Alkyl ether ionizable lipid compound and application thereof

Abstract

The invention discloses an alkyl ether ester ionizable lipid compound and application thereof. The alkyl ether ionizable lipid compound is composed of a head group R1, a linker L and a tail group R2 in sequence. According to the alkyl ether ionizable lipid disclosed by the invention, two tertiary amine centers are connected by adopting a cyclic carbon chain structure, so that the delivery effect is obviously higher than that of ionizable lipid linearly connected with the two tertiary amine centers, and by adding an ether bond-containing tail structure in the alkyl ether ionizable lipid, the cell endocytosis and the cell endosome escape effect of LNP are improved; therefore, more mRNA is delivered into a living body and translated and expressed into corresponding proteins. Therefore, compared with the ionizable lipid in the prior art, the alkyl ether ionizable lipid disclosed by the invention has a better delivery effect.

Description

Technical Field

[0001] The invention belongs to the field of new medicinal materials and relates to an alkyl ether ionizable lipid compound and application thereof. Background Art

[0002] As we all know, the COVID-19 outbreak has significantly advanced the development of mRNA vaccines. Compared to conventional vaccines, mRNA vaccines offer advantages such as low cost, high production efficiency, and superior safety. They also have the potential to synthesize any protein. Therefore, they hold enormous potential for use against emerging infectious viruses that traditional vaccines are unable to combat. However, the application of mRNA vaccines has been limited due to the instability of mRNA molecules, their susceptibility to degradation by RNases, and their low in vivo delivery efficiency. To achieve widespread application of mRNA vaccines, addressing the delivery system is crucial. mRNA vaccines require a suitable delivery vehicle to deliver them into the body for optimal immune efficacy, so developing an efficient and stable delivery vehicle is crucial to their success.

[0003] Lipid nanoparticles (LNPs), an advanced delivery vehicle for mRNA, typically contain at least an ionizable lipid, a neutral auxiliary lipid, and a phospholipid polyethylene glycol derivative. The ionizable lipid compound plays a key role in the physical and chemical properties and delivery efficacy of the LNP. The structure of an ionizable lipid compound primarily consists of a head, a linker, and a tail. Many classic ionizable lipid compounds, such as C12-200 (CAS Number 1220890-25-4), are prepared by reacting primary amines with alkylene oxides. They have an alkyl tail structure, resulting in limited delivery efficacy in animals, restricting their commercial applications. Summary of the Invention

[0004] The purpose of the present invention is to provide an alkyl ether ionizable lipid compound and its application in view of the above-mentioned deficiencies in the prior art.

[0005] Another object of the present invention is to provide a lipid nanoparticle composition.

[0006] Another object of the present invention is to provide applications of the lipid nanoparticle composition.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] An alkyl ether ionizable lipid compound is sequentially composed of a head group tertiary amine group R1, a linker L, and a tail group R2, characterized in that the structure is as follows: 、 、 、 ; Wherein, R4 is selected from ; R5 is selected from 、 ; R3 is selected from -L-R2, R6, L is selected from -CH2CH(OH)CH2-, R2 is selected from -OC 12 H 25 、-OC 14 H 29 、-OC 16 H 33 The linear alkyl ether, R6 is selected from cyclohexyl, C 1-6 alkyl, and in an alkyl ether ionizable lipid compound, there are at most two R3 selected from R6; q = an integer from 1 to 4.

[0009] As a preferred embodiment of the present invention, the alkyl ether ionizable lipid compound is selected from any one of the following: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 .

[0010] A composition of alkyl ether ionizable lipid compounds, comprising and composition.

[0011] The invention relates to an application of the alkyl ether ionizable lipid compound in preparing lipid nanoparticles for delivering nucleic acid.

[0012] The composition is used in preparing lipid nanoparticles for delivering nucleic acids.

[0013] A lipid nanoparticle composition comprising the alkyl ether ionizable lipid compound according to claim 1 or the composition according to claim 3.

[0014] As a preference of the present invention, the lipid nanoparticle composition further comprises one or more of neutral lipids and PEG lipids.

[0015] As a preferred embodiment of the present invention, the neutral lipid is selected from any one or two of DSPC and cholesterol; and the PEG lipid is selected from DMG-PEG2000.

[0016] As a preferred embodiment of the present invention, the molar ratio of the alkyl ether ionizable lipid compound according to claim 1 or the composition according to claim 3: DSPC: cholesterol: DMG-PEG2000 is 45-55:10-15:35-40:1-3.

[0017] Use of the lipid nanoparticle composition of the present invention in the preparation of mRNA drugs or vaccines.

[0018] Unless otherwise specified, the terms used in the present invention have the following definitions: “C 1-6 The term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, pentyl, neopentyl, hexyl, etc. The alkyl group may be substituted or unsubstituted, and may have multiple substituents.

[0019] "Alkyl ether" refers to an ether compound in which at least one of the two hydrocarbon groups connecting the oxygen atom in the ether molecule is an alkyl group (i.e., a saturated hydrocarbon group).

[0020] Beneficial effects: The alkyl ether ionizable lipid compound of the present invention has good physicochemical properties and excellent in vivo mRNA delivery efficiency.

[0021] The alkyl ether ionizable lipid compound of the present invention has a simple synthesis process, high synthesis efficiency, low production cost, and is convenient for commercial production.

[0022] The alkyl ether ionizable lipids of the present invention utilize a cyclic carbon chain structure to connect two tertiary amine centers, resulting in significantly higher delivery efficiency than ionizable lipids with a linear structure connecting two tertiary amine centers. Furthermore, the ether-bonded tail structure in the alkyl ether ionizable lipids enhances LNP endocytosis and endosomal escape, allowing more mRNA to be delivered into the body and translated into the corresponding protein. Therefore, the alkyl ether ionizable lipids of the present invention exhibit superior delivery efficiency compared to prior art ionizable lipids. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Lipid nanoparticles prepared with alkyl ether ionizable lipids O-7 to 0-15 were injected into mice muscles and photographed by animal imaging equipment.

[0024] Figure 2 Lipid nanoparticles prepared with alkyl ether ionizable lipids 0-34 to 0-39 and alkyl ether ionizable lipids 0-45 and 0-46 were injected into mice muscles and photographed with animal imaging equipment.

[0025] Figure 3 Lipid nanoparticles prepared with control ionizable lipids D-1 to D-5 were injected into mice intramuscularly and photographed using an animal imaging device. DETAILED DESCRIPTION

[0026] Example 1 Synthesis of Alkyl Ether Ionizable Lipid O-7:

[0027] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0028] To a 100 mL reaction flask equipped with a magnetic rod, 5 mmol of 1,3-cyclohexanediamine, 15 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a yellow oily product in a yield of 87%. 1 HNMR (400 MHz, CDCl3) δ 3.98-3.24 (m, 34H), 2.73-2.39 (m, 8H), 1.67-1.50 (m,9H), 1.39-1.16 (m, 79H), 0.90 (t, 12H).

[0029] The specific reaction formula is as follows:

[0030]

[0031] Example 2 Synthesis of Alkyl Ether Ionizable Lipid O-8:

[0032] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0033] To a 100 mL reaction flask equipped with a magnetic bar, 5 mmol of 1,2-cyclohexanediamine, 30 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a light yellow oily product in a yield of 89%. 1 HNMR (400 MHz, CDCl3) δ 4.01-3.88 (m, 3H), 3.63-3.31 (m, 25H), 1.65-1.51 (m,8H), 1.41-1.15 (m, 95H), 0.88 (t, 12H).

[0034] The specific reaction formula is as follows:

[0035]

[0036] Example 3 Synthesis of Alkyl Ether Ionizable Lipid O-9:

[0037] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0038] To a 100 mL reaction flask equipped with a magnetic bar, 5 mmol of cis-1,3-bis(aminomethyl)cyclohexane, 30 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a yellow oily product in a yield of 88%. 1 H NMR (400 MHz, CDCl3) δ 3.91-3.29 (m, 37H), 2.73-2.20 (m, 11H), 1.87-1.67 (m, 4H), 1.65-1.51 (m, 10H), 1.40-1.17 (m, 89H), 0.90 (t, 12H).

[0039] The specific reaction formula is as follows:

[0040]

[0041] Example 4 Synthesis of Alkyl Ether Ionizable Lipid O-10:

[0042] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0043] To a 100 mL reaction flask equipped with a magnetic bar, 5 mmol of trans-1,4-bis(aminomethyl)cyclohexane, 30 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a yellow oily product in a yield of 77%. 1 H NMR (400 MHz, CDCl3) δ 3.89-3.33 (m, 24H), 2.65-2.19 (m, 8H), 1.87-1.51 (m, 10H), 1.45-1.20 (m, 67H), 0.90 (t, 12H).

[0044] The specific reaction formula is as follows:

[0045]

[0046] Example 5 Synthesis of Alkyl Ether Ionizable Lipid O-11:

[0047] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0048] To a 100 mL reaction flask equipped with a magnetic bar, 5 mmol of cis-1,4-bis(aminomethyl)cyclohexane, 30 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a yellow oily product in a yield of 81%. 1 H NMR (400 MHz, CDCl3) δ 3.90-3.31 (m, 30H), 2.66-2.28 (m, 11H), 1.70-1.46 (m, 16H), 1.42-1.18 (m, 83H), 0.90 (t, 12H).

[0049] The specific reaction formula is as follows:

[0050]

[0051] Example 6 Synthesis of Alkyl Ether Ionizable Lipid O-12:

[0052] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0053] To a 100 mL reaction flask equipped with a magnetic bar, 5 mmol of cis-1,4-cyclohexanediamine, 30 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added in sequence. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a yellow oily product with a yield of 80%. 1 H NMR (400 MHz, CDCl3) δ 4.00-3.27 (m, 33H), 2.85-2.38 (m, 9H), 1.67-1.50 (m, 13H), 1.41-1.16 (m, 81H), 0.90 (t, 12H).

[0054] The specific reaction formula is as follows:

[0055]

[0056] Example 7 Synthesis of Alkyl Ether Ionizable Lipid O-13:

[0057] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0058] To a 100 mL reaction flask equipped with a magnetic bar, 5 mmol of 1,4-cyclohexanediamine, 30 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a yellow oily product in a yield of 91%. 1 HNMR (400 MHz, CDCl3) δ 3.94-3.28 (m, 32H), 2.85-2.35 (m, 10H), 1.64-1.49 (m,11H), 1.39-1.19 (m, 81H), 0.90 (t, 12H).

[0059] The specific reaction formula is as follows:

[0060]

[0061] Example 8 Synthesis of Alkyl Ether Ionizable Lipid O-14:

[0062] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0063] To a 100 mL reaction flask equipped with a magnetic bar, 5 mmol of trans-1,3-cyclohexanediamine, 30 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a yellow oily product with a yield of 85%. 1 H NMR (400 MHz, CDCl3) δ 3.88-3.24 (m, 35H), 2.73-2.38 (m, 7H), 1.64-1.49 (m, 8H), 1.39-1.19 (m, 84H), 0.90 (t, 12H).

[0064] The specific reaction formula is as follows:

[0065]

[0066] Example 9 Synthesis of Alkyl Ether Ionizable Lipid O-15:

[0067] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0068] To a 100 mL reaction flask equipped with a magnetic bar, 5 mmol of cis-1,3-cyclohexanediamine, 30 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a yellow oily product in a yield of 87%. 1 H NMR (400 MHz, CDCl3) δ 3.95-3.25 (m, 44H), 1.66-1.47 (m, 16H), 1.39-1.18 (m, 97H), 0.90 (t, 12H).

[0069] The specific reaction formula is as follows:

[0070] .

[0071] Example 10 Synthesis of Alkyl Ether Ionizable Lipid O-34:

[0072] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0073] To a 100 mL reaction flask equipped with a magnetic rod, 5 mmol of bicyclo[2.2.1]heptanedimethylamine, 30 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After completion of the reaction as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a brown oily product in a yield of 74%. 1 H NMR (400 MHz, CDCl3) δ 3.93-3.31 (m, 30H), 2.83-2.13 (m, 13H), 1.65-1.49 (m, 9H), 1.45-1.11 (m, 81H), 0.90 (t, 12H).

[0074] The specific reaction formula is as follows:

[0075] Example 11 Synthesis of Alkyl Ether Ionizable Lipid O-35:

[0076] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0077] To a 100 mL reaction flask equipped with a magnetic bar were added 5 mmol of 4-aminomethylpiperidine, 30 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a yellow oily product in a yield of 87%. 1 H NMR (400 MHz, CDCl3) δ 3.98-3.28 (m, 26H), 2.65-2.29 (m, 6H), 1.64-1.49 (m, 7H), 1.39-1.17 (m, 62H), 0.90 (t, 9H).

[0078] The specific reaction formula is as follows:

[0079]

[0080] Example 12 Synthesis of Alkyl Ether Ionizable Lipid O-36:

[0081] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0082] To a 100 mL reaction flask equipped with a magnetic rod were added 5 mmol of N-aminoethylpiperazine, 30 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a yellow oily product in a yield of 83%. 1 H NMR (400 MHz, CDCl3) δ 3.98-3.31 (m, 24H), 2.79-2.23 (m, 14H), 1.65-1.49 (m, 6H), 1.39-1.17 (m, 59H), 0.90 (t, 9H).

[0083] The specific reaction formula is as follows:

[0084]

[0085] Example 13 Synthesis of Alkyl Ether Ionizable Lipid O-37:

[0086] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0087] To a 100 mL reaction flask equipped with a magnetic rod, 5 mmol of 1,4-piperazinediethylamine, 30 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a brown oily product in a yield of 81%. 1 HNMR (400 MHz, CDCl3) δ 3.90-3.33 (m, 32H), 2.66-2.42 (m, 11H), 1.66-1.15 (m,91H), 0.90 (t, 12H).

[0088] The specific reaction formula is as follows:

[0089]

[0090] Example 14 Synthesis of Alkyl Ether Ionizable Lipid O-38:

[0091] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0092] To a 100 mL reaction flask equipped with a magnetic bar, 5 mmol of N-(2-aminoethyl)-1,4-piperazinediyldiethylamine, 40 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature and the solvent was removed using a rotary evaporator. The product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a brown oily product in a yield of 73%. 1 H NMR (400 MHz, CDCl3) δ 3.93-3.27 (m, 38H), 2.89-2.22 (m,30H), 1.65-1.49 (m, 11H), 1.39-1.18 (m, 98H), 0.90 (t, 15H).

[0093] The specific reaction formula is as follows:

[0094]

[0095] Example 15 Synthesis of Alkyl Ether Ionizable Lipid O-39:

[0096] Synthesis of 2-[(tetradecyloxy)methyl]oxirane: Same as Example 1.

[0097] To a 100 mL reaction flask equipped with a magnetic rod, 5 mmol of 1,4-bis(3-aminopropyl)piperazine, 30 mmol of 2-[(tetradecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a brown oily product in a yield of 82%. 1 H NMR (400 MHz, CDCl3) δ 4.01-3.28 (m, 34H), 2.94-2.30 (m, 15H), 1.69-1.47 (m, 11H), 1.37-1.20 (m, 79H), 0.90 (t, 12H).

[0098] The specific reaction formula is as follows:

[0099]

[0100] Example 16 Synthesis of Alkyl Ether Ionizable Lipid O-45:

[0101] Synthesis of 2-[(Dodecyloxy)methyl]oxirane: To a 50mL reaction flask equipped with a magnetic separator, add 5mL of n-hexane, 12-ol (0.3mmol), and NaOH (0.3mmol) in sequence. Stir vigorously. When the solution temperature reaches 60°C, add 3-chloro-1,2-propylene oxide (0.2mmol) and water (0.1mmol). After reacting for 4 hours, filter the product. Cool the reaction to room temperature, wash the solid phase three times with n-hexane (3 x 5mL). The eluate is concentrated under reduced pressure and separated by thin-layer chromatography (silica gel column, eluent: dichloromethane) to obtain the product as a white solid in a 59% yield. The specific reaction formula is as follows:

[0102]

[0103] To a 100 mL reaction flask equipped with a magnetic rod, 5 mmol of 1,2-cyclohexanediamine, 30 mmol of 2-[(dodecyloxy)methyl]oxirane, and 50 mL of anhydrous ethanol were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature, the solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: ethyl acetate: petroleum ether: triethylamine = 20:15:1) to obtain a yellow oily product in a yield of 84%. 1 H NMR (400 MHz, CDCl3) δ 4.18-4.05 (m, 4H), 4.02-3.88 (m, 3H), 3.61-3.37 (m, 22H), 1.65-1.49 (m, 8H), 1.38-1.13 (m, 80H), 0.88 (t, 12H).

[0104] The specific reaction formula is as follows:

[0105]

[0106] Example 17 Synthesis of Alkyl Ether Ionizable Lipid Composition O-46:

[0107] The synthetic products O-8 and O-45 of Example 8 and Example 45 were mixed at a molar ratio of 1:1, and the mixture was the alkyl ether ionizable lipid composition O-46.

[0108] Comparative Example 1 Synthesis of ionizable lipid D-1:

[0109] To a 100 mL reaction flask equipped with a magnetic bar, 5 mmol of 1,8-diamino-3,6-dioxaoctane, 30 mmol of 1,2-epoxytetradecane, and 30 mL of dichloromethane were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature. The solvent was removed using a rotary evaporator, and the product was separated by thin-layer chromatography (silica gel column, eluent: dichloromethane:methanol:triethylamine (volume ratio) = 93:6:1) to obtain a pale yellow oily product D-1 in a yield of 89%. 1 H NMR (400 MHz, CDCl3) δ 3.86-3.35 (m, 14H), 2.93-2.28 (m, 11H), 1.54-1.14 (m, 88H), 0.90 (t, 12H).

[0110] The specific reaction formula is as follows:

[0111]

[0112] Comparative Example 2 Synthesis of control ionizable lipid D-2:

[0113] To a 100 mL reaction flask equipped with a magnetic rod, 5 mmol of 1,2-diaminocyclohexane, 30 mmol of 1,2-epoxytetradecane, and 30 mL of dichloromethane were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature and the solvent was removed using a rotary evaporator. The product was separated by thin-layer chromatography (silica gel column, eluent: dichloromethane:methanol:triethylamine, volume ratio = 93:6:1) to obtain a yellow oily product D-1 in a yield of 90%. 1 H NMR (400 MHz, CDCl3) δ 3.83-3.16 (m, 9H), 1.53-1.14 (m, 93H), 0.90 (t, 12H).

[0114] The specific reaction formula is as follows:

[0115]

[0116] Comparative Example 3 Synthesis of ionizable lipid D-3:

[0117] To a 100 mL reaction flask equipped with a magnetic rod, 5 mmol of N-(2-aminoethyl)-1,4-piperazinediyldiethylamine, 40 mmol of 1,2-epoxytetradecane, and 30 mL of dichloromethane were added sequentially. The reaction tube was heated and stirred at 80°C for 72 hours. After the reaction was complete as monitored by TLC, the reaction was cooled to room temperature and the solvent was removed using a rotary evaporator. The product was separated by thin-layer chromatography (silica gel column, eluent: dichloromethane:methanol:triethylamine, volume ratio = 93:6:1) to obtain a brown oily product D-1 in a yield of 84%. 1 H NMR (400 MHz, CDCl3) δ 3.78-3.48 (m, 7H), 2.92-2.18 (m, 23H), 1.55-1.17 (m, 93H), 0.90 (t, 12H).

[0118] The specific reaction formula is as follows:

[0119]

[0120] Comparative Example 4

[0121] Other control ionizable lipids:

[0122] The control ionizable lipid D-4: C12-200, CAS number: 1220890-25-4, was purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd., and the structural formula is as follows.

[0123]

[0124] The control ionizable lipid D-5: SM-102, CAS No.: 2089251-47-6, was purchased from Xiamen Sinobond Biotechnology Co., Ltd., and the structural formula is as follows.

[0125]

[0126] Example 18: Preparation and Detection of Lipid Nanoparticles (LNP Formulations)

[0127] S1. Prepare a 5 mM pH 3.0 citric acid buffer solution containing firefly luciferase (fluc) mRNA as the aqueous phase.

[0128] S2. Prepare an anhydrous ethanol solution of the ionizable lipids of the above Examples and Comparative Examples, DSPC, cholesterol, and DMG-PEG 2000. Prepare a lipid mixed solution as the organic phase according to a molar ratio of ionizable lipid compound lipid: DSPC: cholesterol: DMG-PEG 2000 of 50:10:38.5:1.5.

[0129] S3, through the microfluidic device, the above two solutions are mixed in the microfluidic chip, the aqueous phase and organic phase solutions enter from the two sides and the middle flow path of the microfluidic chip respectively, the total flow rate of the aqueous phase is controlled at 3ml / min, the organic phase flow rate is controlled at 1ml / min, the aqueous phase: organic phase flow rate ratio is 3:1, the two phases flow path converge in the chip and then mix, the weight ratio of total lipids to mRNA is about 15~30:1, and mRNA lipid nanoparticles (mRNA-LNP) are prepared by combining positively charged lipids with negatively charged mRNA.

[0130] In step S4, the obtained mRNA-LNPs were diluted 10-fold with 10 mM PBS buffer (pH 7.0) and purified by ultrafiltration through a 100 kDa pore size ultrafiltration tube. The ultrafiltration method was to concentrate the mRNA to 1 / 10 of its volume using a 30-degree fixed-angle rotor, a centrifugal force of 2000 g, and room temperature at 25°C.

[0131] S5, mRNA-LNP was filtered through a 0.22 μm filter membrane and stored at 2-8 °C.

[0132] S6. The average particle size and polydispersity index (PDI) of the mRNA-LNP sample were determined using a dynamic light scattering nanoparticle size analyzer. The test results are shown in Table 2.

[0133] S7. Determine the encapsulation efficiency of lipid nanoparticles using the Quant-it Ribogreen RNA Quantification Assay Kit (ThermoFisher Scientific, UK) according to the manufacturer's instructions. Specific steps are: (1) Dilute the prepared mRNA-LNP suspension and PBS (negative control, equal volume of TE buffer) to 4 ng / μl using the TE buffer in the kit to obtain the mRNA-LNP working solution.

[0134] (2) Continue to use TE buffer (or TE buffer containing 2% Triton-X100) to further dilute the mRNA-LNP working solution to an equal volume, mix well and let it stand at 37°C for 10 minutes (TE buffer without Triton-X100 is used to measure the unencapsulated free mRNA, while TE buffer containing 2% Triton-X100 is used to measure the total mRNA in the mRNA-LNP working solution, which includes free mRNA and mRNA encapsulated in lipid nanoparticles).

[0135] (3) After calibrating the fluorescence intensity:concentration standard curve with the standard sample, pipette an appropriate amount of Quant-it™ RiboGreen RNA reagent nucleic acid dye into each group of samples according to the kit instructions and stain for 5 minutes. After staining, transfer each group of samples into a microplate reader for detection and use the software to accurately quantify the mRNA in the sample.

[0136] (4) The mRNA encapsulation efficiency in lipid nanoparticles was calculated using the following formula: Encapsulation efficiency = [1-m (free mRNA): m (total mRNA)] × 100%. The test results are shown in Table 1.

[0137] Table 1

[0138] Example 19 Verification of delivery effect through in vivo animal experiments The mRNA lipid nanoparticles prepared in Example 18 were injected into the leg muscles of mice at a dose of 5 μg mRNA per mouse. Six hours later, the delivery effect was detected by taking a picture with an animal imaging device and calculating the fluorescence intensity of the injection site. The test results are shown in Tables 2 and Figure 1-3 .

[0139] Table 2

[0140] According to the data in Table 2, the delivery effect of mRAN-LNPs prepared from the alkyl ether ionizable lipids 0-7 to 0-15, 0-34 to 0-39, 0-45 and composition 0-46 of the present invention in animals reached the level of commercial ionizable lipid SM-102 (control ionizable lipid D-5), and most of them exceeded the delivery effect of ionizable lipid SM-102.

[0141] Compared with the control ionizable lipid D-1, the alkyl ether ionizable lipid of the present invention has a significantly higher delivery effect than the ionizable lipid with a linear structure connecting two tertiary amine centers due to the use of a cyclic carbon chain structure. In addition, the tail structure containing an ether bond in the alkyl ether ionizable lipid improves the endocytosis and endosome escape of the LNP, allowing more mRNA to be delivered into the body and translated into the corresponding protein. Therefore, the alkyl ether ionizable lipid of the present invention has a better delivery effect than the ionizable lipid of the prior art.

[0142] Comparing the alkyl ether ionizable lipids 0-8, 0-45, and 0-46 with the control ionizable lipid D-2, the ionizable lipids with alkyl ether tails, despite having the same head tertiary amine structure, showed significantly higher delivery efficacy than the control ionizable lipid D-2. Furthermore, analysis revealed that ionizable lipids with C12 alkyl ether tails (0-45), C14 alkyl ether tails (0-8), and a mixed C12 and C14 alkyl ether tail (0-46) all exhibited excellent in vivo delivery efficacy.

[0143] Comparing the alkyl ether ionizable lipid 0-38 with the control ionizable lipids D-3 and D-4, the alkyl ether tail ionizable lipid 0-38, with the same head tertiary amine structure, showed significantly higher delivery efficiency than the control ionizable lipids D-3 and D4. Both the C12 alkyl tail ionizable lipid compound (D-3) and the commercial ionizable lipid C12-200 (C10 alkyl tail) showed significantly lower delivery efficiency than the alkyl ether tail ionizable lipids with the same tertiary amine core structure.

Claims

1. An alkyl ether ionizable lipid compound, consisting of a head group tertiary amine group R1, a linker L and a tail group R2, characterized in that: The structure is as follows: 、 、 、 ； Wherein, R4 is selected from ; R5 is selected from 、 ; R3 is selected from -L-R2, R6, L is selected from -CH2CH(OH)CH2-, R2 is selected from -OC 12 H 25 、-OC 14 H 29 、-OC 16 H 33 The linear alkyl ether, R6 is selected from cyclohexyl, C 1-6 alkyl, and in an alkyl ether ionizable lipid compound, there are at most two R3 selected from R6; q=an integer from 1 to 4.

2. The alkyl ether ionizable lipid compound according to claim 1, characterized in that The alkyl ether ionizable lipid compound is selected from any one of the following: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 3. A composition of an alkyl ether ionizable lipid compound, characterized in that: According to claim 2 and composition.

4. Use of the alkyl ether ionizable lipid compound according to claim 1 in preparing lipid nanoparticles for delivering nucleic acids.

5. Use of the composition according to claim 3 in preparing lipid nanoparticles for delivering nucleic acids.

6. A lipid nanoparticle composition, characterized in that Containing the alkyl ether ionizable lipid compound according to claim 1 or the composition according to claim 3.

7. The lipid nanoparticle composition according to claim 6, characterized in that The lipid nanoparticle composition further comprises one or more of neutral lipids and PEG lipids.

8. The lipid nanoparticle composition according to claim 7, characterized in that The neutral lipid is selected from any one or two of DSPC and cholesterol; and the PEG lipid is selected from DMG-PEG2000.

9. The lipid nanoparticle composition according to claim 7, characterized in that The molar ratio of the alkyl ether ionizable lipid compound according to claim 1 or the composition according to claim 3: DSPC: cholesterol: DMG-PEG2000 is 45-55:10-15:35-40:1-3.

10. Use of the lipid nanoparticle composition according to any one of claims 6 to 9 in the preparation of mRNA drugs or vaccines.

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