 ¹ The H NMR spectrum is shown in FIG. 8, and the nuclear magnetic data is ¹ H NMR(300MHz,DMSO-d6)δ4.49-4.42(m,1H),4.04(t,J＝6.3Hz,2H),3.79(s,1H),3.62(s,1H),3.37-3.24(m,2H),3.04(s,9H),2.46-2.07(m,6H),2.04-1.94(m,1H),1.85-1.68(m,7H),1.65-1.42(m,8H),1.43-1.16(m,38H),1.01-0.89(m,5H),0.88-0.84(m,6H),0.59(s,3H).

¹⁹ F-NMR(400MHz,DMSO-d6)δ-74.46。

Example 9

This example provides compound 9, the synthetic route of which is as follows:

the preparation method comprises the following steps:

synthesis of Compound 9-1: the compound cholic acid (1.5 g,3.671mmol,1 eq) was dissolved in N, N-dimethylformamide (30 mL), and dimethylaminohexanol (799.9 mg,5.506mmol,1.5 eq), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.06 g,5.506mmol,1.5 eq) and 4-dimethylaminopyridine (134.6 mg,1.101mmol,0.3 eq) were added sequentially. Stir at room temperature overnight. The reaction was directly purified by reverse phase column (C18 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 (1.4 g, 71% yield).

Synthesis of Compound 9-2: compound 9-1 (1.3 g,2.426mmol,1 eq) was dissolved in dichloromethane (13 mL) and 4-dimethylaminopyridine (326.1 mg, 2.81mmol, 1.1 eq) was added. A solution of octadecanoyl chloride (808.5 mg, 2.81mmol, 1.1 eq) in methylene chloride (13 mL) was slowly added dropwise to the reaction solution after cooling to 0deg.C. Stir at room temperature for 2 hours. 100mL of 6wt% aqueous acetic acid was added and extracted three times with methylene chloride. The organic phases were combined, washed with saturated aqueous sodium chloride solution, then dried over anhydrous sodium sulfate and concentrated. The crude product was purified by column chromatography on silica gel (volume ratio dichloromethane/methanol 1:0-20:1) to give the product as a white solid (1 g, 51% yield).

Synthesis of compound 9: compound 9-2 (900 mg,1.122mmol,1 eq) was dissolved in dichloromethane (18 mL) and methyl trifluoroacetate (430.8 mg, 3.365 mmol,3 eq) and triethylamine (113.5 mg,1.122mmol,1 eq) were added. Stir at room temperature for 2 hours. The reaction was concentrated and the crude product purified using a reverse phase column (C18 column). The eluate was spin-dried to give the product as a yellow semisolid (586.4 mg, 56% yield).

Compound 9 ¹ The H NMR spectrum is shown in FIG. 9, and the nuclear magnetic data is ¹ H NMR(400MHz,DMSO-d6)δ4.50-4.37(m,1H),4.01(t,J＝6.6Hz,2H),3.79(s,1H),3.62(s,1H),3.32-3.20(m,2H),3.04(s,9H),2.47-2.27(m,2H),2.27-2.07(m,4H),2.03-1.92(m,1H),1.87-1.76(m,2H),1.74-1.61(m,6H),1.61-1.43(m,7H),1.43-1.32(m,8H),1.30-1.07(m,34H),1.02-0.79(m,11H),0.59(s,3H)。

¹⁹ F-NMR(400MHz,DMSO-d6)δ-74.20。

Example 10

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

(1) ALC-0315 (cationic lipid, available from Xiaomenobang Biotechnology Co., ltd.), structural lipid compound or 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 structural lipid compounds are respectively compounds 1 to 9 and 2-1;

(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; 2mL 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).

Physical property test:

the particle size, PDI (dispersibility index) encapsulation efficiency and mRNA concentration of the mRNA-LNP provided in example 8 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%.

mRNA concentration: mRNA concentration was determined by Stuner ultraviolet spectrophotometry. 2. Mu.L of the sample was added to the sample cell, and the total concentration of mRNA in the sample was measured by selecting the type of measurement to be RNA-LNP at a wavelength of 260 nm.

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

TABLE 1

From the test results of Table 1, it can be seen that mRNA-LNP prepared with compounds 1 to 9 and compound 2-1 each exhibited similar particle size (75 to 120 nm), similar or smaller PDI (0.02 to 0.19), similar encapsulation efficiency (75 to 98%), and similar mRNA concentration, as compared to DSPC.

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 8 was added 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. 10.

As can be seen from fig. 10, the transfection efficiency of Luciferase-mRNA-LNP prepared with compound 1, compound 2-1, compound 4, compound 6, and compound 7 was similar to that of DSPC, and the transfection efficiency of Luciferase-mRNA-LNP prepared with compound 2, compound 3, compound 5, compound 8, and compound 9 was significantly higher than that of DSPC.

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 structural lipid compound, characterized in that the structural lipid compound has a structure represented by the following formula I:

in the formula I, R ₁ 、R ₂ 、R ₃ Each independently selected from the group consisting of hydroxy, One of the following;

R ₄ selected from the group consisting ofOne of the following;

wherein L is ₁ Is C1-C19 alkyl or C9-C21 alkenyl, L ₂ Is C3-C13 alkylene, L ₃ Is C2-C14 alkyl, L ₄ Is a C2-C6 alkylene group,anions to satisfy chemical environment;

represents a bond of a group.

2. The structural lipid compound according to claim 1, wherein R ₁ 、R ₂ 、R ₃ Each independently selected from one of the following groups:

wherein — represents a bond of a group.

3. The structural lipid compound according to claim 1 or 2, wherein R ₁ 、R ₂ 、R ₃ Each independently selected from one of the following groups:

and R is ₁ 、R ₂ 、R ₃ Not all are selected from

Wherein — represents a bond of a group.

4. A structural lipid compound according to any one of claims 1-3, wherein theIs CF (CF) ₃ COO ^- 、I ^- Or Cl ^- Preferably CF ₃ COO ^- Or I ^- 。

5. The structural lipid compound according to any one of claims 1 to 4, wherein the structural lipid compound is selected from the group consisting of the following compounds 1 to 9 and 2 to 1:

6. a method of preparing a structured lipid compound according to any one of claims 1 to 5, comprising the steps of:

combining cholic acid with a compoundDissolving in an organic solvent, and reacting under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to generate an intermediate 1;combining said intermediate 1 with R ₁ 、R ₂ And/or R ₃ To produce intermediate 2 as said structural lipid compound; or the intermediate 2 is further reacted with a compound M-CH ₃ Reacting to generate the structural lipid compound;

or comprises the following steps:

dissolving cholic acid in an organic solvent, adding potassium carbonate, and dropwise adding benzyl bromide for reaction to generate an intermediate 3; combining said intermediate 3 with R ₁ 、R ₂ And/or R ₃ Reacting the acid chloride compound of (a) to form intermediate 4; the intermediate 4 is reduced in a hydrogen environment under the catalysis of palladium-carbon to generate an intermediate 5; combining said intermediate 5 with a compoundDissolving in an organic solvent, and reacting under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to generate an intermediate 2 serving as the structural lipid compound; or the intermediate 2 is further reacted with a compound M-CH ₃ And (3) reacting to generate the structural lipid compound.

7. Use of a structured 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 and a structured lipid compound according to any one of claims 1-5;

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

preferably, the cationic lipid is selected from one or more of 4- (N, N-dimethylamino) butanoic acid (diiodo) methyl ester, [ (4-hydroxybutyl) azadialkyl ] bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) and heptadec-9-yl-8- [ (2-hydroxyethyl) (6-oxo-6- ((decyloxy) hexyl) amino) octanoate ];

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

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

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.