Structural lipid compounds, methods for their preparation and uses thereof

Abstract

The application provides a structural lipid compound and a preparation method and application thereof. The structural lipid compound has a structure shown in formula I. The structural lipid compound provided by the application can replace traditional neutral phospholipids to prepare drug-loaded lipid nanoparticles. The drug-loaded lipid nanoparticles containing the structural lipid compound have small particle sizes, uniform particle size distribution, and good loading and delivery effects on nucleic acid drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a structural lipid compound, its preparation method, and its uses. Background Technology

[0002] Since the outbreak of the COVID-19 pandemic, mRNA vaccines developed by Pfizer-BioNTech and Moderna have been successfully commercialized in the United States, and mRNA drugs have quickly become a hot topic in the biopharmaceutical field. It has been reported that both vaccines offer protection against COVID-19 infection exceeding 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 traditional vaccines, mRNA vaccines have many advantages, including simple production processes, rapid development, quick response to variant strains, and rapid scaling up of production; furthermore, mRNA degrades rapidly within cells, posing no risk of integration into the host genome. However, mRNA also has disadvantages such as instability, high innate immunogenicity, low in vivo delivery efficiency, and easy 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].

[0003] Lipid nanoparticles (LNPs) delivery systems refer to nanoparticles formed by the self-assembly of various lipid components to encapsulate and deliver nucleic acid drugs. They can effectively improve the stability of nucleic acid drugs, reduce their immunogenicity, and enhance their in vivo delivery efficiency. The first commercial application of LNPs was Onpattro, which was approved in the US and EU in 2018 for the treatment of amyloidosis. Since then, LNPs have received widespread attention as nucleic acid delivery carriers. In particular, since 2020, Moderna and BioNTech's novel coronavirus mRNA vaccines have both adopted LNP delivery systems. LNP delivery systems generally contain four lipid components [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]: 1) Ionizable cationic lipids, used to bind to negatively charged mRNA; 2) Cholesterol: mediates LNP endocytosis and stabilizes LNP structure; 3) Neutral phospholipids: helper lipids that can accelerate the release of mRNA during endocytosis; 4) PEG phospholipids: prolong metabolic time, improve LNP stability, and control particle size.

[0004] Neutral phospholipids can modulate the fluidity of nanoparticles and enhance their delivery efficiency by promoting lipid phase transitions that facilitate membrane-endosome fusion. Common neutral phospholipids include DSPC, DOPC, and DSPE, among which DSPC... Neutral phospholipids have been used in the FDA-approved SARS-CoV-2 vaccines mRNA-1273 and BNT162b2. Neutral phospholipids play a crucial role in the self-assembly process, liposome stability, in vivo delivery, endocytosis, and intracellular transfection and release of LNP delivery systems.

[0005] However, there is currently limited research on neutral phospholipids in LNP delivery systems, and there are significant patent protection barriers. Therefore, there is an urgent need to develop more effective compounds with similar functions to promote the development of the nucleic acid drug industry. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a structured lipid compound, its preparation method, and its uses. This structured lipid compound can replace traditional neutral phospholipids for the preparation of drug-loaded lipid nanoparticles. Drug-loaded lipid nanoparticles containing this structured lipid compound have small particle sizes and uniform particle size distribution, exhibiting excellent loading and delivery effects for nucleic acid drugs.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a structural lipid compound having the structure shown in Formula I:

[0009]

[0010] In Formula I, R1, R2, and R3 are each independently selected from hydroxyl groups, One of them;

[0011] R4 is selected from One of them;

[0012] Wherein, L1 is a C1-C19 alkyl or C9-C21 alkenyl, L2 is a C3-C13 alkylene, L3 is a C2-C14 alkyl, and L4 is a C2-C6 alkylene. To meet the requirements of anions in the chemical environment;

[0013] -* represents the linking bond of a group.

[0014] It should be noted that the alkenyl group described in this invention may contain one carbon-carbon double bond or multiple carbon-carbon double bonds.

[0015] In some embodiments of the present invention, R1, R2, and R3 are each independently selected from one of the following groups:

[0016]

[0017]

[0018]

[0019] Here, -* represents the linking bond of a group.

[0020] In some embodiments of the present invention, R1, R2, and R3 are each independently selected from one of the following groups:

[0021]

[0022]

[0023] And R1, R2, and R3 are not all selected from

[0024] Here, -* represents the linking bond of a group.

[0025] In some embodiments of the present invention, the For CF3COO - I - or Cl - Preferably CF3COO - or I - .

[0026] In some embodiments of the present invention, the structural lipid compound is selected from compounds 1 to 9 and compound 2-1:

[0027]

[0028]

[0029] In a second aspect, the present invention provides a method for preparing the structural lipid compound as described in the first aspect, the method comprising the following steps:

[0030]

[0031] Bile acid and compounds Dissolved in an organic solvent, the mixture is reacted under the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) to generate intermediate 1; intermediate 1 is reacted with acyl chloride compounds of R1, R2 and / or R3 to generate intermediate 2 as the structural lipid compound; or intermediate 2 is further reacted with compound M-CH3 to generate the structural lipid compound.

[0032] Or it may include the following steps:

[0033]

[0034] Cholic acid is dissolved in an organic solvent, potassium carbonate is added, and benzyl bromide is added dropwise to react and generate intermediate 3. Intermediate 3 is reacted with acyl chloride compounds of R1, R2, and / or R3 to generate intermediate 4. Intermediate 4 is reduced in a hydrogen atmosphere under the catalysis of palladium on carbon to generate intermediate 5. Intermediate 5 is then reacted with a compound... Dissolved in an organic solvent, the mixture is reacted under the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to generate intermediate 2 as the structural lipid compound; or intermediate 2 is further reacted with compound M-CH3 to generate the structural lipid compound.

[0035] It should be noted that the acyl chloride compounds R1, R2, and R3 mentioned in this invention refer to compounds in which the acyl oxygen group in R1, R2, and R3 is replaced by an acyl chloride group. For example, when R1 is... When, the acyl chloride compound of R1 is

[0036] Thirdly, the present invention provides the use of the structural lipid compound as described in the first aspect for preparing a drug carrier, preferably for preparing a nucleic acid drug carrier.

[0037] Fourthly, the present invention provides lipid nanoparticles comprising cationic lipids and structural lipid compounds as described in the first aspect.

[0038] In some embodiments of the present invention, the lipid nanoparticles further include cholesterol and polymer-modified lipids.

[0039] In some embodiments of the present invention, the cationic lipid is selected from one or more of the following: methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester (abbreviated as DLin-MC3-DMA), [(4-hydroxybutyl)azadialkyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (abbreviated as ALC-0315), and heptadecan-9-yl-8-[(2-hydroxyethyl)(6-oxo-6-((decoxy)hexyl)amino)octanoate] (abbreviated as SM-102).

[0040] In some embodiments of the present invention, the polymer-modified lipid is a polyethylene glycol-modified lipid.

[0041] In some embodiments of the present invention, the lipid nanoparticles comprise the following components in molar percentages: 30-50% cationic lipids, 5-25% structural lipid compounds as described in the first aspect, 28.5-48.5% cholesterol, and 0.5-3% polyethylene glycol-modified lipids.

[0042] Fifthly, the present invention provides a drug-loaded lipid nanoparticle, comprising: the lipid nanoparticle as described in the fourth aspect and a nucleic acid drug loaded in the lipid nanoparticle.

[0043] Sixthly, the present invention provides a method for preparing drug-loaded lipid nanoparticles as described in the fifth aspect, the method comprising the following steps:

[0044] An oil phase containing the components of the lipid nanoparticles and an aqueous phase containing nucleic acid drugs were prepared separately. The oil phase and the aqueous phase were mixed using a microfluidic device to self-assemble the drug-loaded lipid nanoparticles.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] The structural lipid compounds provided by this invention can replace traditional neutral phospholipids (such as DSPC) for the preparation of drug-loaded lipid nanoparticles. Drug-loaded lipid nanoparticles containing these structural lipid compounds have small particle sizes and uniform particle size distribution, exhibiting good encapsulation efficiency and transfection efficiency. They can effectively load nucleic acid drugs and deliver them to cells and animals for expression.

[0047] Furthermore, the structured lipid compounds provided by this invention have a simpler synthesis process and higher yield compared to traditional neutral phospholipids (such as DSPC), which helps to reduce production costs. Attached Figure Description

[0048] Figure 1 Compound 1 of the present invention 1 H NMR spectrum;

[0049] Figure 2 Compound 2 of the present invention 1 H NMR spectrum;

[0050] Figure 3 Compound 3 of the present invention 1 H NMR spectrum;

[0051] Figure 4 Compound 4 of the present invention 1 H NMR spectrum;

[0052] Figure 5 Compound 5 of the present invention 1 H NMR spectrum;

[0053] Figure 6 Compound 6 of the present invention 1 H NMR spectrum;

[0054] Figure 7 Compound 7 of the present invention 1 H NMR spectrum;

[0055] Figure 8 Compound 8 of the present invention 1 H NMR spectrum;

[0056] Figure 9 Compound 9 of the present invention 1 H NMR spectrum;

[0057] Figure 10 This is a diagram showing the cell transfection results of the drug-loaded lipid nanoparticles prepared in Example 10 of this invention. Detailed Implementation

[0058] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art should understand that the specific embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0059] Example 1

[0060] This embodiment provides compound 1, whose synthetic route is as follows:

[0061]

[0062] The specific preparation method is as follows:

[0063] Synthesis of Compound 1-1: Cholic acid (30 g, 73.529 mmol, 1 eq) was dissolved in N,N-dimethylformamide (600 mL), followed by the addition of dimethylaminoethanol (13.088 g, 147.059 mmol, 2 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21.066 g, 110.294 mmol, 1.5 eq), and 4-dimethylaminopyridine (2.691 g, 22.059 mmol, 0.3 eq). The mixture was stirred overnight at room temperature. The reaction solution was collected and purified by reverse-phase column chromatography. The eluent was extracted twice with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and concentrated to give the product as a white solid (30 g, yield 85%, purity 99%).

[0064] Synthesis of compounds 1-2: Compound 1-1 (3 g, 7.255 mmol, 1 eq) and glutaric anhydride (927 mg, 8.133 mmol, 1.3 eq) were dissolved in pyridine solution and stirred overnight at 80 °C. The reaction solution was cooled to room temperature, concentrated, and the pyridine was evaporated to dryness. The crude product was diluted with ethyl acetate and washed with saturated sodium chloride aqueous solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give a pale yellow oil (2 g, yield 54%).

[0065] Synthesis of compounds 1-3: Compounds 1-2 (2 g, 3.373 mmol, 1 eq) were dissolved in dichloromethane (20 mL). Then, n-dodecyl alcohol (754 mg, 4.384 mmol, 1.3 eq), EDCI (971 mg, 5.057 mmol, 1.5 eq), and DMAP (123 mg, 1.008 mmol, 0.3 eq) were added sequentially, and the mixture was stirred at room temperature for 16 hours. The solution was first diluted with dichloromethane, then washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by silica gel column chromatography (dichloromethane to methanol volume ratio 1:0-20:1). The eluent was concentrated to give the product as a yellow solid (1 g, yield 39%).

[0066] Synthesis of Compound 1: Compounds 1-3 (1 g, 1.314 mmol, 1 eq) were dissolved in dichloromethane (20 mL), and iodomethane (560 mg, 3.944 mmol, 3 eq) was added. The mixture was left to stand overnight at room temperature. The solution was directly evaporated to dryness, and the crude product was purified by silica gel column chromatography (dichloromethane / methanol volume ratio 1:0-10:1). The eluent was concentrated to give the product as a yellow solid (504.9 mg, yield 49%).

[0067] Compound 1 1 H NMR spectrum as shown Figure 1 As shown, the NMR data are 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).

[0068] Example 2

[0069] This embodiment provides compound 2, whose synthetic route is as follows:

[0070]

[0071] The specific preparation method is as follows:

[0072] Synthesis of Compound 2-1: Compound 1-1 (3 g, 6.254 mmol, 1 eq) was dissolved in dichloromethane (30 mL), and 4-dimethylaminopyridine (840.5 mg, 6.879 mmol, 1.1 eq) was added. A solution of octadecanoyl chloride (2.084 g, 6.879 mmol, 1.1 eq) in dichloromethane (30 mL) was slowly added dropwise to the reaction mixture. The reaction was carried out at room temperature for 2 hours, and the reaction mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol v / v = 20:1) to give a white solid (1.6 g, yield 34%, purity 99%).

[0073] Synthesis of Compound 2: Compound 2-1 (1.4 g, 0.899 mmol, 1 eq) was dissolved in dichloromethane (23 mL), and iodomethane (534 mg, 3.752 mmol, 2 eq) was added. The reaction was carried out at room temperature for 16 hours, and the reaction solution was concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol volume ratio = 10:1) to give a yellow solid (1.1243 g, yield 66%, purity 98%).

[0074] Compound 2 1 H NMR spectrum as shown Figure 2 As shown, the NMR data are 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),2.0 4-1.91(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).

[0075] Example 3

[0076] This embodiment provides compound 3, whose synthetic route is as follows:

[0077]

[0078] The specific preparation method is as follows:

[0079] Synthesis of compound 3-1: Compound 1-1 (10 g, 20.846 mmol, 1 eq) was dissolved in tetrahydrofuran (200 mL), imidazole (2.84 g, 41.692 mmol, 2 eq) was added, and tert-butyldiphenylchlorosilane (13.088 g, 147.059 mmol, 2 eq) was slowly added. The reaction was carried out at room temperature for 4 hours. The reaction solution was collected and diluted with dichloromethane. The organic phases were combined and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol v / v = 20:1). The eluent was concentrated to give a yellow oily substance (9.0 g, yield 60%, purity 87%).

[0080] Synthesis of compound 3-2: Compound 3-1 (2.5 g, 4.209 mmol, 1 eq) was dissolved in pyridine (40 mL), and octadecanoyl chloride (3.19 g, 10.522 mmol, 2.5 eq) was added. The mixture was stirred at 110 °C for 3 hours. The reaction solution was diluted with dichloromethane (100 mL), and the organic phases were combined and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate volume ratio = 1:1). The eluent was concentrated to give a yellow oily substance (2.0 g, yield 53%).

[0081] Synthesis of compound 3-3: Compound 3-2 (1.6 g, 1.279 mmol, 1 eq) was dissolved in tetrahydrofuran (24 mL), and tetrabutylammonium fluoride (1.27 mg, 8.953 mmol, 7 eq) was added. The mixture was stirred at 40 °C for 4 hours under nitrogen protection. The reaction solution was diluted with ethyl acetate (100 mL) and washed once with saturated sodium chloride aqueous solution (100 mL). The organic phases were combined and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol v / v = 20:1), and the eluent was concentrated to give a colorless oily product (1.0 g, yield 77%).

[0082] Synthesis of Compound 3: Compound 3-3 (800 mg, 0.790 mmol, 1 eq) was dissolved in dichloromethane (1.6 mL), and iodomethane (336.40 mg, 2.370 mmol, 3 eq) was added. The reaction was carried out at room temperature for 4 hours. After concentration, the reaction solution was purified by silica gel column chromatography (dichloromethane:methanol volume ratio = 20:1). The eluent was concentrated to give a yellow oily product (506.4 mg, yield 55%, purity 97%).

[0083] Compound 3 1 H NMR spectrum as shown Figure 3 As shown, the NMR data are 1¹H NMR (300 MHz, chloroform-d) δ 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).

[0084] Example 4

[0085] This embodiment provides compound 4, whose synthetic route is as follows:

[0086]

[0087] The specific preparation method is as follows:

[0088] Synthesis of compound 4-1: Compound 1-2 (1 g, 1.686 mmol, 1 eq) was dissolved in dichloromethane (20 mL), followed by the sequential addition of dodecylamine (406 mg, 2.195 mmol, 1.3 eq), HATU (0.96 g, 2.52 mmol, 1.5 eq), and DIEA (N,N-diisopropylethylamine, 650 mg, 5.04 mmol, 3 eq). The mixture was stirred overnight at room temperature. The reaction solution was diluted with dichloromethane and washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by silica gel column chromatography (dichloromethane to methanol volume ratio 1:0–6:1). The eluent was concentrated to give a pale yellow oil (500 mg, yield 39%).

[0089] Synthesis of Compound 4: Compound 4-1 (500 mg, 0.657 mmol, 1 eq) was dissolved in dichloromethane (10 mL), and methyl trifluoroacetate (252 mg, 1.971 mmol, 3 eq) was added. After reacting for 1 hour, triethylamine (7 mg, 0.066 mmol, 0.1 eq) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated, and the crude product was purified using a reverse-phase column (C18 column). The eluent was concentrated to give the product as a pale white solid (258.0 mg, 50% yield).

[0090] Compound 4 1 H NMR spectrum as shown Figure 4 As shown, the NMR data are 1H 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).

[0091] 19 F-NMR(400MHz,DMSO-d6)δ-74.38.

[0092] Example 5

[0093] This embodiment provides compound 5, whose synthetic route is as follows:

[0094]

[0095] The specific preparation method is as follows:

[0096] Synthesis of compound 5-1: Compound 1-1 (4 g, 8.351 mmol, 1 eq) was dissolved in dichloromethane (40 mL), followed by the addition of dimethylaminopyridine (1.019 g, 8.352 mmol, 1 eq) and dropwise addition of petroyl chloride (2.513 g, 8.371 mmol, 1 eq). The mixture was stirred at room temperature for 2 h and extracted three times with dichloromethane (100 mL). The organic phases were combined, washed once with saturated sodium chloride aqueous solution (300 mL), and the organic phase was collected and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol v / v = 20:1). The eluent was concentrated under reduced pressure to give a grayish-white oil (1.444 g, yield 23%, purity 95.6%).

[0097] Synthesis of Compound 5: Compound 5-1 (1.3 g, 1.747 mmol, 1 eq) was dissolved in dichloromethane (26 mL), and iodomethane (744 mg, 5.239 mmol, 3 eq) was added. The reaction was carried out at room temperature for 16 hours. The crude product was concentrated and purified by silica gel column chromatography (dichloromethane:methanol v / v = 20:1). The eluent was concentrated to give the product as a yellow solid (0.7098 g, yield 54%, purity 97%).

[0098] Compound 5 1 H NMR spectrum as shown Figure 5 As shown, the NMR data are 1¹H NMR (400 MHz, chloroform-d) δ 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 = 5.2 Hz, 3H), 0.90–0.86 (m, 6H), 0.70 (s, 3H).

[0099] Example 6

[0100] This embodiment provides compound 6, whose synthetic route is as follows:

[0101]

[0102] The specific preparation method is as follows:

[0103] Synthesis of compound 6-1: Compound 1-1 (2 g, 13.947 mmol, 1 eq) was dissolved in pyridine (20 mL), and triethylbenzylammonium bromide (1.588 g, 6.972 mmol, 2.5 eq) was added. The mixture was refluxed for 3 h. The reaction mixture was diluted with 100 mL of dichloromethane and washed once with 100 mL of saturated sodium chloride aqueous solution. The organic phase was collected and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane:methanol v / v = 10:1). The eluent was concentrated under reduced pressure to give the product as a white solid (2 g, yield 58%).

[0104] Synthesis of compound 6-2: Compound 6-1 (2 g, 1.605 mmol, 1 eq) was dissolved in tetrahydrofuran (20 mL), and tetrabutylammonium fluoride (2.536 g, 8.025 mmol, 5 eq) was added. The mixture was stirred at 40 °C for 16 hours. The reaction solution was diluted with dichloromethane (100 mL), washed once with saturated sodium chloride aqueous solution (100 mL), and the organic phase was collected and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate volume ratio = 1:1). The eluent was concentrated under reduced pressure to give a grayish-white oil (1 g, yield 62%, purity 92.8%).

[0105] Synthesis of Compound 6: Compound 6-2 (1 g, 0.992 mmol, 1 eq) was dissolved in dichloromethane (10 mL), and iodomethane (442 mg, 2.976 mmol, 3 eq) was added. The mixture was stirred at room temperature for 16 hours. The reaction solution was directly purified by silica gel column chromatography (dichloromethane:methanol v / v = 30:1). After concentration of the eluent, the product was given as a yellow solid (505.5 mg, yield 49%, purity 95.043%).

[0106] Compound 6 1 H NMR spectrum as shown Figure 6 As shown, the NMR data are 1 ¹H NMR (300 MHz, chloroform-d) δ 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 = 6.0 Hz, 3H), 0.73 (s, 3H).

[0107] Example 7

[0108] This embodiment provides compound 7, whose synthetic route is as follows:

[0109]

[0110] The specific preparation method is as follows:

[0111] Synthesis of Compound 7-1: Compound 1-1 (3 g, 6.254 mmol, 1 eq) was dissolved in pyridine (60 mL), and benzyltriethylammonium chloride (427.3 mg, 1.876 mmol, 0.3 eq) and oleoyl chloride (9.4 g, 31.270 mmol, 5 eq) were added sequentially. The mixture was stirred and refluxed for 3 hours. After cooling to room temperature, it was diluted with dichloromethane. The product was washed with saturated sodium chloride, and the organic phase was collected and concentrated. The crude product was purified by reverse-phase column chromatography (40%–80% tetrahydrofuran, 30 min). The target eluent was collected and concentrated to give a colorless oil (2.5 g, yield 29%, purity 95%).

[0112] Synthesis of compound 7: Compound 7-1 (2.3 g, 1.716 mmol, 1 eq) was dissolved in dichloromethane (46 mL), and iodomethane (487.2 mg, 3.432 mmol, 2 eq) was added. The reaction mixture was reacted at room temperature for 16 hours. After concentration, the crude product was purified by silica gel column chromatography (dichloromethane:methanol v / v = 10:1) to give a yellow solid (1.3042 g, yield 54%, purity 95%). MS m / z [M]+ (ESI): 1287.05.

[0113] Compound 7 1 H NMR spectrum as shown Figure 7 As shown, the NMR data are 1 ¹H NMR (300 MHz, chloroform-d) δ 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).

[0114] Example 8

[0115] This embodiment provides compound 8, whose synthetic route is as follows:

[0116]

[0117] The specific preparation method is as follows:

[0118] Synthesis of compound 8-1: Cholic acid (2 g, 4.902 mmol, 1 eq) was dissolved in N,N-dimethylamide (40 mL) solution, followed by the sequential addition of dimethylaminobutanol (746 mg, 4.337 mmol, 1.3 eq), EDCI (1.4 g, 7.292 mmol, 1.5 eq), and DMAP (179 mg, 1.467 mmol, 0.3 eq) at room temperature and stirred overnight. The reaction solution was purified directly using a reverse-phase column (C18 column), and the eluent 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).

[0119] Synthesis of Compound 8-2: Compound 8-1 (1.5 g, 2.959 mmol, 1 eq) and DMAP (433 mg, 3.549 mmol, 1.2 eq) were dissolved in dichloromethane (15 mL). A solution of octadecanoyl chloride (983 mg, 3.255 mmol, 1.1 eq) in dichloromethane (15 mL) was slowly added dropwise to the reaction mixture at 0 °C, and the reaction was carried out at room temperature for 2 hours. 100 mL of 6 wt% aqueous acetic acid was added, and the mixture was extracted three times with dichloromethane. The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane to methanol volume ratio 1:0–20:1). The eluent was concentrated to give the product as a yellow solid (600 mg, yield 26%).

[0120] Synthesis of Compound 8: Compound 8-2 (600 mg, 0.775 mmol, 1 eq) was dissolved in dichloromethane (20 mL), and methyl trifluoroacetate (297.6 mg, 2.325 mmol, 3 eq) was added. After reacting for 1 hour, triethylamine (8 mg, 0.078 mmol, 0.1 eq) was added. The mixture was stirred at room temperature for 2 hours. The crude product was purified using a reverse-phase column (C18 column). The eluent was concentrated to give the product as a white semi-solid (548.5 mg, yield 89%).

[0121] Compound 8 1 H NMR spectrum as shown Figure 8 As shown, the NMR data are 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).

[0122] 19 F-NMR(400MHz,DMSO-d6)δ-74.46.

[0123] Example 9

[0124] This embodiment provides compound 9, whose synthetic route is as follows:

[0125]

[0126] The specific preparation method is as follows:

[0127] Synthesis of Compound 9-1: Cholic acid (1.5 g, 3.671 mmol, 1 eq) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of dimethylaminohexanol (799.9 mg, 5.506 mmol, 1.5 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.06 g, 5.506 mmol, 1.5 eq), and 4-dimethylaminopyridine (134.6 mg, 1.101 mmol, 0.3 eq). The mixture was stirred overnight at room temperature. The reaction solution was purified directly using a reverse-phase column (C18 column), and the eluent 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).

[0128] Synthesis of compound 9-2: Compound 9-1 (1.3 g, 2.426 mmol, 1 eq) was dissolved in dichloromethane (13 mL), and 4-dimethylaminopyridine (326.1 mg, 2.811 mmol, 1.1 eq) was added. The mixture was cooled to 0 °C, and a solution of octadecanoyl chloride (808.5 mg, 2.811 mmol, 1.1 eq) in dichloromethane (13 mL) was slowly added dropwise to the reaction mixture. The mixture was stirred at room temperature for 2 hours. 100 mL of 6 wt% aqueous acetic acid was added, and the mixture was extracted three times with dichloromethane. The organic phases were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol volume ratio 1:0–20:1) to give a white solid (1 g, yield 51%).

[0129] Synthesis of Compound 9: Compound 9-2 (900 mg, 1.122 mmol, 1 eq) was dissolved in dichloromethane (18 mL), and methyl trifluoroacetate (430.8 mg, 3.366 mmol, 3 eq) and triethylamine (113.5 mg, 1.122 mmol, 1 eq) were added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated, and the crude product was purified by reverse-phase column chromatography (C18 column). The eluent was evaporated to dryness to give the product as a yellow semi-solid (586.4 mg, yield 56%).

[0130] Compound 9 1 H NMR spectrum as shown Figure 9 As shown, the NMR data are 1H 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).

[0131] 19 F-NMR(400MHz,DMSO-d6)δ-74.20.

[0132] Example 10

[0133] This embodiment provides a series of drug-loaded lipid nanoparticles, the preparation method of which is as follows:

[0134] (1) ALC-0315 (cationic lipid, purchased from Xiamen Sainobange Biotechnology Co., Ltd.), structural lipid compounds or DSPC (distearate phosphatidylcholine), CHOL (cholesterol) and ALC-0159 (polyethylene glycol modified lipid, purchased from Xiamen Sainobange Biotechnology Co., Ltd.) were dissolved in anhydrous ethanol at 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;

[0135] Among them, the structural lipid compounds are compounds 1 to 9 and compound 2-1, respectively;

[0136] (2) Dissolve mRNA or Luciferase-mRNA (luciferase-labeled mRNA) in citrate buffer (pH = 4.5, 50 mM) to prepare an aqueous phase with an mRNA concentration of 0.133 mg / mL (0.4 mmol / L);

[0137] (3) The above oil phase and aqueous phase were introduced into a microfluidic nanofabrication system, and the oil-water volume ratio was controlled to be 1:3, so that the N / P ratio of lipid to mRNA (the molar ratio of nitrogen in lipid to phosphorus in mRNA) was 6:1, the total flow rate was 12 mL / min, the waste was discharged at the beginning and 0.1 mL at the end, and the prothrombus was prepared. 2 mL of prothrombus was loaded into a 10 kD dialysis card, the magnetic stirring speed was set to 120 rpm, and the mixture was dialyzed in 500 mL PBS dialysis solution for 4 h to obtain drug-loaded lipid nanoparticles (named mRNA-LNP or Luciferase-mRNA-LNP).

[0138] Physical property testing:

[0139] The particle size, PDI (dispersion index) encapsulation efficiency, and mRNA concentration of the mRNA-LNP provided in Example 8 were tested using the following methods:

[0140] Particle size and PDI: Mix 100 μL of mRNA-LNP sample with 900 μL of PBS and add it to the sample cell. Place the sample cell into the sample chamber of the Malvern Zetasizer Ultra nanoparticle size potentiometry instrument, select liposomes as the sample type, set the equilibration time to 30 seconds, and measure the particle size and PDI.

[0141] Encapsulation efficiency: The encapsulation efficiency of mRNA was determined using the Ribogreen fluorescence assay. Two mRNA-LNP samples were taken. One sample was diluted 50-fold with 1×TE buffer and then bound to Ribogreen fluorescent dye. The content of unencapsulated mRNA (F) was measured using a microplate reader (excitation wavelength 480 nm and emission wavelength 520 nm). free Another sample was demulsified with 2% Triton X-100 solution (polyethylene glycol octylphenyl ether), then diluted to 1 / 50 of the original mRNA concentration, bound to Ribogreen fluorescent dye, and the total mRNA concentration was determined using a microplate reader (excitation wavelength 480 nm and emission wavelength 520 nm). total According to the formula EE% = (F total -F free ) / F total Calculate the encapsulation efficiency of mRNA-LNP by multiplying by 100%.

[0142] mRNA concentration: mRNA concentration was determined using Stunner ultraviolet spectrophotometry. 2 μL of sample was added to the sample cell, the assay type was selected as RNA-LNP, and the wavelength was 260 nm to determine the total mRNA concentration in the sample.

[0143] The results of the above tests are shown in Table 1 below:

[0144] Table 1

[0145]

[0146] As can be seen from the test results in Table 1, compared with DSPC, the mRNA-LNPs prepared using compounds 1-9 and compound 2-1 all exhibited similar particle size (75-120 nm), similar or smaller PDI (0.02-0.19), similar encapsulation efficiency (75-98%), and similar mRNA concentration.

[0147] Cell transfection efficiency test:

[0148] 293T cells were added to a 96-well plate at a cell density of 2×10⁻⁶. 4 Each well contains 100 μL of enzyme-linked immunosorbent assay (ELISA) substrate and is incubated overnight at 37°C with 5% CO2. Luciferase-mRNA-LNP prepared in Example 8 is added to control the mRNA concentration at 50 ng / well. After 18 h of incubation, 100 μL of Luciferase fluorescent substrate is added to each well, and the reaction is allowed to proceed for 5 min. The fluorescence value of each well is detected using a fluorescence microplate reader, and the results are as follows: Figure 10 As shown.

[0149] from Figure 10 It can be seen that the transfection efficiency of Luciferase-mRNA-LNP prepared using compounds 1, 2-1, 4, 6, and 7 is similar to that of DSPC, while the transfection efficiency of Luciferase-mRNA-LNP prepared using compounds 2, 3, 5, 8, and 9 is significantly higher than that of DSPC.

[0150] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A structural lipid compound, characterized in that, The structural lipid compound has the structure shown in Formula I: Equation I; In formula I, R1 and R3 are each independently selected from one of the following groups: ； R2 is selected from one of the following groups: ； And R1, R2, and R3 are not all selected from , and ; R4 is selected from and One of them; wherein L4 is a C2~C6 alkylene group, For CF3COO - I - or Cl - ; Represents the connecting bond of a group.

2. The structural lipid compound according to claim 1, characterized in that, The structural lipid compound is selected from compounds 1 to 9 and compound 2-1: Compound 1 Compound 2 Compound 2-1 Compound 3 Compound 4 Compound 5 Compound 6 Compound 7 Compound 8 Compound 9.

3. A method for preparing the structural lipid compound as described in claim 1 or 2, characterized in that, The preparation method includes the following steps: Bile acid and compounds Dissolved in an organic solvent, the mixture is reacted under the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to generate intermediate 1; intermediate 1 is reacted with acyl chloride compounds of R1, R2 and / or R3 to generate intermediate 2 as the structural lipid compound; or intermediate 2 is further reacted with compound M-CH3 to generate the structural lipid compound; wherein the acyl chloride compounds of R1, R2 and / or R3 refer to compounds in which the acyl oxygen group in R1, R2 and / or R3 is replaced by an acyl chloride group.

4. Use of a structural lipid compound as described in claim 1 or 2 for the preparation of a drug carrier.

5. The use according to claim 4, characterized in that, The structural lipid compound is used to prepare nucleic acid drug carriers.

6. A lipid nanoparticle, characterized in that, This includes cationic lipids and structural lipid compounds as described in claim 1 or 2.

7. The lipid nanoparticles according to claim 6, characterized in that, The lipid nanoparticles also include cholesterol and polymer-modified lipids.

8. The lipid nanoparticles according to claim 7, characterized in that, The cationic lipid is selected from one or more of the following: methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester, [(4-hydroxybutyl)azadialkyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) ester, and heptadecan-9-yl-8-[(2-hydroxyethyl)(6-oxo-6-((decoxy)hexyl)amino)octanoate].

9. The lipid nanoparticles according to claim 7, characterized in that, The polymer-modified lipid is a polyethylene glycol-modified lipid.

10. A lipid nanoparticle, characterized in that, The lipid nanoparticles comprise the following components in molar percentage: 30-50% cationic lipids, 5-25% structural lipid compounds as described in claim 1 or 2, 28.5-48.5% cholesterol, and 0.5-3% polyethylene glycol-modified lipids.

11. A drug-loaded lipid nanoparticle, characterized in that, include: The lipid nanoparticles as described in any one of claims 6-10 and the nucleic acid drug loaded in the lipid nanoparticles.

12. A method for preparing drug-loaded lipid nanoparticles as described in claim 11, characterized in that, The preparation method includes the following steps: An oil phase containing the components of the lipid nanoparticles and an aqueous phase containing nucleic acid drugs were prepared separately. The oil phase and the aqueous phase were mixed using a microfluidic device to self-assemble the drug-loaded lipid nanoparticles.

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