Lipid compound of L-malic acid as well as preparation method and application of lipid compound

Abstract

The invention discloses a lipid compound of L-malic acid as well as a preparation method and application thereof, and belongs to the technical field of ionized lipid. The structure of the lipid compound of L-malic acid is shown as a formula 1, a is selected from 1-10, and b is selected from 1-5. The lipid compounds of L-malic acid can be used to prepare vaccine formulations of nanoparticle compositions. The preparation method of the lipid compound of L-malic acid is mild in condition, high in product yield, safe, reliable and suitable for large-scale industrial production. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of ionizable lipids, and particularly to a lipid compound of L - malic acid, its preparation method and application. Background Art

[0002] Ionizable lipids are lipids that can be ionized under specific conditions and are commonly used in lipid nanoparticle (LNPs) systems for nucleic acid delivery. These lipids have unique chemical structures that can be protonated in an acidic environment, thereby enhancing the nucleic acid release ability. When preparing nanoparticle compositions, ionizable lipids can interact electrostatically with nucleic acid molecules to help form stable nanoparticles. In addition, they usually include hydrophobic tails to enhance the membrane fusion ability of the lipids, assist the binding of lipid nanoparticles to cell membranes, and thus improve the cell uptake efficiency. Ionizable lipids have wide application values in fields such as mRNA vaccines, gene therapy, and RNA interference (RNAi) drugs, and are one of the key components of the LNP delivery system.

[0003] Since ionizable lipids play important roles in various biological systems, such as in the construction of cell membrane structure and function, signal transduction, cell division, cell migration, and immune responses, they have received extensive attention from researchers in the biological and pharmaceutical fields. Moreover, when applied to vaccine formulations of nanoparticle compositions, they can be made into the most advanced type of RNA delivery carriers. In addition, the global market sales of lipid drug delivery reached 3.3 billion US dollars in 2021 and are expected to reach 6.5 billion US dollars in 2028, with a compound annual growth rate (CAGR) of 10.0% (2022 - 2028).

[0004] Therefore, the research and development of new ionizable lipids is of great significance for their further application in biological systems. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a lipid compound of L - malic acid, its preparation method and application. The lipid compound of L - malic acid can be used to prepare vaccine formulations of nanoparticle compositions. The preparation method of the lipid compound of L - malic acid has mild conditions, high product yield, and is safe and reliable, and is suitable for large - scale industrial production.

[0006] To achieve the above - mentioned purpose, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a lipid compound of L - malic acid, and its structure is shown in Formula 1:

[0008]

[0009] Wherein, a is selected from 1 - 10, and b is selected from 1 - 5.

[0010] Preferably, a is selected from 1, 2, 3, 8 or 10;

[0011] Preferably, b is selected from 1, 2, 3 or 5.

[0012] Preferably, the lipid compound of L-malic acid of the present invention is selected from the structures shown in Formula 1-1 or Formula 1-2:

[0013]

[0014] The present invention also provides a method for preparing the above lipid compound of L-malic acid, comprising the following steps:

[0015] (1) Mix and react L-malic acid, Compound A, a condensing agent and a base to obtain an intermediate product shown in Formula 2;

[0016] (2) Mix and react the intermediate product shown in Formula 2 with an acid to obtain an intermediate product shown in Formula 3;

[0017] (3) Mix and react the intermediate product shown in Formula 3, the aldehyde of C 3 -C 12 and a solvent to obtain a mixed system S1, and then add a reducing agent and an acidic catalyst to react to obtain a lipid compound of L-malic acid;

[0018] Compound A is selected from tert-butyl N-(6-aminohexyl)carbamate, N-Boc-1,2-ethylenediamine, N-Boc-1,3-propanediamine or N-Boc-1,4-butanediamine;

[0019]

[0020] Among them, b is selected from 1-5.

[0021] Preferably, the condensing agent in step (1) is selected from one or more of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), 1H-benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP); more preferably HATU or TBTU.

[0022] Preferably, the base in step (1) is selected from triethylamine or N,N-diisopropylethylamine (DIEA);

[0023] Preferably, the temperature of the mixing reaction in step (1) is 0 to 40 °C; more preferably 0 to 10 °C or 20 to 30 °C.

[0024] After the reaction in step (1) is completed, post-extraction and other treatments are also included.

[0025] The present invention does not have any special limitation on the solvent for extraction, and any extraction solvent well-known to those skilled in the art can be used.

[0026] In some specific embodiments of the present invention, the extraction solvent is ethyl acetate and water, or dichloromethane and water.

[0027] The solvent for the mixing reaction in step (1) is selected from one or more of dichloromethane, DMF, and tetrahydrofuran; more preferably DMF or dichloromethane.

[0028] Preferably, in the present invention, the acid in step (2) is selected from trifluoroacetic acid or hydrogen chloride;

[0029] Preferably, the temperature of the mixing reaction in step (2) is 0 to 80 °C;

[0030] Preferably, the solvent for the mixing reaction in step (2) is selected from one or more of methanol, ethanol, dioxane, and ethyl acetate; more preferably ethyl acetate.

[0031] Preferably, in the present invention, the temperature of the mixing reaction in step (3) is 0 to 25 °C, and the time is 15 min to 30 min;

[0032] Preferably, the reducing agent in step (3) is selected from sodium triacetoxyborohydride, sodium cyanoborohydride, or sodium borohydride; more preferably sodium triacetoxyborohydride or sodium borohydride.

[0033] Preferably, the acidic catalyst in step (3) is selected from acetic acid;

[0034] Preferably, after adding the reducing agent and the acidic catalyst in step (3), the reaction temperature is 0 to 60 °C, and the time is 6 h to 24 h. More preferably 20 to 30 °C.

[0035] Preferably, after the reaction in step (3) is completed, post-treatments such as extraction and column chromatography are also included.

[0036] The present invention does not have any special limitation on the solvent for extraction, and any extraction solvent well-known to those skilled in the art can be used.

[0037] In some specific embodiments of the present invention, the extraction solvent is dichloromethane and sodium bicarbonate solution.

[0038] The solvent for the column chromatography is preferably dichloromethane and methanol, and the volume ratio of the two is 100:1 to 10:1.

[0039] Preferably in the present invention, in the step (3), C 3 -C 12 The aldehyde is selected from propionaldehyde, n-butanal, n-pentanal, decanal or dodecanal.

[0040] Preferably in the present invention, the solvent for the mixed reaction in the step (3) is selected from one or more of methanol, dichloromethane, and ethyl acetate; more preferably dichloromethane.

[0041] The present invention also provides the use of the lipid compound of L-malic acid as described above or the lipid compound of L-malic acid prepared by the preparation method as described above in a vaccine preparation of a nanoparticle composition.

[0042] Compared with the prior art, the lipid compound of L-malic acid provided by the present invention has a structure as shown in formula 1, wherein a is selected from 1-10 and b is selected from 1-5. The lipid compound of L-malic acid can be used to prepare a vaccine preparation of a nanoparticle composition. The preparation method of the lipid compound of L-malic acid has mild conditions, high product yield, and is safe and reliable, and is suitable for large-scale industrial production. Description of the Drawings

[0043] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of compound V prepared in Example 1. Detailed Description of the Invention

[0044] In order to further illustrate the present invention, the lipid compound of L-malic acid provided by the present invention, its preparation method and application will be described in detail below in conjunction with examples.

[0045] Example 1

[0046] 1) Add 8.00 g of L-malic acid, 28.39 g of tert-butyl N-(6-aminohexyl)carbamate, 45.98 g of TBTU, and 160 mL of DMF to a reaction flask. After adding, start stirring and cool down to 0 °C; keep the internal temperature at 0-10 °C and start dropping 18.51 g of N,N-diisopropylethylamine; after dropping, keep the reaction at 0-10 °C for 2 hours; add ethyl acetate and water, stir, let stand, and separate layers. Collect the upper organic phase, and concentrate the organic phase under reduced pressure to dryness at 35-45 °C; recrystallize with methanol and water to obtain 29.82 g of compound II, with a yield of 94.4%.

[0047] 2) Add 18.62 g of compound II and 140.5 mL of a 2 mol / L hydrochloric acid solution in ethyl acetate to a reaction flask, and keep the reaction at 20-30 °C for 2 hours. Filter and dry to obtain 13.51 g of compound III, with a yield of 95.5%.

[0048] 3) Add 12.21 g of Compound III to the reaction flask, 240 mL of dichloromethane. Add 11.00 g of DIEA at 20 - 30 °C, then add 31.37 g of dodecanal, and stir for 15 min at 20 - 30 °C; add 36.08 g of sodium triacetoxyborohydride; then add 8.58 g of acetic acid, and react at 20 - 30 °C for 16 h; add dichloromethane and sodium bicarbonate solution for extraction, and concentrate the organic phase to dryness; obtain 22.15 g of Compound V as a transparent oily substance through silica gel column chromatography (eluent: dichloromethane:methanol = 100:1 - 10:1), with a yield of 73.0%. The hydrogen spectrum data is as Figure 1 shown.

[0049]

[0050] Example 2

[0051] 1) Add 8.00 g of L - malic acid and 21.03 g of N - tert - butoxycarbonyl - 1,2 - ethylenediamine, 49.91 g of HATU, and 160 mL of dichloromethane to the reaction flask. After adding, start stirring; keep the internal temperature at 20 - 30 °C and start dropping 14.49 g of triethylamine; after dropping, react at 20 - 30 °C for 2 h; add dichloromethane and water, stir, let stand, separate layers, collect the organic phase, and concentrate the organic phase to dryness under reduced pressure at 35 - 45 °C; obtain 22.15 g of Compound VI through recrystallization with methanol and water, with a yield of 88.7%.

[0052] 2) Add 19.25 g of Compound VI and 112 mL of ethanol solution of 2 mol / L hydrogen chloride to the reaction flask, react at 20 - 30 °C for 2 h, filter and dry to obtain 11.41 g of Compound VII, with a yield of 85.1%.

[0053] 3) Add 10.15 g of Compound VII, 220 mL of dichloromethane to the reaction flask. Add 10.6 g of triethylamine at 20 - 30 °C, then add 32.69 g of decanal, and stir for 15 min at 20 - 30 °C; add 7.91 g of sodium borohydride; then add 10.46 g of acetic acid, and react at 20 - 30 °C for 20 h; add dichloromethane and sodium bicarbonate solution for extraction, and concentrate the organic phase to dryness; obtain 21.29 g of Compound VIII as a transparent oily substance through silica gel column chromatography (eluent: dichloromethane:methanol = 100:1 - 10:1), with a yield of 78.4%.

[0054]

[0055] The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A lipid compound of L-malic acid, characterized in that Its structure is shown in formula 1: Among them, a is selected from 1-10, and b is selected from 1-5.

2. The lipid compound of L-malic acid according to claim 1, characterized in that The a is selected from 1, 2, 3, 8 or 10; The b is selected from 1, 2, 3 or 5.

3. The lipid compound of L-malic acid according to claim 1, characterized in that The lipid compound of L-malic acid is selected from the structure shown in Formula 1-1 or Formula 1-2:

4. The method for preparing a lipid compound of L-malic acid according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) mixing L-malic acid, compound A, a condensing agent and a base to react to obtain an intermediate product shown in formula 2; (2) mixing the intermediate product of Formula 2 with an acid to obtain an intermediate product of Formula 3; (3) The intermediate product shown in Formula 3, C3-C 12 The aldehyde and the solvent are mixed to obtain a mixed system S1, and then a reducing agent and an acid catalyst are added to react to obtain a lipid compound of L-malic acid; The compound A is selected from tert-butyl N-(6-aminohexyl)carbamate, N-tert-butoxycarbonyl-1,2-ethylenediamine, N-tert-butoxycarbonyl-1,3-propylenediamine or N-tert-butoxycarbonyl-1,4-butanediamine; Wherein, b is selected from 1-5.

5. The preparation method according to claim 4, characterized in that: The condensing agent in step (1) is selected from one or more of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate, 2-(1H-benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate; The base in step (1) is selected from triethylamine or N,N-diisopropylethylamine; The temperature of the mixing reaction in step (1) is 0 to 40°C.

6. The preparation method according to claim 4, characterized in that: The acid in step (2) is selected from trifluoroacetic acid or hydrogen chloride; The temperature of the mixed reaction in step (2) is 0 to 80° C. The solvent for the mixed reaction in step (2) is selected from one or more of methanol, ethanol, dioxane and ethyl acetate.

7. The preparation method according to claim 4, characterized in that: The temperature of the mixed reaction in step (3) is 0 to 25°C and the time is 15 to 30 minutes; The reducing agent in step (3) is selected from sodium triacetoxyborohydride, sodium cyanoborohydride or sodium borohydride; The acidic catalyst in step (3) is selected from acetic acid; After adding the reducing agent and the acid catalyst in step (3), the reaction temperature is 0 to 60° C. and the reaction time is 6 to 24 hours.

8. The preparation method according to claim 4, characterized in that: In the step (3), C3-C 12 The aldehyde is selected from propionaldehyde, n-butyraldehyde, n-valeraldehyde, decanal or dodecanal.

9. The preparation method according to claim 4, characterized in that: The solvent for the mixed reaction in step (3) is selected from one or more of methanol, dichloromethane and ethyl acetate.

10. Use of the lipid compound of L-malic acid according to any one of claims 1 to 3 or the lipid compound of L-malic acid prepared by the preparation method according to any one of claims 4 to 9 in a vaccine preparation of a nanoparticle composition.

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